Clinical Focus


  • Pathology and Laboratory Medicine
  • Pathology
  • Clinical Biochemical Genetics

Academic Appointments


Administrative Appointments


  • Program Director, Clinical Biochemical Genetics Fellowship (2019 - Present)
  • Board of Directors, Society of Inherited Metabolic Disorders (2015 - Present)
  • Board of Directors, American College of Medical Genetics and Genomics (2015 - Present)
  • Board of Directors, American Board of Medical Genetics (2006 - 2011)
  • Faculty Senate, Stanford University School of Medicine (2005 - 2008)
  • Laboratory Quality Assurance Committee, American College of American Genetics and Genomics (2003 - 2007)

Boards, Advisory Committees, Professional Organizations


  • Member, American Society of Human Genetics (1986 - Present)
  • Member, Society for the Study of Inborn Errors of Metabolism (2002 - Present)
  • Member, Association of Biochemistry Educators (2013 - Present)

Professional Education


  • Board Certification: American Board of Medical Genetics and Genomics, Clinical Biochemical/ Molecular Genetics (1990)
  • PhD Training: UCLA David Geffen School Of Medicine Registrar (1986) CA
  • Fellowship: University of Maryland Baltimore (1989)
  • Board Certification: American Board of Medical Genetics and Genomics, Medical Genetics (1990)
  • B.A., UCLA, Biology (1979)
  • Ph.D., UCLA, Genetics (1986)

Patents


  • Tina Cowan, Anthony Le. "United States Patent 8,945,933 Liquid Chromatography-Mass Spectrometry Methods For Multiplexed Detection and Quantitation of Free Amino Acids", Stanford, Feb 3, 2015

Current Research and Scholarly Interests


Translational research activities are aimed at the development of new laboratory testing approaches for the diagnosis and monitoring of patients with inborn errors of metabolism. In particular, a tandem-mass spectrometry (MS/MS)-based test for glutathione, an important antioxidant, is being applied to the evaluation of patients with various metabolic disorders including mitochondrial myopathies. Results of these studies will further our understanding of the pathophysiology of metabolic diseases, with the ultimate aim of improving patient testing and developing new theraputic strategies.

2024-25 Courses


Graduate and Fellowship Programs


  • Human Genetics and Genetic Counseling (Masters Program)
  • Medical Genetics (Fellowship Program)

All Publications


  • Biochemical genetics decoded: A comparative study of ChatGPT and IEMbase Zapata, L., Cowan, T. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2024
  • A Phase 1 Study of Oral Vitamin D3 in Boys and Young Men With X-Linked Adrenoleukodystrophy. Neurology. Genetics Van Haren, K. P., Cunanan, K., Awani, A., Gu, M., Peña, D., Chromik, L. C., Považan, M., Rossi, N. C., Goodman, J., Sundaram, V., Winterbottom, J., Raymond, G. V., Cowan, T., Enns, G. M., Waubant, E., Steinman, L., Barker, P. B., Spielman, D., Fatemi, A. 2023; 9 (2): e200061

    Abstract

    There are no therapies for preventing cerebral demyelination in X-linked adrenoleukodystrophy (ALD). Higher plasma vitamin D levels have been linked to lower risk of inflammatory brain lesions. We assessed the safety and pharmacokinetics of oral vitamin D dosing regimens in boys and young men with ALD.In this open-label, multicenter, phase 1 study, we recruited boys and young men with ALD without brain lesions to a 12-month study of daily oral vitamin D3 supplementation. Our primary outcome was attainment of plasma 25-hydroxyvitamin D levels in target range (40-80 ng/mL) at 6 and 12 months. Secondary outcomes included safety and glutathione levels in the brain, measured with magnetic resonance spectroscopy, and blood, measured via mass spectrometry. Participants were initially assigned to a fixed dosing regimen starting at 2,000 IU daily, regardless of weight. After a midstudy safety assessment, we modified the dosing regimen, so all subsequent participants were assigned to a weight-stratified dosing regimen starting as low as 1,000 IU daily.Between October 2016 and June 2019, we enrolled 21 participants (n = 12, fixed-dose regimen; n = 9, weight-stratified regimen) with a median age of 6.7 years (range: 1.9-22 years) and median weight of 20 kg (range: 11.7-85.5 kg). The number of participants achieving target vitamin D levels was similar in both groups at 6 months (fixed dose: 92%; weight stratified: 78%) and 12 months (fixed dose: 67%; weight stratified: 67%). Among the 12 participants in the fixed-dose regimen, half had asymptomatic elevations in either urine calcium:creatinine or plasma 25-hydroxyvitamin D; no laboratory deviations occurred with the weight-stratified regimen. Glutathione levels in the brain, but not the blood, increased significantly between baseline and 12 months.Our vitamin D dosing regimens were well tolerated and achieved target 25-hydroxyvitamin D levels in most participants. Brain glutathione levels warrant further study as a biomarker for vitamin D and ALD.This study provides Class IV evidence that fixed or weight-stratified vitamin D supplementation achieved target levels of 25-hydroxyvitamin D in boys and young men with X-ALD without brain lesions.

    View details for DOI 10.1212/NXG.0000000000200061

    View details for PubMedID 37090939

    View details for PubMedCentralID PMC10117697

  • MT-ATP6 mitochondrial disease identified by newborn screening reveals a distinct biochemical phenotype. American journal of medical genetics. Part A Tise, C. G., Verscaj, C. P., Mendelsohn, B. A., Woods, J., Lee, C. U., Enns, G. M., Stander, Z., Hall, P. L., Cowan, T. M., Cusmano-Ozog, K. P. 2023

    Abstract

    Although decreased citrulline is used as a newborn screening (NBS) marker to identify proximal urea cycle disorders (UCDs), it is also a feature of some mitochondrial diseases, including MT-ATP6 mitochondrial disease. Here we describe biochemical and clinical features of 11 children born to eight mothers from seven separate families who were identified with low citrulline by NBS (range 3-5muM; screening cutoff >5) and ultimately diagnosed with MT-ATP6 mitochondrial disease. Follow-up testing revealed a pattern of hypocitrullinemia together with elevated propionyl-(C3) and 3-hydroxyisovaleryl-(C5-OH) acylcarnitines, and a homoplasmic pathogenic variant in MT-ATP6 in all cases. Single and multivariate analysis of NBS data from the 11 cases using Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu) demonstrated citrulline <1st percentile, C3>50th percentile, and C5-OH >90th percentile when compared with reference data, as well as unequivocal separation from proximal UCD cases and false-positive low citrulline cases using dual scatter plots. Five of the eight mothers were symptomatic at the time of their child(ren)'s diagnosis, and all mothers and maternal grandmothers evaluated molecularly and biochemically had a homoplasmic pathogenic variant in MT-ATP6, low citrulline, elevated C3, and/or elevated C5-OH. All molecularly confirmed individuals (n=17) with either no symptoms (n=12), migraines (n=1), or a neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) phenotype (n=3) were found to have an A or U mitochondrial haplogroup, while one child with infantile-lethal Leigh syndrome had a B haplogroup.

    View details for DOI 10.1002/ajmg.a.63159

    View details for PubMedID 36883293

  • Host-microbe co-metabolism via MCAD generates circulating metabolites including hippuric acid. Nature communications Pruss, K. M., Chen, H., Liu, Y., Van Treuren, W., Higginbottom, S. K., Jarman, J. B., Fischer, C. R., Mak, J., Wong, B., Cowan, T. M., Fischbach, M. A., Sonnenburg, J. L., Dodd, D. 2023; 14 (1): 512

    Abstract

    The human gut microbiota produces dozens of small molecules that circulate in blood, accumulate to comparable levels as pharmaceutical drugs, and influence host physiology. Despite the importance of these metabolites to human health and disease, the origin of most microbially-produced molecules and their fate in the host remains largely unknown. Here, we uncover a host-microbe co-metabolic pathway for generation of hippuric acid, one of the most abundant organic acids in mammalian urine. Combining stable isotope tracing with bacterial and host genetics, we demonstrate reduction of phenylalanine to phenylpropionic acid by gut bacteria; the host re-oxidizes phenylpropionic acid involving medium-chain acyl-CoA dehydrogenase (MCAD). Generation of germ-free male and female MCAD-/- mice enabled gnotobiotic colonization combined with untargeted metabolomics to identify additional microbial metabolites processed by MCAD in host circulation. Our findings uncover a host-microbe pathway for the abundant, non-toxic phenylalanine metabolite hippurate and identify β-oxidation via MCAD as a novel mechanism by which mammals metabolize microbiota-derived metabolites.

    View details for DOI 10.1038/s41467-023-36138-3

    View details for PubMedID 36720857

  • Validation of a targeted metabolomics panel for improved second-tier newborn screening. Journal of inherited metabolic disease Mak, J., Peng, G., Le, A., Gandotra, N., Enns, G. M., Scharfe, C., Cowan, T. M. 2023

    Abstract

    Improved second-tier assays are needed to reduce the number of false positives in newborn screening (NBS) for inherited metabolic disorders including those on the Recommended Uniform Screening Panel (RUSP). We developed an expanded metabolite panel for second-tier testing of dried blood spot (DBS) samples from screen-positive cases reported by the California NBS program, consisting of true- and false-positives from four disorders: glutaric acidemia type 1 (GA1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). This panel was assembled from known disease markers and new features discovered by untargeted metabolomics and applied to second-tier analysis of single DBS punches using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a 3-min run. Additionally, we trained a Random Forest (RF) machine learning classifier to improve separation of true- and false positive cases. Targeted metabolomic analysis of 121 analytes from DBS extracts in combination with RF classification at a sensitivity of 100% reduced false positives for GA1 by 83%, MMA by 84%, OTCD by 100%, and VLCADD by 51%. This performance was driven by a combination of known disease markers (3-hydroxyglutaric acid, methylmalonic acid, citrulline, C14:1), other amino acids and acylcarnitines, and novel metabolites identified to be isobaric to several long-chain acylcarnitine and hydroxy-acylcarnitine species. These findings establish the effectiveness of this second-tier test to improve screening for these four conditions and demonstrate the utility of supervised machine learning in reducing false-positives for conditions lacking clearly discriminating markers, with future studies aimed at optimizing and expanding the panel to additional disease targets. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/jimd.12591

    View details for PubMedID 36680545

  • Metabolic diversity in human populations and correlation with genetic and ancestral geographic distances. Molecular genetics and metabolism Peng, G., Pakstis, A. J., Gandotra, N., Cowan, T. M., Zhao, H., Kidd, K. K., Scharfe, C. 2022; 137 (3): 292-300

    Abstract

    DNA polymorphic markers and self-defined ethnicity groupings are used to group individuals with shared ancient geographic ancestry. Here we studied whether ancestral relationships between individuals could be identified from metabolic screening data reported by the California newborn screening (NBS) program. NBS data includes 41 blood metabolites measured by tandem mass spectrometry from singleton babies in 17 parent-reported ethnicity groupings. Ethnicity-associated differences identified for 71% of NBS metabolites (29 of 41, Cohen's d > 0.5) showed larger differences in blood levels of acylcarnitines than of amino acids (P < 1e-4). A metabolic distance measure, developed to compare ethnic groupings based on metabolic differences, showed low positive correlation with genetic and ancient geographic distances between the groups' ancestral world populations. Several outlier group pairs were identified with larger genetic and smaller metabolic distances (Black versus White) or with smaller genetic and larger metabolic distances (Chinese versus Japanese) indicating the influence of genetic and of environmental factors on metabolism. Using machine learning, comparison of metabolic profiles between all pairs of ethnic groupings distinguished individuals with larger genetic distance (Black versus Chinese, AUC = 0.96), while genetically more similar individuals could not be separated metabolically (Hispanic versus Native American, AUC = 0.51). Additionally, we identified metabolites informative for inferring metabolic ancestry in individuals from genetically similar populations, which included biomarkers for inborn metabolic disorders (C10:1, C12:1, C3, C5OH, Leucine-Isoleucine). This work sheds new light on metabolic differences in healthy newborns in diverse populations, which could have implications for improving genetic disease screening.

    View details for DOI 10.1016/j.ymgme.2022.10.002

    View details for PubMedID 36252453

  • MITOCHONDRIAL-ATP6-ASSOCIATED DISEASE PRESENTS WITH DISTINCT PATTERN ON NEWBORN SCREENING: SHOULD IT BE INCLUDED AS A SECONDARY CONDITION? Tise, C., Mendelsohn, B., Lee, C., Woods, J., Hall, P., Tang, H., Rinaldo, P., Cowan, T., Cusmano-Ozog, K. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2022: 247-248
  • Targeted plasma metabolomics combined with machine learning for the diagnosis of severe acute respiratory syndrome virus type 2. Frontiers in microbiology Le, A. T., Wu, M., Khan, A., Phillips, N., Rajpurkar, P., Garland, M., Magid, K., Sibai, M., Huang, C., Sahoo, M. K., Bowen, R., Cowan, T. M., Pinsky, B. A., Hogan, C. A. 2022; 13: 1059289

    Abstract

    The routine clinical diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely restricted to real-time reverse transcription quantitative PCR (RT-qPCR), and tests that detect SARS-CoV-2 nucleocapsid antigen. Given the diagnostic delay and suboptimal sensitivity associated with these respective methods, alternative diagnostic strategies are needed for acute infection.We studied the use of a clinically validated liquid chromatography triple quadrupole method (LC/MS-MS) for detection of amino acids from plasma specimens. We applied machine learning models to distinguish between SARS-CoV-2-positive and negative samples and analyzed amino acid feature importance.A total of 200 samples were tested, including 70 from individuals with COVID-19, and 130 from negative controls. The top performing model overall allowed discrimination between SARS-CoV-2-positive and negative control samples with an area under the receiver operating characteristic curve (AUC) of 0.96 (95%CI 0.91, 1.00), overall sensitivity of 0.99 (95%CI 0.92, 1.00), and specificity of 0.92 (95%CI 0.85, 0.95).This approach holds potential as an alternative to existing methods for the rapid and accurate diagnosis of acute SARS-CoV-2 infection.

