- Pathology and Laboratory Medicine
- Clinical Pathology
- Molecular Genetic Pathology
Director, Molecular Pathology laboratory, Stanford University (2003 - Present)
Director, Molecular Genetic Pathology fellowship program, Stanford University (2003 - Present)
Associate Program Director for Clinical Pathology residency training, Stanford University (2008 - Present)
Medical Director (license holder), Stanford Clinical Laboratory at North Campus, Stanford University (2013 - Present)
Medical Director (license holder), Point-of-Care testing (Stanford North Campus), Stanford University (2013 - Present)
Honors & Awards
Lifetime Achievement Award, College of American Pathologists (2013)
Young Clinical Scientist Award Association of Clinical Scientists, Association of Clinical Scientists (2007)
Sheard Sanford Pathology Resident Award, American Society for Clinical Pathology (2001)
Medical Education:University of Utrecht (1994) Netherlands
Residency:Stanford University School of Medicine (2002) CA
Internship:Stanford University School of Medicine (2000) CA
Board Certification: Clinical Pathology, American Board of Pathology (2002)
Board Certification: Clinical Molecular Genetics, American Board of Medical Genetics (1999)
MD, Utrecht University, Medicine (1994)
Current Research and Scholarly Interests
Cystic fibrosis (CF) is a serious genetic condition caused by two disease-causing changes (mutations) in the CFTR gene. Additional genes have been implicated in CF-like disease. The types of mutations that cause CF and CF-like disease in individuals with African, Native American, Asian, or Middle Eastern backgrounds are not well known. The Schrijver laboratory and collaborators seek to reduce the disparity in mutation detection between Caucasian individuals and those of other or mixed extraction by adequately determining the mutation composition and individual frequencies in previously understudied populations. Such improved diagnostics are expected to allow earlier diagnosis and therapeutic management and could affect prognosis. This is an important step towards correlation of mutations (or combinations of mutations) with the clinical severity of symptoms. The knowledge of genotype-phenotype correlations can affect results reporting, counseling, prognosis predictions