    View details for DOI 10.3389/fmicb.2022.1059289

    View details for PubMedID 37063449

    View details for PubMedCentralID PMC10092816

  • Biochem for the Win! The added value of biochemical genetic testing for diagnosis and variant interpretation in the genomic era Tise, C., Grand, K., Corado, J., Gates, R., Graham, J., Enns, G., Gomez-Ospina, N., Mak, J., Cowan, T., Cusmano-Ozog, K. ELSEVIER SCIENCE INC. 2022: S24
  • GlcNAc-Asn (GNA) is a biomarker for NGLY1 deficiency. Journal of biochemistry Mueller, W. F., Zhu, L., Tan, B., Dwight, S., Beahm, B., Wilsey, M., Wechsler, T., Mak, J., Cowan, T., Pritchett, J., Taylor, E., Crawford, B. E. 2021

    Abstract

    Substrate derived biomarkers are necessary in slowly progressing monogenetic diseases caused by single enzyme deficiencies to identify affected patients and serve as surrogate markers for therapy response. N-glycanase 1 (NGLY1) deficiency is an ultra-rare autosomal recessive disorder characterized by developmental delay, peripheral neuropathy, elevated liver transaminases, hyperkinetic movement disorder, and (hypo)-alacrima. We demonstrate that N-acetylglucosamine-asparagine (GlcNAc-Asn; GNA), is the analyte most closely associated with NGLY1 deficiency, showing consistent separation in levels between patients and controls. GNA accumulation is directly linked to the absence of functional NGLY1, presenting strong potential for its use as a biomarker. In agreement, a quantitative LC-MS/MS assay, developed to assess GNA from 3 to 3000 ng/mL, showed it is conserved as a marker for loss of NGLY1 function in NGLY1 deficient cell lines, rodents (urine, cerebrospinal fluid, plasma, and tissues), and patients (plasma and urine). Elevated GNA levels differentiate patients from controls, are stable over time, and correlate with changes in NGLY1 activity. GNA as a biomarker has the potential to identify and validate patients with NGLY1 deficiency, act as a direct pharmacodynamic marker, and serve as a potential surrogate endpoint in clinical trials.

    View details for DOI 10.1093/jb/mvab111

    View details for PubMedID 34697629

  • Variable clinical severity in TANGO2 deficiency: Case series and literature review. American journal of medical genetics. Part A Schymick, J., Leahy, P., Cowan, T., Ruzhnikov, M. R., Gates, R., Fernandez, L., Pramanik, G., Undiagnosed Diseases Network, Yarlagadda, V., Wheeler, M., Bernstein, J. A., Enns, G. M., Lee, C. 2021

    Abstract

    Biallelic pathogenic variants in the TANGO2 (transport and Golgi organization 2 homolog) gene have been identified as causing a rare metabolic disorder characterized by susceptibility to recurrent rhabdomyolysis, lactic acidosis, encephalopathy, and life-threatening tachyarrhythmias. Recently published reports suggest variable clinical severity and phenotypes. This study details five new patients from two families with biallelic pathogenic variants in the TANGO2 gene identified by whole exome sequencing and includes the largest number of affected individuals from a single family reported to date. We document significant intrafamilial variability and highlight that milder phenotypes may be underrecognized. We present biochemical and clinical data to help highlight the features that aid in consideration of this condition in the differential with disorders of fatty acid oxidation. We also present a comprehensive literature review summarizing the molecular, clinical, and biochemical findings for 92 individuals across 13 publications. Of the 27 pathogenic variants reported to date, the recurrent exons 3-9 deletion represents the most common variant seen in 42% of individuals with TANGO2 deficiency. Common clinical features seen in >70% of all individuals include acute metabolic crisis, rhabdomyolysis, neurologic abnormalities, developmental delay, and intellectual disability. Findings such as elevated creatine kinase, hypothyroidism, ketotic hypoglycemia, QT prolongation, or abnormalities of long-chain acylcarnitines and urine dicarboxylic acids should raise clinical suspicion for this life-threatening condition.

    View details for DOI 10.1002/ajmg.a.62543

    View details for PubMedID 34668327

  • Detecting lysosomal storage disorders by glycomic profiling using liquid chromatography mass spectrometry. Molecular genetics and metabolism Mak, J., Cowan, T. M. 2021

    Abstract

    BACKGROUND: Urine and plasma biomarker testing for lysosomal storage disorders by liquid chromatography mass spectrometry (LC-MS) currently requires multiple analytical methods to detect the abnormal accumulation of oligosaccharides, mucopolysaccharides, and glycolipids. To improve clinical testing efficiency, we developed a single LC-MS method to simultaneously identify disorders of oligosaccharide, mucopolysaccharide, and glycolipid metabolism with minimal sample preparation.METHODS: We created a single chromatographic method for separating free glycans and glycolipids in their native form, using an amide column and high pH conditions. We used this glycomic profiling method both in untargeted analyses of patient and control urines using LC ion-mobility high-resolution MS (biomarker discovery), and targeted analyses of urine, serum, and dried blood spot samples by LC-MS/MS (clinical validation).RESULTS: Untargeted glycomic profiling revealed twenty biomarkers that could identify and subtype mucopolysaccharidoses. We incorporated these with known oligosaccharide and glycolipid biomarkers into a rapid test that identifies at least 27 lysosomal storage disorders, including oligosaccharidoses, mucopolysaccharidoses, sphingolipidoses, glycogen storage disorders, and congenital disorders of glycosylation and de-glycosylation. In a validation set containing 115 urine samples from patients with lysosomal storage disorders, all were unambiguously distinguished from normal controls, with correct disease subtyping for 88% (101/115) of cases. Glucosylsphingosine was reliably elevated in dried blood spots from Gaucher disease patients with baseline resolution from galactosylsphingosine.CONCLUSION: Glycomic profiling by liquid chromatography mass spectrometry identifies a range of lysosomal storage disorders. This test can be used in clinical evaluations to rapidly focus a diagnosis, as well as to clarify or support additional gene sequencing and enzyme studies.

    View details for DOI 10.1016/j.ymgme.2021.08.006

    View details for PubMedID 34474962

  • Nasopharyngeal metabolomics and machine learning approach for the diagnosis of influenza. EBioMedicine Hogan, C. A., Rajpurkar, P., Sowrirajan, H., Phillips, N. A., Le, A. T., Wu, M., Garamani, N., Sahoo, M. K., Wood, M. L., Huang, C., Ng, A. Y., Mak, J., Cowan, T. M., Pinsky, B. A. 2021; 71: 103546

    Abstract

    BACKGROUND: Respiratory virus infections are significant causes of morbidity and mortality, and may induce host metabolite alterations by infecting respiratory epithelial cells. We investigated the use of liquid chromatography quadrupole time-of-flight mass spectrometry (LC/Q-TOF) combined with machine learning for the diagnosis of influenza infection.METHODS: We analyzed nasopharyngeal swab samples by LC/Q-TOF to identify distinct metabolic signatures for diagnosis of acute illness. Machine learning models were performed for classification, followed by Shapley additive explanation (SHAP) analysis to analyze feature importance and for biomarker discovery.FINDINGS: A total of 236 samples were tested in the discovery phase by LC/Q-TOF, including 118 positive samples (40 influenza A 2009 H1N1, 39 influenza H3 and 39 influenza B) as well as 118 age and sex-matched negative controls with acute respiratory illness. Analysis showed an area under the receiver operating characteristic curve (AUC) of 1.00 (95% confidence interval [95% CI] 0.99, 1.00), sensitivity of 1.00 (95% CI 0.86, 1.00) and specificity of 0.96 (95% CI 0.81, 0.99). The metabolite most strongly associated with differential classification was pyroglutamic acid. Independent validation of a biomarker signature based on the top 20 differentiating ion features was performed in a prospective cohort of 96 symptomatic individuals including 48 positive samples (24 influenza A 2009 H1N1, 5 influenza H3 and 19 influenza B) and 48 negative samples. Testing performed using a clinically-applicable targeted approach, liquid chromatography triple quadrupole mass spectrometry, showed an AUC of 1.00 (95% CI 0.998, 1.00), sensitivity of 0.94 (95% CI 0.83, 0.98), and specificity of 1.00 (95% CI 0.93, 1.00). Limitations include lack of sample suitability assessment, and need to validate these findings in additional patient populations.INTERPRETATION: This metabolomic approach has potential for diagnostic applications in infectious diseases testing, including other respiratory viruses, and may eventually be adapted for point-of-care testing.

    View details for DOI 10.1016/j.ebiom.2021.103546

    View details for PubMedID 34419924

  • Timing of newborn blood collection alters screening performance for metabolic disorders Peng, G., Tang, Y., Gandotra, N., Cowan, T., Zhao, H., Scharfe, C. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2021: S26
  • A conserved role for AMP-activated protein kinase in NGLY1 deficiency. PLoS genetics Han, S. Y., Pandey, A., Moore, T., Galeone, A., Duraine, L., Cowan, T. M., Jafar-Nejad, H. 2020; 16 (12): e1009258

    Abstract

    Mutations in human N-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known as NGLY1 deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and its in vivo consequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, here we report that loss of NGLY1 in the visceral muscle of the Drosophila larval intestine results in a severe reduction in the level of AMP-activated protein kinase alpha (AMPKalpha), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality. Ngly1-/-mouse embryonic fibroblasts and NGLY1 deficiency patient fibroblasts also show reduced AMPKalpha levels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKalpha level and impaired energy metabolism observed in NGLY1 deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects in NGLY1 deficiency and suggest AMPK signaling as a therapeutic target in this disease.

    View details for DOI 10.1371/journal.pgen.1009258

    View details for PubMedID 33315951

  • A conserved role for AMP-activated protein kinase in NGLY1 deficiency Han, S., Panday, A., Moore, T., Galeone, A., Duraine, L., Cowan, T. M., Hamed, J. OXFORD UNIV PRESS INC. 2020: 1057–58
  • Exploring the mechanisms underlying the NGLY1 loss-of-function phenotypes in Drosophila Pandey, A., Han, S., Mueller, W. F., Story, B. A., Moore, T., Galeone, A., Cowan, T. M., Steinmetz, L. OXFORD UNIV PRESS INC. 2020: 1026
  • AAV9-Mediated Gene Therapy for NGLY1 Deficiency and Assessment of GNA Biomarker Changes in a Rat Disease Model Mueller, W. F., Zhu, L., Dwight, S. S., Beahm, B., Lee, K. J., Wilsey, M., Mak, J., Pollard, L., Wood, T., Cowan, T., Crawford, B., Wechsler, T. CELL PRESS. 2020: 124–25
  • Whole genome sequencing for mutation discovery in a single case of lysosomal storage disease (MPS type 1) in the dog. Scientific reports Mansour, T. A., Woolard, K. D., Vernau, K. L., Ancona, D. M., Thomasy, S. M., Sebbag, L., Moore, B. A., Knipe, M. F., Seada, H. A., Cowan, T. M., Aguilar, M., Titus Brown, C., Bannasch, D. L. 2020; 10 (1): 6558

    Abstract

    Mucopolysaccharidosis (MPS) is a metabolic storage disorder caused by the deficiency of any lysosomal enzyme required for the breakdown of glycosaminoglycans. A 15-month-old Boston Terrier presented with clinical signs consistent with lysosomal storage disease including corneal opacities, multifocal central nervous system disease and progressively worsening clinical course. Diagnosis was confirmed at necropsy based on histopathologic evaluation of multiple organs demonstrating accumulation of mucopolysaccharides. Whole genome sequencing was used to uncover a frame-shift insertion affecting the alpha-L-iduronidase (IDUA) gene (c.19_20insCGGCCCCC), a mutation confirmed in another Boston Terrier presented 2 years later with a similar clinical picture. Both dogs were homozygous for the IDUA mutation and shared coat colors not recognized as normal for the breed by the American Kennel Club. In contrast, the mutation was not detected in 120 unrelated Boston Terriers as well as 202 dogs from other breeds. Recent inbreeding to select for recessive and unusual coat colors may have concentrated this relatively rare allele in the breed. The identification of the variant enables ante-mortem diagnosis of similar cases and selective breeding to avoid the spread of this disease in the breed. Boston Terriers carrying this variant represent a promising model for MPS I with neurological abnormalities in humans.

    View details for DOI 10.1038/s41598-020-63451-4

    View details for PubMedID 32300136

  • Metabolic profiling by reversed-phase/ion-exchange mass spectrometry. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Le, A., Mak, J., Cowan, T. M. 2020; 1143: 122072

    Abstract

    Metabolic profiling is commonly achieved by mass spectrometry (MS) following reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) either performed independently, leading to overlapping datasets, or in a coupled configuration, requiring multiple liquid chromatography (LC) systems. To overcome these limitations, we developed a single, 20-minute chromatographic method using an in-line RP-ion-exchange (IEX) column arrangement and a single LC system. This configuration separates clinically significant polar and non-polar compounds without derivatization or ion-pairing reagents, allowing ionization in both polarities. An in-house library was created with 397 authentic standards, including acylcarnitines, amino acids, bile acids, nucleosides, organic acids, steroid hormones, and vitamins. Analysis of pooled plasma and urine samples revealed 5445 and 4111 ion features, leading to 88 and 82 confirmed metabolite identifications, respectively. Metabolites were detected at clinically relevant concentrations with good precision, and good chromatographic separation was demonstrated for clinically significant isomers including methylmalonic acid and succinic acid, as well as alloisoleucine and isoleucine/leucine. Evaluation of the samples by unsupervised principal component analysis showed excellent analytical quality.

    View details for DOI 10.1016/j.jchromb.2020.122072

    View details for PubMedID 32220802

  • Reducing False-Positive Results in Newborn Screening Using Machine Learning. International journal of neonatal screening Peng, G., Tang, Y., Cowan, T. M., Enns, G. M., Zhao, H., Scharfe, C. 2020; 6 (1)

    Abstract

    Newborn screening (NBS) for inborn metabolic disorders is a highly successful public health program that by design is accompanied by false-positive results. Here we trained a Random Forest machine learning classifier on screening data to improve prediction of true and false positives. Data included 39 metabolic analytes detected by tandem mass spectrometry and clinical variables such as gestational age and birth weight. Analytical performance was evaluated for a cohort of 2777 screen positives reported by the California NBS program, which consisted of 235 confirmed cases and 2542 false positives for one of four disorders: glutaric acidemia type 1 (GA-1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Without changing the sensitivity to detect these disorders in screening, Random Forest-based analysis of all metabolites reduced the number of false positives for GA-1 by 89%, for MMA by 45%, for OTCD by 98%, and for VLCADD by 2%. All primary disease markers and previously reported analytes such as methionine for MMA and OTCD were among the top-ranked analytes. Random Forest's ability to classify GA-1 false positives was found similar to results obtained using Clinical Laboratory Integrated Reports (CLIR). We developed an online Random Forest tool for interpretive analysis of increasingly complex data from newborn screening.

    View details for DOI 10.3390/ijns6010016

    View details for PubMedID 32190768

  • Timing of Newborn Blood Collection Alters Metabolic Disease Screening Performance. Frontiers in pediatrics Peng, G., Tang, Y., Cowan, T. M., Zhao, H., Scharfe, C. 2020; 8: 623184

    Abstract

    Blood collection for newborn genetic disease screening is preferably performed within 24-48 h after birth. We used population-level newborn screening (NBS) data to study early postnatal metabolic changes and whether timing of blood collection could impact screening performance. Newborns were grouped based on their reported age at blood collection (AaBC) into early (12-23 h), standard (24-48 h), and late (49-168 h) collection groups. Metabolic marker levels were compared between the groups using effect size analysis, which controlled for group size differences and influence from the clinical variables of birth weight and gestational age. Metabolite level differences identified between groups were correlated to NBS data from false-positive cases for inborn metabolic disorders including carnitine transport defect (CTD), isovaleric acidemia (IVA), methylmalonic acidemia (MMA), and phenylketonuria (PKU). Our results showed that 56% of the metabolites had AaBC-related differences, which included metabolites with either decreasing or increasing levels after birth. Compared to the standard group, the early-collection group had elevated marker levels for PKU (phenylalanine, Cohen's d = 0.55), IVA (C5, Cohen's d = 0.24), MMA (C3, Cohen's d = 0.23), and CTD (C0, Cohen's d = 0.23). These findings correlated with higher false-positive rates for PKU (P < 0.05), IVA (P < 0.05), and MMA (P < 0.001), and lower false-positive rate for CTD (P < 0.001) in the early-collection group. Blood collection before 24 h could affect screening performance for some metabolic disorders. We have developed web-based tools integrating AaBC and other variables for interpretive analysis of screening data.

    View details for DOI 10.3389/fped.2020.623184

    View details for PubMedID 33553077

  • AMP-independent activator of AMPK for treatment of mitochondrial disorders. PloS one Moore, T., Yanes, R. E., Calton, M. A., Vollrath, D., Enns, G. M., Cowan, T. M. 2020; 15 (10): e0240517

    Abstract

    Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

    View details for DOI 10.1371/journal.pone.0240517

    View details for PubMedID 33052980

  • Ethnic Variability in Newborn Metabolic Screening Markers Associated with False-Positive Outcomes. Journal of inherited metabolic disease Peng, G. n., Tang, Y. n., Gandotra, N. n., Enns, G. M., Cowan, T. M., Zhao, H. n., Scharfe, C. n. 2020

    Abstract

    Newborn screening (NBS) programs utilize information on a variety of clinical variables such as gestational age, sex and birth weight to reduce false-positive screens for inborn metabolic disorders. Here we study the influence of ethnicity on metabolic marker levels in a diverse newborn population. NBS data from screen-negative singleton babies (n = 100 000) were analyzed, which included blood metabolic markers measured by tandem mass spectrometry and ethnicity status reported by the parents. Metabolic marker levels were compared between major ethnic groups (Asian, Black, Hispanic, White) using effect size analysis, which controlled for group size differences and influence from clinical variables. Marker level differences found between ethnic groups were correlated to NBS data from 2532 false-positive cases for four metabolic diseases: glutaric acidemia type 1 (GA-1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). In result, 79% of the metabolic markers (34 of 43) had ethnicity-related differences. Compared to the other groups, Black infants had elevated GA-1 markers (C5DC, Cohen's d = 0.37, P < .001), Hispanics had elevated MMA markers (C3, Cohen's d = 0.13, P < .001, and C3/C2, Cohen's d = 0.27, P < .001); and Whites had elevated VLCADD markers (C14, Cohen's d = 0.28, P < .001, and C14:1, Cohen's d = 0.22, P < .001) and decreased OTCD markers (citrulline, Cohen's d = -0.26, P < .001). These findings correlated with the higher false-positive rates in Black infants for GA-1, in Hispanics for MMA, and in Whites for OTCD and for VLCADD. Web-based tools are available to analyze ethnicity-related changes in newborn metabolism and to support developing methods to identify false-positives in metabolic screening. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/jimd.12236

    View details for PubMedID 32216101

  • LOCALIZING NEUROLOGIC FEATURES AT PRESENTATION OF VLCAD DEFICIENCY Leahy, P., Matalon, D., Ruzhnikov, M., Cowan, T., Enns, G. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2019: 282
  • Biomarkers of oxidative stress, inflammation, and vascular dysfunction in inherited cystathionine -synthase deficient homocystinuria and the impact of taurine treatment in a phase 1/2 human clinical trial JOURNAL OF INHERITED METABOLIC DISEASE Van Hove, J. K., Freehauf, C. L., Ficicioglu, C., Pena, L. M., Moreau, K. L., Henthorn, T. K., Christians, U., Jiang, H., Cowan, T. M., Young, S. P., Hite, M., Friederich, M. W., Stabler, S. P., Spector, E. B., Kronquist, K. E., Thomas, J. A., Emmett, P., Harrington, M. J., Pyle, L., Creadon-Swindell, G., Wempe, M. F., MacLean, K. N. 2019; 42 (3): 424–37

    View details for DOI 10.1002/jimd.12085

    View details for Web of Science ID 000465530700007

  • Combining newborn metabolic and DNA analysis for second-tier testing of methylmalonic acidemia GENETICS IN MEDICINE Peng, G., Shen, P., Gandotra, N., Le, A., Fung, E., Jelliffe-Pawlowski, L., Davis, R. W., Enns, G. M., Zhao, H., Cowan, T. M., Scharfe, C. 2019; 21 (4): 896–903
  • Biomarkers of Oxidative Stress, Inflammation, and Vascular Dysfunction in Inherited Cystathionine beta-synthase Deficient Homocystinuria and the Impact of Taurine Treatment in a Phase 1/2 human Clinical Trial. Journal of inherited metabolic disease Van Hove, J. L., Freehauf, C. L., Ficicioglu, C., Pena, L. D., Moreau, K. L., Henthorn, T. K., Christians, U., Jiang, H., Cowan, T. M., Young, S. P., Hite, M., Friederich, M. W., Stabler, S. P., Spector, E. B., Kronquist, K. E., Thomas, J. A., Emmett, P., Harrington, M. J., Pyle, L., Creadon-Swindell, G., Wempe, M. F., MacLean, K. N. 2019

    Abstract

    STUDY OBJECTIVE: A phase 1/2 clinical trial was performed in individuals with cystathionine beta synthase (CBS) deficient homocystinuria with aims to: 1) assess pharmacokinetics and safety of taurine therapy 2) evaluate oxidative stress, inflammation, and vascular function in CBS deficiency and 3) evaluate the impact of short-term taurine treatment.METHODS: Individuals with pyridoxine-nonresponsive CBS deficiency with homocysteine >50 muM, without inflammatory disorder or on antioxidant therapy were enrolled. Biomarkers of oxidative stress and inflammation, endothelial function (brachial artery flow-mediated dilation FMD), and disease-related metabolites obtained at baseline were compared to normal values. While maintaining current treatment, patients were treated with 75 mg/kg taurine twice daily, and treatment response assessed after 4 hours and 4 days.RESULTS: Fourteen patients (8-35 years; 8 males, 6 females) were enrolled with baseline homocysteine levels 161±67 muM. The study found high-dose taurine to be safe when excluding preexisting hypertriglyceridemia. Taurine pharmacokinetics showed a rapid peak level returning to near normal levels at 12 hours, but had slow accumulation and elevated predosing levels after 4 days of treatment. Only a single parameter of oxidative stress, 2,3-dinor-iPF2alpha, was elevated at baseline, with no elevated inflammatory parameters, and no change in FMD values overall. Taurine had no effect on any of these parameters. However, the effect of taurine was strongly related to pretreatment FMD values; and taurine significantly improved FMD in the subset of individuals with pretreatment FMD values <10% and in individuals with homocysteine levels >125 muM, pertinent to endothelial function.CONCLUSION: Taurine improves endothelial function in CBS deficient homocystinuria in patients with preexisting reduced function. This article is protected by copyright. All rights reserved.

    View details for PubMedID 30873612

  • LOCALIZING NEUROLOGIC FEATURES AT PRESENTATION OF VLCAD DEFICIENCY Leahy, P., Matalon, D., Ruzhnikov, M., Cowan, T., Enns, G. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2019: 311
  • SETD3 is an actin histidine methyltransferase that prevents primary dystocia NATURE Wilkinson, A. W., Diep, J., Dai, S., Liu, S., Ooi, Y., Song, D., Li, T., Horton, J. R., Zhang, X., Liu, C., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J., Cowan, T., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X., Carette, J. E., Gozani, O. 2019; 565 (7739): 372-+
  • Quantitative Analysis of Underivatized Amino Acids by Liquid Chromatography-Tandem Mass Spectrometry. Methods in molecular biology (Clifton, N.J.) Mak, J. n., Cowan, T. M., Le, A. n. 2019; 2030: 85–109

    Abstract

    Quantitative amino acid analysis has diverse applications in clinical diagnostics, biomedical research, and agriculture. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables more rapid and specific detection of amino acids in comparison to traditional, gold-standard ninhydrin-based methods. However, triple quadrupole mass spectrometers are unable to definitively differentiate isomers and are susceptible to ion suppression, both of which prevent accurate quantitation. Therefore, appropriate chromatography must be applied before ionization.We have shown that two-dimensional LC enables rapid and specific amino acid quantitation without derivatization by resolving isomers, such as alloisoleucine, isoleucine, and leucine, and reducing matrix effects (Le et al., J Chromatogr B Analyt Technol Biomed Life Sci 944:166-174, 2014). In this clinically validated protocol, we provide an updated description of the chromatographic setup and selected reaction monitoring (SRM) transitions. Then, we describe sample processing for serum, plasma, urine, cerebral spinal fluid, and dried blood spots. Most importantly, we outline a singular quantitative design for efficient data analysis of the listed sample types and quality assurance strategies to ensure test fidelity. Lastly, we share extensive knowledge critical to the success of this method. A liquid sample can be processed and be ready for injection within 5 min, and each sample is analyzed by the MS in 14.5 min.

    View details for DOI 10.1007/978-1-4939-9639-1_8

    View details for PubMedID 31347112

  • Quantitative Analysis of Underivatized Amino Acids by Liquid Chromatography-Tandem Mass Spectrometry AMINO ACID ANALYSIS, 2 EDITION Mak, J., Cowan, T. M., Le, A., Alterman, M. A. 2019; 2030: 85-109
  • Elevated methylmalonic acidemia (MMA) screening markers in Hispanic and preterm newborns MOLECULAR GENETICS AND METABOLISM Peng, G., de Fontnouvelle, C. A., Enns, G. M., Cowan, T. M., Zhao, H., Scharfe, C. 2019; 126 (1): 39–42
  • Medical Biochemistry Without Rote Memorization: Multi-Institution Implementation and Student Perceptions of a Nationally Standardized Metabolic Map for Learning and Assessment. Medical science educator Spicer, D. B., Thompson, K. H., Tong, M. S., Cowan, T. M., Fulton, T. B., Lindsley, J. E. 2019; 29 (1): 87-92

    Abstract

    Despite the growing number of patients worldwide with metabolism-related chronic diseases, medical biochemistry education is commonly perceived as focusing on recall of facts irrelevant for patient care. The authors suggest that this focus on rote memorization of pathways creates excessive cognitive load that may interfere with learners' development of an integrated understanding of metabolic regulation and dysregulation. This cognitive load can be minimized by providing appropriate references during learning and assessment. Biochemistry educators collaborated to develop a medically relevant pathways of human metabolism map (MetMap) that is now being used at many medical schools as a nationally standardized resource during learning and assessments. To assess impact, students from three medical schools were surveyed about its benefits and disadvantages. Responses were obtained from 481 students (84%) and were examined using thematic analysis. Five main themes emerged as perceived benefits of using the MetMap: (1) aids visual and mental organization, (2) promotes deep learning and applied understanding, (3) decreases emphasis on memorization, (4) reduces anxiety on exams, and (5) aids recall. Perceived disadvantages were (1) fear of underpreparation for licensing exams, (2) overwhelming nature of the map, and (3) reduced motivation for and time spent studying. Results affirm that students' perceive use of the MetMap promotes focus on broader metabolic concepts and deep versus surface learning, supporting a shift in cognitive load toward desired goals. Although the long-term impact on learning needs to be further studied, the use of the MetMap represents a step toward open-reference exams that reflect "real-world" practice.

    View details for DOI 10.1007/s40670-018-00631-y

    View details for PubMedID 34457455

    View details for PubMedCentralID PMC8360249

  • Elevated methylmalonic acidemia (MMA) screening markers in Hispanic and preterm newborns. Molecular genetics and metabolism Peng, G., de Fontnouvelle, C. A., Enns, G. M., Cowan, T. M., Zhao, H., Scharfe, C. 2018

    Abstract

    Analysis of California newborn screening (NBS) data revealed a high prevalence of Hispanic infants testing positive for methylmalonic acidemia (MMA), a trend seen for both true- and false-positive cases. Here we show that Hispanic infants have significantly higher levels of MMA screening markers than non-Hispanics. Preterm birth and increased birth weight were found to be associated with elevated MMA marker levels but could not entirely explain these differences. While the preterm birth rate was higher in Blacks than Hispanics, Black infants had on average the lowest MMA marker levels. Preterm birth was associated with lower birth weight and increased MMA marker levels suggesting that gestational age is the stronger predictive covariate compared to birth weight. These findings could help explain why MMA false-positive results are more likely in Hispanic than in Black infants, which could inform screening and diagnostic procedures for MMA and potentially other disorders in newborns.

    View details for PubMedID 30448007

  • Rapid Underivatized Method for Quantitative Methylmalonic Acid by Liquid Chromatography-Tandem Mass Spectrometry. The journal of applied laboratory medicine Mak, J., Wong, B. T., Kwan, T., Le, A., Cowan, T. M. 2018; 3 (3): 408-417

    Abstract

    Increased methylmalonic acid (MMA) levels can aid in assessing vitamin B12 deficiency or abnormal propionate metabolism. MMA analysis by LC-MS/MS is challenging because of both the nanomolar reference range and potential interference from succinic acid, an endogenous isomer. We show that ultrafiltration followed by gradient chromatography permits rapid, sensitive, and selective quantification that is essentially devoid of matrix effects.Fifty microliters of serum or plasma were mixed with 50 μL of MMA-d3 and deproteinized by ultrafiltration. Filtrates were analyzed by reversed-phase LC-MS/MS. The clinical performance of the MMA assay was validated using guidelines from both the College of American Pathologists and the Clinical and Laboratory Standards Institute. Matrix effects were examined by postcolumn infusion, phospholipid analysis, and peak area comparisons.The analytical measurement range was 0.05 to 100 μmol/L. The resolution between physiological succinic acid and MMA was >2.3. Recovery of MMA averaged 92%, and MMA eluted away from ion suppressants. Direct correlation with our earlier method and with consensus data from external proficiency testing yielded an R2 ≥ 0.9409 and average biases less than ±5%. In the production environment, ongoing correlation with external proficiency testing yielded an R2 of 0.9980 and a mean bias of 0.36%. Over 1.7 years, the imprecision of 2 quality control levels was <6.4%.We combined ultrafiltration, a simple sample extraction method, with gradient chromatography to exclude matrix effects to accurately and precisely quantify MMA.

    View details for DOI 10.1373/jalm.2018.026724

    View details for PubMedID 33636915

  • Rapid Underivatized Method for Quantitative Methylmalonic Acid by Liquid Chromatography-Tandem Mass Spectrometry JOURNAL OF APPLIED LABORATORY MEDICINE Mak, J., Wong, B. T., Kwan, T., Le, A., Cowan, T. M. 2018; 3 (3): 408–17
  • Combining newborn metabolic and DNA analysis for second-tier testing of methylmalonic acidemia. Genetics in medicine : official journal of the American College of Medical Genetics Peng, G., Shen, P., Gandotra, N., Le, A., Fung, E., Jelliffe-Pawlowski, L., Davis, R. W., Enns, G. M., Zhao, H., Cowan, T. M., Scharfe, C. 2018

    Abstract

    PURPOSE: Improved second-tier tools are needed to reduce false-positive outcomes in newborn screening (NBS) for inborn metabolic disorders on the Recommended Universal Screening Panel (RUSP).METHODS: We designed an assay for multiplex sequencing of 72 metabolic genes (RUSPseq) from newborn dried blood spots. Analytical and clinical performance was evaluated in 60 screen-positive newborns for methylmalonic acidemia (MMA) reported by the California Department of Public Health NBS program. Additionally, we trained a Random Forest machine learning classifier on NBS data to improve prediction of true and false-positive MMA cases.RESULTS: Of 28 MMA patients sequenced, we found two pathogenic or likely pathogenic (P/LP) variants in a MMA-related gene in 24 patients, and one pathogenic variant and a variant of unknown significance (VUS) in 1 patient. No such variant combinations were detected in MMA false positives and healthy controls. Random Forest-based analysis of the entire NBS metabolic profile correctly identified the MMA patients and reduced MMA false-positive cases by 51%. MMA screen-positive newborns were more likely of Hispanic ethnicity.CONCLUSION: Our two-pronged approach reduced false positives by half and provided a reportable molecular finding for 89% of MMA patients. Challenges remain in newborn metabolic screening and DNA variant interpretation in diverse multiethnic populations.

    View details for PubMedID 30209273

  • Phosphorylation of MCAD selectively rescues PINK1 deficiencies in behavior and metabolism MOLECULAR BIOLOGY OF THE CELL Course, M. M., Scott, A. I., Schoor, C., Hsieh, C., Papakyrikos, A. M., Winter, D., Cowan, T. M., Wang, X. 2018; 29 (10): 1219-1227
  • Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder AMERICAN JOURNAL OF HUMAN GENETICS Olahova, M., Yoon, W., Thompson, K., Jangam, S., Fernandez, L., Davidson, J. M., Kyle, J. E., Grove, M. E., Fisk, D. G., Kohler, J. N., Holmes, M., Dries, A. M., Huang, Y., Zhao, C., Contrepois, K., Zappala, Z., Fresard, L., Waggott, D., Zink, E. M., Kim, Y., Heyman, H. M., Stratton, K. G., Webb-Robertson, B. M., Snyder, M., Merker, J. D., Montgomery, S. B., Fisher, P. G., Feichtinger, R. G., Mayr, J. A., Hall, J., Barbosa, I. A., Simpson, M. A., Deshpande, C., Waters, K. M., Koeller, D. M., Metz, T. O., Morris, A. A., Schelley, S., Cowan, T., Friederich, M. W., McFarland, R., Van Hove, J. K., Enns, G. M., Yamamoto, S., Ashley, E. A., Wangler, M. F., Taylor, R. W., Bellen, H. J., Bernstein, J. A., Wheeler, M. T., Undiagnosed Diseases Network 2018; 102 (3): 494–504

    Abstract

    ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F1FO ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.

    View details for PubMedID 29478781

  • SETD3 is an actin histidine methyltransferase that prevents primary dystocia. Nature Wilkinson, A. W., Diep, J. n., Dai, S. n., Liu, S. n., Ooi, Y. S., Song, D. n., Li, T. M., Horton, J. R., Zhang, X. n., Liu, C. n., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J. n., Cowan, T. n., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X. n., Carette, J. E., Gozani, O. n. 2018

    Abstract

    For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

    View details for PubMedID 30626964

  • CAP/ACMG proficiency testing for biochemical genetics laboratories: a summary of performance GENETICS IN MEDICINE Oglesbee, D., Cowan, T. M., Pasquali, M., Wood, T. C., Weck, K. E., Long, T., Palomaki, G. E., Coll Amer Pathologists, Amer Coll Med Genetics & Genomics, Biochem Mol Genetics Resource Comm 2018; 20 (1): 83–90

    Abstract

    PurposeTesting for inborn errors of metabolism is performed by clinical laboratories worldwide, each utilizing laboratory-developed procedures. We sought to summarize performance in the College of American Pathologists' (CAP) proficiency testing (PT) program and identify opportunities for improving laboratory quality. When evaluating PT data, we focused on a subset of laboratories that have participated in at least one survey since 2010.MethodsAn analysis of laboratory performance (2004 to 2014) on the Biochemical Genetics PT Surveys, a program administered by CAP and the American College of Medical Genetics and Genomics. Analytical and interpretive performance was evaluated for four tests: amino acids, organic acids, acylcarnitines, and mucopolysaccharides.ResultsSince 2010, 150 laboratories have participated in at least one of four PT surveys. Analytic sensitivities ranged from 88.2 to 93.4%, while clinical sensitivities ranged from 82.4 to 91.0%. Performance was higher for US participants and for more recent challenges. Performance was lower for challenges with subtle findings or complex analytical patterns.ConclusionUS clinical biochemical genetics laboratory proficiency is satisfactory, with a minority of laboratories accounting for the majority of errors. Our findings underscore the complex nature of clinical biochemical genetics testing and highlight the necessity of continuous quality management.

    View details for PubMedID 28661487

    View details for PubMedCentralID PMC5763156

  • Oncogenic KRAS Regulates Amino Acid Homeostasis and Asparagine Biosynthesis via ATF4 and Alters Sensitivity to L-Asparaginase CANCER CELL Gwinn, D. M., Lee, A. G., Briones-Martin-del-Campo, M., Conn, C. S., Simpson, D. R., Scott, A. I., Le, A., Cowan, T. M., Ruggero, D., Sweet-Cordero, E. 2018; 33 (1): 91-+

    Abstract

    KRAS is a regulator of the nutrient stress response in non-small-cell lung cancer (NSCLC). Induction of the ATF4 pathway during nutrient depletion requires AKT and NRF2 downstream of KRAS. The tumor suppressor KEAP1 strongly influences the outcome of activation of this pathway during nutrient stress; loss of KEAP1 in KRAS mutant cells leads to apoptosis. Through ATF4 regulation, KRAS alters amino acid uptake and asparagine biosynthesis. The ATF4 target asparagine synthetase (ASNS) contributes to apoptotic suppression, protein biosynthesis, and mTORC1 activation. Inhibition of AKT suppressed ASNS expression and, combined with depletion of extracellular asparagine, decreased tumor growth. Therefore, KRAS is important for the cellular response to nutrient stress, and ASNS represents a promising therapeutic target in KRAS mutant NSCLC.

    View details for PubMedID 29316436

    View details for PubMedCentralID PMC5761662

  • Phosphorylation of MCAD selectively rescuesPINK1deficiencies in behavior and metabolism. Molecular biology of the cell Course, M. M., Scott, A. I., Schoor, C. n., Hsieh, C. H., Papakyrikos, A. M., Winter, D. n., Cowan, T. M., Wang, X. n. 2018

    Abstract

    PINK1 is a mitochondria-targeted kinase, whose mutations are a cause of Parkinson's disease. We set out to better understand PINK1's effects on mitochondrial proteinsin vivoUsing an unbiased phosphoproteomic screen inDrosophila, we found that PINK1 mediates the phosphorylation of MCAD, a mitochondrial matrix protein critical to fatty acid metabolism. By mimicking phosphorylation of this protein in aPINK1null background, we restoredPINK1null's climbing, flight, thorax, and wing deficiencies. Due to MCAD's role in fatty acid metabolism, we examined the metabolic profile ofPINK1null flies, where we uncovered significant disruptions in both acylcarnitines and amino acids. Some of these disruptions were rescued by phosphorylation of MCAD, consistent with MCAD's rescue ofPINK1null's organismal phenotypes. Our work validates and extends the current knowledge of PINK1, identifies a novel function of MCAD, and illuminates the need for and effectiveness of metabolic profiling in models of neurodegenerative disease.

    View details for PubMedID 29563254

  • Prenatal treatment of ornithine transcarbamylase deficiency. Molecular genetics and metabolism Wilnai, Y. n., Blumenfeld, Y. J., Cusmano, K. n., Hintz, S. R., Alcorn, D. n., Benitz, W. E., Berquist, W. E., Bernstein, J. A., Castillo, R. O., Concepcion, W. n., Cowan, T. M., Cox, K. L., Lyell, D. J., Esquivel, C. O., Homeyer, M. n., Hudgins, L. n., Hurwitz, M. n., Palma, J. P., Schelley, S. n., Akula, V. P., Summar, M. L., Enns, G. M. 2018

    Abstract

    Patients with neonatal urea cycle defects (UCDs) typically experience severe hyperammonemia during the first days of life, which results in serious neurological injury or death. Long-term prognosis despite optimal pharmacological and dietary therapy is still poor. The combination of intravenous sodium phenylacetate and sodium benzoate (Ammonul®) can eliminate nitrogen waste independent of the urea cycle. We report attempts to improve outcomes for males with severe ornithine transcarbamylase deficiency (OTCD), a severe X-linked condition, via prenatal intravenous administration of Ammonul and arginine to heterozygous carrier females of OTCD during labor.Two heterozygote OTCD mothers carrying male fetuses with a prenatal diagnosis of OTCD received intravenous Ammonul, arginine and dextrose-containing fluids shortly before birth. Maintenance Ammonul and arginine infusions and high-caloric enteral nutrition were started immediately after birth. Ammonul metabolites were measured in umbilical cord blood and the blood of the newborn immediately after delivery. Serial ammonia and biochemical analyses were performed following delivery.Therapeutic concentrations of Ammonul metabolites were detected in umbilical cord and neonatal blood samples. Plasma ammonia and glutamine levels in the postnatal period were within the normal range. Peak ammonia levels in the first 24-48h were 53mcmol/l and 62mcmol/l respectively. The boys did not experience neurological sequelae secondary to hyperammonemia and received liver transplantation at ages 3months and 5months. The patients show normal development at ages 7 and 3years.Prenatal treatment of mothers who harbor severe OTCD mutations and carry affected male fetuses with intravenous Ammonul and arginine, followed by immediate institution of maintenance infusions after delivery, results in therapeutic levels of benzoate and phenylacetate in the newborn at delivery and, in conjunction with high-caloric enteral nutrition, prevents acute hyperammonemia and neurological decompensation. Following initial medical management, early liver transplantation may improve developmental outcome.

    View details for PubMedID 29396029

  • Correction of hyperleucinemia in MSUD patients on leucine-free dietary therapy MOLECULAR GENETICS AND METABOLISM Scott, A. I., Cusmano-Ozog, K., Enns, G. M., Cowan, T. M. 2017; 122 (4): 156–59
  • Correction of hyperleucinemia in MSUD patients on leucine-free dietary therapy. Molecular genetics and metabolism Scott, A. I., Cusmano-Ozog, K., Enns, G. M., Cowan, T. M. 2017

    Abstract

    PURPOSE: Maple Syrup Urine Disease (MSUD) is a rare disorder of branched-chain amino acid catabolism associated with encephalopathy from accumulation of leucine. Leucine is closely monitored during normal growth and particularly during acute illness. As most hospitals do not have access to rapid plasma amino acid quantification, the initial management is often empirical. A model describing the reduction of plasma leucine in hyperleucinemic patients on leucine-free formula would help to guide management and optimize testing frequency.METHODS: We retrospectively reviewed charts from 15 MSUD patients comprising 29 episodes of hyperleucinemia that were managed with leucine-free formula. Episodes were categorized by clinical presentation.RESULTS: Upon leucine restriction, plasma leucine concentrations fell exponentially at a rate proportional to approximately 50% of the starting value over each 24-hour period. Recovery appears to be sensitive to clinical status and triggering event of the hyperleucinemic episode. Patients with upper respiratory infections generally recovered slowly, while cases of dietary non-adherence resolved more quickly.CONCLUSION: This general model may help anticipate leucine levels during clinical management of MSUD patients when using nutritional support and leucine-free formula. The response of individual patients may vary depending on clinical status and triggering factors.

    View details for PubMedID 29032949

  • Glutathione as a Redox Biomarker in Mitochondrial Disease-Implications for Therapy. Journal of clinical medicine Enns, G. M., Cowan, T. M. 2017; 6 (5)

    Abstract

    Technical advances in the ability to measure mitochondrial dysfunction are providing new insights into mitochondrial disease pathogenesis, along with new tools to objectively evaluate the clinical status of mitochondrial disease patients. Glutathione (l-ϒ-glutamyl-l-cysteinylglycine) is the most abundant intracellular thiol, and the intracellular redox state, as reflected by levels of oxidized (GSSG) and reduced (GSH) glutathione, as well as the GSH/GSSG ratio, is considered to be an important indication of cellular health. The ability to quantify mitochondrial dysfunction in an affected patient will not only help with routine care, but also improve rational clinical trial design aimed at developing new therapies. Indeed, because multiple disorders have been associated with either primary or secondary deficiency of the mitochondrial electron transport chain and redox imbalance, developing mitochondrial therapies that have the potential to improve the intracellular glutathione status has been a focus of several clinical trials over the past few years. This review will also discuss potential therapies to increase intracellular glutathione with a focus on EPI-743 (α-tocotrienol quinone), a compound that appears to have the ability to modulate the activity of oxidoreductases, in particular NAD(P)H:quinone oxidoreductase 1.

    View details for DOI 10.3390/jcm6050050

    View details for PubMedID 28467362

  • Acylcarnitine Profiles Reflect Metabolic Vulnerability for Necrotizing Enterocolitis in Newborns Born Premature. journal of pediatrics Sylvester, K. G., Kastenberg, Z. J., Moss, R. L., Enns, G. M., Cowan, T. M., Shaw, G. M., Stevenson, D. K., Sinclair, T. J., Scharfe, C., Ryckman, K. K., Jelliffe-Pawlowski, L. L. 2017; 181: 80-85 e1

    Abstract

    To evaluate the association between newborn acylcarnitine profiles and the subsequent development of necrotizing enterocolitis (NEC) with the use of routinely collected newborn screening data in infants born preterm.A retrospective cohort study was conducted with the use of discharge records for infants born preterm admitted to neonatal intensive care units in California from 2005 to 2009 who had linked state newborn screening results. A model-development cohort of 94 110 preterm births from 2005 to 2008 was used to develop a risk-stratification model that was then applied to a validation cohort of 22 992 births from 2009.Fourteen acylcarnitine levels and acylcarnitine ratios were associated with increased risk of developing NEC. Each log unit increase in C5 and free carnitine /(C16 + 18:1) was associated with a 78% and a 76% increased risk for developing NEC, respectively (OR 1.78, 95% CI 1.53-2.02, and OR 1.76, 95% CI 1.51-2.06). Six acylcarnitine levels, along with birth weight and total parenteral nutrition, identified 89.8% of newborns with NEC in the model-development cohort (area under the curve 0.898, 95% CI 0.889-0.907) and 90.8% of the newborns with NEC in the validation cohort (area under the curve 0.908, 95% CI 0.901-0.930).Abnormal fatty acid metabolism was associated with prematurity and the development of NEC. Metabolic profiling through newborn screening may serve as an objective biologic surrogate of risk for the development of disease and thus facilitate disease-prevention strategies.

    View details for DOI 10.1016/j.jpeds.2016.10.019

    View details for PubMedID 27836286

  • A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature Dodd, D. n., Spitzer, M. H., Van Treuren, W. n., Merrill, B. D., Hryckowian, A. J., Higginbottom, S. K., Le, A. n., Cowan, T. M., Nolan, G. P., Fischbach, M. A., Sonnenburg, J. L. 2017; 551 (7682): 648–52

    Abstract

    The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

    View details for PubMedID 29168502

  • Laboratory diagnosis of biotinidase deficiency, 2017 update: a technical standard and guideline of the American College of Medical Genetics and Genomics. Genetics in medicine : official journal of the American College of Medical Genetics Strovel, E. T., Cowan, T. M., Scott, A. I., Wolf, B. 2017; 19 (10)

    Abstract

    Disclaimer: These ACMG Standards and Guidelines are intended as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these Standards and Guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of others that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, clinical laboratory geneticists should apply their professional judgment to the specific circumstances presented by the patient or specimen. Clinical laboratory scientists and geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Biotinidase deficiency is an autosomal recessively inherited disorder of biotin recycling that is associated with neurologic and cutaneous consequences if untreated. Fortunately, the clinical features of the disorder can be ameliorated or prevented by administering pharmacological doses of the vitamin biotin. Newborn screening and confirmatory diagnosis of biotinidase deficiency encompasses both enzymatic and molecular testing approaches. These guidelines were developed to define and standardize laboratory procedures for enzymatic biotinidase testing, to delineate situations for which follow-up molecular testing is warranted, and to characterize variables that can influence test performance and interpretation of results.

    View details for PubMedID 28682309

  • Expanding the phenotype of hawkinsinuria: new insights from response to N-acetyl-L-cysteine. Journal of inherited metabolic disease Gomez-Ospina, N., Scott, A. I., Oh, G. J., Potter, D., Goel, V. V., Destino, L., Baugh, N., Enns, G. M., Niemi, A., Cowan, T. M. 2016; 39 (6): 821-829

    Abstract

    Hawkinsinuria is a rare disorder of tyrosine metabolism that can manifest with metabolic acidosis and growth arrest around the time of weaning off breast milk, typically followed by spontaneous resolution of symptoms around 1 year of age. The urinary metabolites hawkinsin, quinolacetic acid, and pyroglutamic acid can aid in identifying this condition, although their relationship to the clinical manifestations is not known. Herein we describe clinical and laboratory findings in two fraternal twins with hawkinsinuria who presented with failure to thrive and metabolic acidosis. Close clinical follow-up and laboratory testing revealed previously unrecognized hypoglycemia, hypophosphatemia, combined hyperlipidemia, and anemia, along with the characteristic urinary metabolites, including massive pyroglutamic aciduria. Treatment with N-acetyl-L-cysteine (NAC) restored normal growth and normalized or improved most biochemical parameters. The dramatic response to NAC therapy supports the idea that glutathione depletion plays a key role in the pathogenesis of hawkinsinuria.

    View details for PubMedID 27488560

  • The antibiotic cefepime interferes with amino acid analysis by ion-exchange chromatography CLINICA CHIMICA ACTA Scott, A. I., Le, A., Cowan, T. M., Mendelsohn, B. A. 2016; 456: 149–50

    View details for PubMedID 26947969

  • RATE OF LEUCINE CLEARANCE IN MSUD Scott, A. I., Cusmano-Ozog, K., Enns, G. M., Cowan, T. M. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2016: 284
  • Treatment of methylmalonic acidemia by liver or combined liver-kidney transplantation. journal of pediatrics Niemi, A., Kim, I. K., Krueger, C. E., Cowan, T. M., Baugh, N., Farrell, R., Bonham, C. A., Concepcion, W., Esquivel, C. O., Enns, G. M. 2015; 166 (6): 1455-61 e1

    Abstract

    To assess biochemical, surgical, and long-term outcomes of liver (LT) or liver-kidney transplantation (LKT) for severe, early-onset methylmalonic acidemia/acid (MMA).A retrospective chart review (December 1997 to May 2012) of patients with MMA who underwent LT or LKT at Lucile Packard Children's Hospital at Stanford.Fourteen patients underwent LT (n = 6) or LKT (n = 8) at mean age 8.2 years (range 0.8-20.7). Eleven (79%) were diagnosed during the neonatal period, including 6 by newborn screening. All underwent deceased donor transplantation; 12 (86%) received a whole liver graft. Postoperative survival was 100%. At a mean follow-up of 3.25 ± 4.2 years, patient survival was 100%, liver allograft survival 93%, and kidney allograft survival 100%. One patient underwent liver re-transplantation because of hepatic artery thrombosis. After transplantation, there were no episodes of hyperammonemia, acidosis, or metabolic decompensation. The mean serum MMA at the time of transplantation was 1648 ± 1492 μmol/L (normal <0.3, range 99-4420). By 3 days, post-transplantation levels fell on average by 87% (mean 210 ± 154 μmol/L), and at 4 months, they were 83% below pre-transplantation levels (mean 305 ± 108 μmol/L). Developmental delay was present in 12 patients (86%) before transplantation. All patients maintained neurodevelopmental abilities or exhibited improvements in motor skills, learning abilities, and social functioning.LT or LKT for MMA eradicates episodes of hyperammonemia, results in excellent long-term survival, and suggests stabilization of neurocognitive development. Long-term follow-up is underway to evaluate whether patients who undergo early LT need kidney transplantation later in life.

    View details for DOI 10.1016/j.jpeds.2015.01.051

    View details for PubMedID 25771389

  • Treatment of Methylmalonic Acidemia by Liver or Combined Liver-Kidney Transplantation JOURNAL OF PEDIATRICS Niemi, A., Kim, I. K., Krueger, C. E., Cowan, T. M., Baugh, N., Farrell, R., Bonham, C. A., Concepcion, W., Esquivel, C. O., Enns, G. M. 2015; 166 (6): 1455-?

    Abstract

    To assess biochemical, surgical, and long-term outcomes of liver (LT) or liver-kidney transplantation (LKT) for severe, early-onset methylmalonic acidemia/acid (MMA).A retrospective chart review (December 1997 to May 2012) of patients with MMA who underwent LT or LKT at Lucile Packard Children's Hospital at Stanford.Fourteen patients underwent LT (n = 6) or LKT (n = 8) at mean age 8.2 years (range 0.8-20.7). Eleven (79%) were diagnosed during the neonatal period, including 6 by newborn screening. All underwent deceased donor transplantation; 12 (86%) received a whole liver graft. Postoperative survival was 100%. At a mean follow-up of 3.25 ± 4.2 years, patient survival was 100%, liver allograft survival 93%, and kidney allograft survival 100%. One patient underwent liver re-transplantation because of hepatic artery thrombosis. After transplantation, there were no episodes of hyperammonemia, acidosis, or metabolic decompensation. The mean serum MMA at the time of transplantation was 1648 ± 1492 μmol/L (normal <0.3, range 99-4420). By 3 days, post-transplantation levels fell on average by 87% (mean 210 ± 154 μmol/L), and at 4 months, they were 83% below pre-transplantation levels (mean 305 ± 108 μmol/L). Developmental delay was present in 12 patients (86%) before transplantation. All patients maintained neurodevelopmental abilities or exhibited improvements in motor skills, learning abilities, and social functioning.LT or LKT for MMA eradicates episodes of hyperammonemia, results in excellent long-term survival, and suggests stabilization of neurocognitive development. Long-term follow-up is underway to evaluate whether patients who undergo early LT need kidney transplantation later in life.

    View details for DOI 10.1016/j.jpeds.2015.01.051

    View details for Web of Science ID 000355018200025

    View details for PubMedID 25771389

  • Analysis of 20 years of urinary mps proficiency data from the college of American pathogists (CAP) Wood, T., Cowan, T., Pasquali, M., Oglesbee, D. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2015: 346
  • Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway GENETICS IN MEDICINE Enns, G. M., Shashi, V., Bainbridge, M., Gambello, M. J., Zahir, F. R., Bast, T., Crimian, R., Schoch, K., Platt, J., Cox, R., Bernstein, J. A., Scavina, M., Walter, R. S., Bibb, A., Jones, M., Hegde, M., Graham, B. H., Need, A. C., Oviedo, A., Schaaf, C. P., Boyle, S., Butte, A. J., Chen, R., Clark, M. J., Haraksingh, R., Cowan, T. M., He, P., Langlois, S., Zoghbi, H. Y., Snyder, M., Gibbs, R. A., Freeze, H. H., Goldstein, D. B. 2014; 16 (10): 751-758

    Abstract

    Purpose:The endoplasmic reticulum-associated degradation pathway is responsible for the translocation of misfolded proteins across the endoplasmic reticulum membrane into the cytosol for subsequent degradation by the proteasome. To define the phenotype associated with a novel inherited disorder of cytosolic endoplasmic reticulum-associated degradation pathway dysfunction, we studied a series of eight patients with deficiency of N-glycanase 1.Methods:Whole-genome, whole-exome, or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data.Results:All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypolacrima or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele.Conclusion:NGLY1 deficiency is a novel autosomal recessive disorder of the endoplasmic reticulum-associated degradation pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a broader range of mutations are detected.Genet Med advance online publication 20 March 2014Genetics in Medicine (2014); doi:10.1038/gim.2014.22.

    View details for DOI 10.1038/gim.2014.22

    View details for Web of Science ID 000342884500005

  • Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway. Genetics in medicine Enns, G. M., Shashi, V., Bainbridge, M., Gambello, M. J., Zahir, F. R., Bast, T., Crimian, R., Schoch, K., Platt, J., Cox, R., Bernstein, J. A., Scavina, M., Walter, R. S., Bibb, A., Jones, M., Hegde, M., Graham, B. H., Need, A. C., Oviedo, A., Schaaf, C. P., Boyle, S., Butte, A. J., Chen, R., Clark, M. J., Haraksingh, R., Cowan, T. M., He, P., Langlois, S., Zoghbi, H. Y., Snyder, M., Gibbs, R. A., Freeze, H. H., Goldstein, D. B. 2014; 16 (10): 751-758

    Abstract

    Purpose:The endoplasmic reticulum-associated degradation pathway is responsible for the translocation of misfolded proteins across the endoplasmic reticulum membrane into the cytosol for subsequent degradation by the proteasome. To define the phenotype associated with a novel inherited disorder of cytosolic endoplasmic reticulum-associated degradation pathway dysfunction, we studied a series of eight patients with deficiency of N-glycanase 1.Methods:Whole-genome, whole-exome, or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data.Results:All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypolacrima or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele.Conclusion:NGLY1 deficiency is a novel autosomal recessive disorder of the endoplasmic reticulum-associated degradation pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a broader range of mutations are detected.Genet Med advance online publication 20 March 2014Genetics in Medicine (2014); doi:10.1038/gim.2014.22.

    View details for DOI 10.1038/gim.2014.22

    View details for PubMedID 24651605

  • Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway (vol 111, pg 236, 2014) GENETICS IN MEDICINE Enns, G. M., Shashi, V., Bainbridge, M., Gambello, M. J., Zahir, F. R., Bast, T., Crimian, R., Schoch, K., Platt, J., Cox, R., Bernstein, J. A., Scavina, M., Walter, R. S., Bibb, A., Jones, M., Hegde, M., Graham, B. H., Need, A. C., Oviedo, A., Schaaf, C. P., Boyle, S., Butte, A. J., Chen, R., Chen, R., Clark, M. J., Haraksingh, R., Cowan, T. M., He, P., Langlois, S., Zoghbi, H. Y., Snyder, M., Gibbs, R. A., Freeze, H. H., Goldstein, D. B., Chen, R., FORGE Canada Consortium 2014; 16 (7): 568
  • Degree of Glutathione Deficiency and Redox Imbalance Depend on Subtype of Mitochondrial Disease and Clinical Status PLOS ONE Enns, G. M., Moore, T., Le, A., Atkuri, K., Shah, M. K., Cusmano-Ozog, K., Niemi, A., Cowan, T. M. 2014; 9 (6)

    Abstract

    Mitochondrial disorders are associated with decreased energy production and redox imbalance. Glutathione plays a central role in redox signaling and protecting cells from oxidative damage. In order to understand the consequences of mitochondrial dysfunction on in vivo redox status, and to determine how this varies by mitochondrial disease subtype and clinical severity, we used a sensitive tandem mass spectrometry assay to precisely quantify whole blood reduced (GSH) and oxidized (GSSG) glutathione levels in a large cohort of mitochondrial disorder patients. Glutathione redox potential was calculated using the Nernst equation. Compared to healthy controls (n = 59), mitochondrial disease patients (n = 58) as a group showed significant redox imbalance (redox potential -251 mV ± 9.7, p<0.0001) with an increased level of oxidation by ∼ 9 mV compared to controls (-260 mV ± 6.4). Underlying this abnormality were significantly lower whole blood GSH levels (p = 0.0008) and GSH/GSSG ratio (p = 0.0002), and significantly higher GSSG levels (p<0.0001) in mitochondrial disease patients compared to controls. Redox potential was significantly more oxidized in all mitochondrial disease subgroups including Leigh syndrome (n = 15), electron transport chain abnormalities (n = 10), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (n = 8), mtDNA deletion syndrome (n = 7), mtDNA depletion syndrome (n = 7), and miscellaneous other mitochondrial disorders (n = 11). Patients hospitalized in metabolic crisis (n = 7) showed the greatest degree of redox imbalance at -242 mV ± 7. Peripheral whole blood GSH and GSSG levels are promising biomarkers of mitochondrial dysfunction, and may give insights into the contribution of oxidative stress to the pathophysiology of the various mitochondrial disorders. In particular, evaluation of redox potential may be useful in monitoring of clinical status or response to redox-modulating therapies in clinical trials.

    View details for DOI 10.1371/journal.pone.0100001

    View details for Web of Science ID 000338508200063

    View details for PubMedCentralID PMC4062483

  • PRENATAL TREATMENT OF ORNITHINE TRANSCARBAMYLASE DEFICIENCY Wilnai, Y., Alcorn, D., Benitz, W., Berquist, W., Bernstein, J., Blumenfeld, Y. J., Castillo, R., Concepcion, W., Cowan, T., Cox, K. L., Cusmano, K., Deirdre, L., Esquival, C., Hintz, S. R., Homeyer, M., Hudgins, L., Palma, J., Summar, M. L., Schelley, S., Vishnu, P., Enns, G. M. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2014: 248
  • CORRELATION OF GLUTATHIONE REDOX POTENTIAL WITH MITOCHONDRIAL DISEASE ETIOLOGY AND CLINICAL SEVERITY Enns, G. M., Moore, T., Le, A., Atkuri, K., Shah, M. K., Cusmano-Ozog, K., Nierni, A., Cowan, T. M. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2014: 268
  • MUTATIONS IN NGLY1 CAUSE AN INHERITED DISORDER OF THE ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION (ERAD) PATHWAY Enns, G. M., Shashi, V., Zahir, F., Gambello, M. J., Bainbridge, M. N., Bast, T., Crimian, R., Schoch, K., Zoghbi, H. Y., Platt, J., Cox, R., Bernstein, J., Scavina, M., Walter, R. S., Need, A. C., Oviedo, A., Langlois, S., Bibb, A., Jones, M., Hedge, M., Gibbs, R. A., Schaaf, C. P., Boyle, S., Butte, A. J., Clark, M., Cowan, T., He, P., Snyder, M., Freeze, H. H., Goldstein, D. B., FORGE Canada Consortium ACADEMIC PRESS INC ELSEVIER SCIENCE. 2014: 236–37
  • QUANTIFICATION OF URINE KERATAN SULFATE LEVELS IN MUCOLIPIDOSIS II AND III PATIENTS BY UPLC-MS/MS Niemi, A., Gomez-Ospina, N., Goel, V., Destino, L., Cowan, T. M. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2014: 253–54
  • A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandem mass spectrometry (LC-MS/MS) JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES Le, A., Ng, A., Kwan, T., Cusmano-Ozog, K., Cowan, T. M. 2014; 944: 166-174

    Abstract

    The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. To overcome these challenges, a improved method for amino acid analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Samples were deproteinized with sulfosalicylic acid and supernatants diluted with tridecafluoroheptanoic acid. Chromatographic separation of amino acids occurred using two columns, with conditions favoring resolution of isobaric compounds and minimizing ion suppression. Eluted compounds were detected by selective reaction monitoring, and quantitated by relating peak areas of amino acids to externally run standards. Validation studies evaluated linearity, within- and between-run imprecision, lower limits of detection and quantification for 33 amino acids, and correlation with the Biochrom 30 Amino Acid Analyzer. Total run time including re-equilibration was 15min per sample. Within-run precision averaged 2.8% for all compounds, with an average linear correlation coefficient of 0.995. The majority of compounds were reliably quantitated at ≤0.1μM. Between-run precision averaged 4.0%. Results showed excellent correlation with the Biochrom 30 amino acid analyzer with an average overall correlation of 0.908. We conclude that our method is extremely sensitive, specific and reproducible and represents an improvement over other currently available technologies.

    View details for DOI 10.1016/j.jchromb.2013.11.017

    View details for Web of Science ID 000329770300027

    View details for PubMedID 24316529

  • Evidence of redox imbalance in a patient with succinic semialdehyde dehydrogenase deficiency. Molecular genetics and metabolism reports Niemi, A., Brown, C., Moore, T., Enns, G. M., Cowan, T. M. 2014; 1: 129-132

    Abstract

    The pathophysiology of succinic semialdehyde dehydrogenase (SSADH) deficiency is not completely understood. Oxidative stress, mitochondrial pathology, and low reduced glutathione levels have been demonstrated in mice, but no studies have been reported in humans. We report on a patient with SSADH deficiency in whom we found low levels of blood reduced glutathione (GSH), and elevations of dicarboxylic acids in urine, suggestive of possible redox imbalance and/or mitochondrial dysfunction. Thus, targeting the oxidative stress axis may be a potential therapeutic approach if our findings are confirmed in other patients.

    View details for PubMedID 27896081

  • Degree of glutathione deficiency and redox imbalance depend on subtype of mitochondrial disease and clinical status. PloS one Enns, G. M., Moore, T., Le, A., Atkuri, K., Shah, M. K., Cusmano-Ozog, K., Niemi, A., Cowan, T. M. 2014; 9 (6)

    Abstract

    Mitochondrial disorders are associated with decreased energy production and redox imbalance. Glutathione plays a central role in redox signaling and protecting cells from oxidative damage. In order to understand the consequences of mitochondrial dysfunction on in vivo redox status, and to determine how this varies by mitochondrial disease subtype and clinical severity, we used a sensitive tandem mass spectrometry assay to precisely quantify whole blood reduced (GSH) and oxidized (GSSG) glutathione levels in a large cohort of mitochondrial disorder patients. Glutathione redox potential was calculated using the Nernst equation. Compared to healthy controls (n = 59), mitochondrial disease patients (n = 58) as a group showed significant redox imbalance (redox potential -251 mV ± 9.7, p<0.0001) with an increased level of oxidation by ∼ 9 mV compared to controls (-260 mV ± 6.4). Underlying this abnormality were significantly lower whole blood GSH levels (p = 0.0008) and GSH/GSSG ratio (p = 0.0002), and significantly higher GSSG levels (p<0.0001) in mitochondrial disease patients compared to controls. Redox potential was significantly more oxidized in all mitochondrial disease subgroups including Leigh syndrome (n = 15), electron transport chain abnormalities (n = 10), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (n = 8), mtDNA deletion syndrome (n = 7), mtDNA depletion syndrome (n = 7), and miscellaneous other mitochondrial disorders (n = 11). Patients hospitalized in metabolic crisis (n = 7) showed the greatest degree of redox imbalance at -242 mV ± 7. Peripheral whole blood GSH and GSSG levels are promising biomarkers of mitochondrial dysfunction, and may give insights into the contribution of oxidative stress to the pathophysiology of the various mitochondrial disorders. In particular, evaluation of redox potential may be useful in monitoring of clinical status or response to redox-modulating therapies in clinical trials.

    View details for DOI 10.1371/journal.pone.0100001

    View details for PubMedID 24941115

  • A new LC-MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Moore, T., Le, A., Niemi, A., Kwan, T., Cusmano-Ozog, K., Enns, G. M., Cowan, T. M. 2013; 929: 51-55

    Abstract

    Reduced levels of glutathione (γ-glutamylcysteinylglycine, GSH) and the ratio of GSH to glutathione disulfide (GSSG) can serve as important indicators of oxidative stress and disease risk. Measured concentrations of GSH and GSSG vary widely between laboratories, largely due to the instability of GSH during sample handling and variables arising from different analytical methods. We have developed a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring whole blood GSH and GSSG that minimizes preanalytic and analytic variability, reliably eliminates interference from ion suppression, and can easily be implemented in clinical laboratories. Samples were deproteinized with sulfosalicylic acid (SSA) and derivatized with N-ethylmaleimide (NEM) in a single preparative step, and the resulting supernatants combined with stable-isotope internal standards (GSH-(13)C, (15)N-NEM and GSSG-(13)C,(15)N), subjected to chromatographic separation using a Hypercarb column, and analyzed by MS/MS in the positive-ion mode. Results showed excellent linearity for both GSH and GSSG over the ranges of physiologic normal, with inter- and intra-assay CV's of 3.1-4.3% and accuracy between 95% and 101%. The lower limits of detection (LLOD) were 0.4μM for GSH and 0.1μM for GSSG and the lower limits of quantitation (LLOQ) were 1.5μM for GSH and 0.1μM for GSSG. Derivatized samples are stable for at least 3 years when stored at -80°C, and underivatized samples for at least 24h at either 4°C or room temperature. Reference intervals were determined for 59 control samples, and were (mean±SD): GSH 900±140μM; GSSG 1.17±0.43μM; GSH/GSSG 880±370.

    View details for DOI 10.1016/j.jchromb.2013.04.004

    View details for PubMedID 23660247

  • Liver transplantation for urea cycle disorders in pediatric patients: A single-center experience PEDIATRIC TRANSPLANTATION Kim, I. K., Niemi, A., Krueger, C., Bonham, C. A., Concepcion, W., Cowan, T. M., Enns, G. M., Esquivel, C. O. 2013; 17 (2): 158-167

    Abstract

    LT has emerged as a surgical treatment for UCDs. We hypothesize that LT can be safely and broadly utilized in the pediatric population to effectively prevent hyperammonemic crises and potentially improve neurocognitive outcomes. To determine the long-term outcomes of LT for UCDs, charts of children with UCD who underwent LT were retrospectively reviewed at an academic institution between July 2001 and May 2012. A total of 23 patients with UCD underwent LT at a mean age of 3.4 yr. Fifteen (65%) patients received a whole-liver graft, seven patients (30%) received a reduced-size graft, and one patient received a living donor graft. Mean five-yr patient survival was 100%, and allograft survival was 96%. Mean peak blood ammonia (NH(3) ) at presentation was 772 μmol/L (median 500, range 178-2969, normal <30-50). After transplantation, there were no episodes of hyperammonemia. Eleven patients were diagnosed with some degree of developmental delay before transplantation, which remained stable or improved after transplantation. Patients without developmental delay before transplantation maintained their cognitive abilities at long-term follow-up. LT was associated with the eradication of hyperammonemia, removal of dietary restrictions, and potentially improved neurocognitive development. Long-term follow-up is underway to evaluate whether LT at an early age (<1 yr) will attain improved neurodevelopmental outcomes.

    View details for DOI 10.1111/petr.12041

    View details for PubMedID 23347504

  • ß-Galactosidosis in Patient with Intermediate GM1 and MBD Phenotype. JIMD reports Moore, T., Bernstein, J. A., Casson-Parkin, S., Cowan, T. M. 2013; 7: 77-79

    Abstract

    A 5-year-old girl with clinical and biochemical phenotypes encompassing both GM1-gangliosidosis (GM1) and Morquio B disease (MBD) is described. Mild generalized skeletal dysplasia and keratan sulfaturia were consistent with a diagnosis of MBD, while developmental delay and GM1-specific oligosacchariduria were consistent with GM1 gangliosidosis. No observable β-galactosidase activity was detected in leukocytes, and two mutations, p.R201H (c.602G>A) and p.G311R (c.931G>A), were identified by gene sequencing. The R201H substitution has been previously reported in patients with both GM1 and MBD, and G311R is a novel mutation. Our patient represents a further example of the clinical heterogeneity that can result from mutations at the β-galactosidase locus.

    View details for DOI 10.1007/8904_2012_145

    View details for PubMedID 23430499

  • The Role of Oxidative Stress in Pediatric Immune Thrombocytopenia Lo, C., Zhang, B., Cusmano-Ozog, K., Wong, W., Jeng, M., Cowan, T., Zehnder, J. L. AMER SOC HEMATOLOGY. 2012
  • Improved redox status after liver transplantation in a patient with MMA mut(0) subtype; functional evidence for EPI-743 therapy Niemi, A., Niemi, A., Moore, T., Cowan, T., Kheifets, V., Enns, G. M. ELSEVIER SCI LTD. 2012: 557–57
  • Increased incidence of profound biotinidase deficiency among Hispanic newborns in California MOLECULAR GENETICS AND METABOLISM Cowan, T. M., Kazerouni, N. N., Dharajiya, N., Lorey, F., Roberson, M., Hodgkinson, C., Schrijver, I. 2012; 106 (4): 485-487

    Abstract

    We report population findings from newborn screening for biotinidase deficiency in California, representing over 2,000,000 newborns. The incidence of profound deficiency was 1/73,629, higher than in other reported populations. Out of 28 patients with profound biotinidase deficiency, 19 were of Hispanic descent, suggesting an increased frequency among this group. Of the 28 patients, 23 underwent mutation analysis of the BTD gene, with one common mutation, 528G>T, found in 43.3% of Hispanic alleles tested.

    View details for DOI 10.1016/j.ymgme.2012.05.017

    View details for Web of Science ID 000307322100015

    View details for PubMedID 22698809

  • An improved LC-MS/MS method for the detection of classic and low excretor glutaric acidemia type 1 JOURNAL OF INHERITED METABOLIC DISEASE Moore, T., Le, A., Cowan, T. M. 2012; 35 (3): 431-435

    Abstract

    Glutaric acidemia type I (GA1) is associated with elevated glutarylcarnitine (C5DC), typically measured as its butylester by acylcarnitine profile analysis using tandem mass spectrometry (MS/MS) and the precursor-product ion pair of m/z 388-85. This method neither distinguishes between C5DC and its isomer 3-hydroxydecanoylcarnitine (C10-OH) nor reliably detects the low-excretor variant of GA1, leading to both false-positive and false-negative results when testing for GA1. To overcome these limitations, we developed an LC-MS/MS method that discriminates C5DC from C10-OH by the use of precursor-product ion pairs specific for butylated C5DC (m/z 388-115) and underivatized C10-OH (m/z 332-85). The C5DC method was validated over the linearity range of 0.025-20 μM with a lower limit of quantification (LOQ) of 0.025 μM. Excellent precision and accuracy were also observed. We tested plasma samples from 10 patients with confirmed GA1 (including 3 with the low-excretor variant), 21 patients with mild elevations of C5DC or C10-OH by routine acylcarnitine analysis for which GA1 ultimately was excluded, and 29 normal controls. By using the m/z 388-115 ion pair, all cases of GA1, including the low-excretor variant, were reliably distinguished from normal controls. By using the m/z 388-85 pair, patients with ambiguous elevations of C5DC or C10-OH demonstrated clearly elevated levels of C10-OH (m/z 332-85) but normal C5DC (m/z 388-115), confirming that the apparent elevation of C5DC is due to interference by C10-OH. Our method results in excellent detection of GA1, including the low-excretor variant, and also provides a means to discriminate C5DC and C10-OH in follow-up testing and routine acylcarnitine studies.

    View details for DOI 10.1007/s10545-011-9405-6

    View details for Web of Science ID 000302492300007

    View details for PubMedID 22005781

  • Low glutathione levels in a patient with succinic semialdehyde dehydrogenase (SSADH) deficiency 35th Annual Meeting of the Society-for-Inherited-Metabolic-Disorders (SIMD) Niemi, A., Brown, C., Moore, T., Enns, G. M., Cowan, T. M. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2012: 345–45
  • A Novel Mutation in the HSD17B10 Gene of a 10-Year-Old Boy with Refractory Epilepsy, Choreoathetosis and Learning Disability PLOS ONE Seaver, L. H., He, X., Abe, K., Cowan, T., Enns, G. M., Sweetman, L., Philipp, M., Lee, S., Malik, M., Yang, S. 2011; 6 (11)

    Abstract

    Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency, whereas a silent mutation results in mental retardation, X-linked, syndromic 10 (MRXS10). Here we report a novel missense mutation found in the HSD17B10 gene, namely c.194T>C transition (rs104886492), brought about by the loss of two forked methyl groups of valine 65 in the HSD10 active site. The affected boy, who possesses mutant HSD10 (p.V65A), has a neurological syndrome with metabolic derangements, choreoathetosis, refractory epilepsy and learning disability. He has no history of acute decompensation or metabolic acidosis whereas his urine organic acid profile, showing elevated levels of 2-methyl-3-hydroxybutyrate and tiglylglycine, is characteristic of HSD10 deficiency. His HSD10 activity was much lower than the normal control level, with normal β-ketothiolase activity. The c.194T>C mutation in HSD17B10 can be identified by the restriction fragment polymorphism analysis, thereby facilitating the screening of this novel mutation in individuals with intellectual disability of unknown etiology and their family members much easier. The patient's mother is an asymptomatic carrier, and has a mixed ancestry (Hawaiian, Japanese and Chinese). This demonstrates that HSD10 deficiency patients are not confined to a particular ethnicity although previously reported cases were either Spanish or German descendants.

    View details for DOI 10.1371/journal.pone.0027348

    View details for Web of Science ID 000297792400006

    View details for PubMedID 22132097

    View details for PubMedCentralID PMC3222643

  • The role of vanin-1 and oxidative stress-related pathways in distinguishing acute and chronic pediatric ITP BLOOD Zhang, B., Lo, C., Shen, L., Sood, R., Jones, C., Cusmano-Ozog, K., Park-Snyder, S., Wong, W., Jeng, M., Cowan, T., Engleman, E. G., Zehnder, J. L. 2011; 117 (17): 4569-4579

    Abstract

    Pediatric immune thrombocytopenia (ITP) is usually self-limited. However, approximately 20% of children develop chronic ITP, which can be associated with significant morbidity because of long-term immunosuppression and splenectomy in refractory cases. To explore the molecular mechanism of chronic ITP compared with acute ITP, we studied 63 pediatric patients with ITP. Gene expression analysis of whole blood revealed distinct signatures for acute and chronic ITP. Oxidative stress-related pathways were among the most significant chronic ITP-associated pathways. Overexpression of VNN1, an oxidative stress sensor in epithelial cells, was most strongly associated with progression to chronic ITP. Studies of normal persons demonstrated VNN1 expression in a variety of blood cells. Exposure of blood mononuclear cells to oxidative stress inducers elicited dramatic up-regulation of VNN1 and down-regulation of PPARγ, indicating a role for VNN1 as a peripheral blood oxidative stress sensor. Assessment of redox state by tandem mass spectrometry demonstrated statistically significant lower glutathione ratios in patients with ITP versus healthy controls; lower glutathione ratios were also seen in untreated patients with ITP compared with recently treated patients. Our work demonstrates distinct patterns of gene expression in acute and chronic ITP and implicates oxidative stress pathways in the pathogenesis of chronic pediatric ITP.

    View details for DOI 10.1182/blood-2010-09-304931

    View details for PubMedID 21325602

  • Commentary. Clinical chemistry Cowan, T. M. 2011; 57 (4): 548-549

    View details for DOI 10.1373/clinchem.2010.160333

    View details for PubMedID 21444739

  • Technical standards and guidelines for the diagnosis of biotinidase deficiency GENETICS IN MEDICINE Cowan, T. M., Blitzer, M. G., Wolf, B. 2010; 12 (7): 464-470

    Abstract

    Biotinidase deficiency is an autosomal recessively inherited disorder of biotin recycling that is associated with neurologic and cutaneous consequences if untreated. Fortunately, the clinical features of the disorder can be ameliorated or prevented by administering pharmacological doses of the vitamin biotin. Newborn screening and confirmatory diagnosis of biotinidase deficiency encompasses both enzymatic and molecular testing approaches. These guidelines were developed to define and standardize laboratory procedures for enzymatic biotinidase testing, to delineate situations for which follow-up molecular testing is warranted, and to characterize variables that can influence test performance and interpretation of results.

    View details for DOI 10.1097/GIM.0b013e3181e4cc0f

    View details for Web of Science ID 000279891300009

    View details for PubMedID 20539236

  • Biomarkers of redox abnormalities in mitochondrial disorders and organic acidemias Atkuri, K. R., Cowan, T. M., Procaccio, V., Herzenberg, L. A., Enns, G. M. ELSEVIER SCI LTD. 2010: 206–7
  • Mapping Gene Associations in Human Mitochondria using Clinical Disease Phenotypes PLOS COMPUTATIONAL BIOLOGY Scharfe, C., Lu, H. H., Neuenburg, J. K., Allen, E. A., Li, G., Klopstock, T., Cowan, T. M., Enns, G. M., Davis, R. W. 2009; 5 (4)

    Abstract

    Nuclear genes encode most mitochondrial proteins, and their mutations cause diverse and debilitating clinical disorders. To date, 1,200 of these mitochondrial genes have been recorded, while no standardized catalog exists of the associated clinical phenotypes. Such a catalog would be useful to develop methods to analyze human phenotypic data, to determine genotype-phenotype relations among many genes and diseases, and to support the clinical diagnosis of mitochondrial disorders. Here we establish a clinical phenotype catalog of 174 mitochondrial disease genes and study associations of diseases and genes. Phenotypic features such as clinical signs and symptoms were manually annotated from full-text medical articles and classified based on the hierarchical MeSH ontology. This classification of phenotypic features of each gene allowed for the comparison of diseases between different genes. In turn, we were then able to measure the phenotypic associations of disease genes for which we calculated a quantitative value that is based on their shared phenotypic features. The results showed that genes sharing more similar phenotypes have a stronger tendency for functional interactions, proving the usefulness of phenotype similarity values in disease gene network analysis. We then constructed a functional network of mitochondrial genes and discovered a higher connectivity for non-disease than for disease genes, and a tendency of disease genes to interact with each other. Utilizing these differences, we propose 168 candidate genes that resemble the characteristic interaction patterns of mitochondrial disease genes. Through their network associations, the candidates are further prioritized for the study of specific disorders such as optic neuropathies and Parkinson disease. Most mitochondrial disease phenotypes involve several clinical categories including neurologic, metabolic, and gastrointestinal disorders, which might indicate the effects of gene defects within the mitochondrial system. The accompanying knowledgebase (http://www.mitophenome.org/) supports the study of clinical diseases and associated genes.

    View details for DOI 10.1371/journal.pcbi.1000374

    View details for PubMedID 19390613

  • Clinical and Molecular Heterogeneity in Patients with the CblD Inborn Error of Cobalamin Metabolism JOURNAL OF PEDIATRICS Miousse, I. R., Watkins, D., Coelho, D., Rupar, T., Crombez, E. A., Vilain, E., Bernstein, J. A., Cowan, T., Lee-Messer, C., Enns, G. M., Fowler, B., Rosenblatt, D. S. 2009; 154 (4): 551-556

    Abstract

    To describe 3 patients with the cblD disorder, a rare inborn error of cobalamin metabolism caused by mutations in the MMADHC gene that can result in isolated homocystinuria, isolated methylmalonic aciduria, or combined homocystinuria and methylmalonic aciduria.Patient clinical records were reviewed. Biochemical and somatic cell genetic studies were performed on cultured fibroblasts. Sequence analysis of the MMADHC gene was performed on patient DNA.Patient 1 presented with isolated methylmalonic aciduria, patient 3 with isolated homocystinuria, and patient 2 with combined methylmalonic aciduria and homocystinuria. Studies of cultured fibroblasts confirmed decreased synthesis of adenosylcobalamin in patient 1, decreased synthesis of methylcobalamin in patient 3, and decreased synthesis of both cobalamin derivatives in patient 2. The diagnosis of cblD was established in each patient by complementation analysis. Mutations in the MMADHC gene were identified in all patients.The results emphasize the heterogeneous clinical, cellular and molecular phenotype of the cblD disorder. The results of molecular analysis of the MMADHC gene are consistent with the hypothesis that mutations affecting the N terminus of the MMADHC protein are associated with methylmalonic aciduria, and mutations affecting the C terminus are associated with homocystinuria.

    View details for DOI 10.1016/j.jpeds.2008.10.043

    View details for Web of Science ID 000264808000020

    View details for PubMedID 19058814

  • Inherited disorders affecting mitochondrial function are associated with glutathione deficiency and hypocitrullinemia PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Atkuri, K. R., Cowan, T. M., Kwan, T., Ng, A., Herzenberg, L. A., Herzenberg, L. A., Enns, G. M. 2009; 106 (10): 3941-3945

    Abstract

    Disorders affecting mitochondria, including those that directly affect the respiratory chain function or result from abnormalities in branched amino acid metabolism (organic acidemias), have been shown to be associated with impaired redox balance. Almost all of the evidence underlying this conclusion has been obtained from studies on patient biopsies or animal models. Since the glutathione (iGSH) system provides the main protection against oxidative damage, we hypothesized that untreated oxidative stress in individuals with mitochondrial dysfunction would result in chronic iGSH deficiency. We confirm this hypothesis here in studies using high-dimensional flow cytometry (Hi-D FACS) and biochemical analysis of freshly obtained blood samples from patients with mitochondrial disorders or organic acidemias. T lymphocyte subsets, monocytes and neutrophils from organic acidemia and mitochondrial patients who were not on antioxidant supplements showed low iGSH levels, whereas similar subjects on antioxidant supplements showed normal iGSH. Measures of iROS levels in blood were insufficient to reveal the chronic oxidative stress in untreated patients. Patients with organic acidemias showed elevated plasma protein carbonyls, while plasma samples from all patients tested showed hypocitrullinemia. These findings indicate that measurements of iGSH in leukocytes may be a particularly useful biomarker to detect redox imbalance in mitochondrial disorders and organic acidemias, thus providing a relatively non-invasive means to monitor disease status and response to therapies. Furthermore, studies here suggest that antioxidant therapy may be useful for relieving the chronic oxidative stress that otherwise occurs in patients with mitochondrial dysfunction.

    View details for DOI 10.1073/pnas.0813409106

    View details for PubMedID 19223582

  • Redox Abnormalities in Inherited Mitochondrial Diseases Atkuri, K. R., Moore, T., Cowan, T. M., Herzenberg, L. A., Herzenberg, L. A., Procaccio, V., Enns, G. M. ELSEVIER SCIENCE INC. 2009: S85
  • Management and quality assurance in the biochemical genetics laboratory. Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.] Cowan, T. M., Strovel, E. T. 2008; Chapter 17: Unit 17 7-?

    Abstract

    High-quality biochemical genetics testing is critical for proper diagnosis and management of patients with inborn errors of metabolism. An accurate diagnosis is a prerequisite for proper treatment, ongoing management, and ultimately, for optimal clinical outcome. Quality testing in the biochemical genetics laboratory is managed via adherence to federal regulations that govern clinical laboratory testing. However, because these were not specifically written for biochemical genetics laboratories, a number of professional organizations have developed practice guidelines to address gaps in the federal code. This unit reviews these regulations and guidelines as they apply to quality management of the biochemical genetics laboratory, including test validation, personnel standards, proficiency testing, and overall quality management (including quality assurance, quality control, and quality improvement). It also provides examples of protocols and forms that can be adapted for the documentation of test validation, personnel training, quality control, and quality assurance.

    View details for DOI 10.1002/0471142905.hg1707s59

    View details for PubMedID 18972371

  • Acylcarnitine profile analysis GENETICS IN MEDICINE Rinaldo, P., Cowan, T. M., Matern, D. 2008; 10 (2): 151-156

    Abstract

    These Technical Standards and Guidelines were developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular standard or guidelines was adopted, and to consider other relevant medical and scientific information that becomes available after that date.

    View details for DOI 10.1097/GIM.0013e3181614289

    View details for Web of Science ID 000253277400011

    View details for PubMedID 18281923

  • An unusual case of Pompe disease presenting as muscular dystrophy Cusmano-Ozog, K., Vogel, H., Cowan, T., Enns, G. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S18
  • Progressive cerebral vascular degeneration with mitochondrial encephalopathy AMERICAN JOURNAL OF MEDICAL GENETICS PART A Longo, N., Schrijver, I., Vogel, H., Pique, L. M., Cowan, T. M., Pasquali, M., Steinberg, G. K., Hedlund, G. L., Ernst, S. L., Gallagher, R. C., Enns, G. M. 2008; 146A (3): 361-367

    Abstract

    MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) is a maternally inherited disorder characterized by recurrent cerebral infarctions that do not conform to discreet vascular territories. Here we report on a patient who presented at 7 years of age with loss of consciousness and severe metabolic acidosis following vomiting and dehydration. She developed progressive sensorineural hearing loss, myopathy, ptosis, short stature, and mild developmental delays after normal early development. Biochemical testing identified metabolites characteristic of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (hexanoylglycine and suberylglycine), but also severe lactic acidemia (10-25 mM) and, in urine, excess of lactic acid, intermediates of the citric cycle, and marked ketonuria, suggesting mitochondrial dysfunction. She progressed rapidly to develop temporary cortical blindness. Brain imaging indicated generalized atrophy, more marked on the left side, in addition to white matter alterations consistent with a mitochondrial disorder. Magnetic resonance angiography indicated occlusion of the left cerebral artery with development of collateral circulation (Moyamoya syndrome). This process worsened over time to involve the other side of the brain. A muscle biopsy indicated the presence of numerous ragged red fibers. Molecular testing confirmed compound heterozygosity for the common mutation in the MCAD gene (985A>G) and a second pathogenic mutation (233T>C). MtDNA testing indicated that the muscle was almost homoplasmic for the 3243A>T mutation in tRNALeu, with a lower mutant load (about 50% heteroplasmy) in blood and skin fibroblasts. These results indicate that mitochondrial disorders may be associated with severe vascular disease resulting in Moyamoya syndrome. The contribution of the concomitant MCAD deficiency to the development of the phenotype in this case is unclear.

    View details for DOI 10.1002/ajmg.a.31841

    View details for Web of Science ID 000253008300014

  • Importance of culturing primary lymphocytes at physiological oxygen levels PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Atkuri, K. R., Herzenberg, L. A., Niemi, A., Cowan, T., Herzenberg, L. A. 2007; 104 (11): 4547-4552

    Abstract

    Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%), the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.

    View details for DOI 10.1073/pnas.0611732104

    View details for Web of Science ID 000244972700055

    View details for PubMedID 17360561

    View details for PubMedCentralID PMC1838638

  • Glutaryl-CoA dehydrogenase deficiency and newborn screening: Retrospective analysis of a low excretor provides further evidence that some cases may be missed MOLECULAR GENETICS AND METABOLISM Gallagher, R. C., Cowan, T. M., Goodman, S. I., Enns, G. A. 2005; 86 (3): 417-420

    Abstract

    Glutaryl-CoA dehydrogenase deficiency (GA-I) is associated with the onset of irreversible, disabling dystonia between 3 and 18 months of age. Presymptomatic identification and treatment can prevent the devastating disability associated with this disorder. We report the retrospective analysis of the newborn blood spot of an affected child with a low excretor phenotype. The level of glutarylcarnitine was below the newborn screening program cut-off. This suggests that some cases of GA-I may be missed by newborn screening by tandem mass spectrometry.

    View details for DOI 10.1016/j.ymgme.2005.08.005

    View details for PubMedID 16183314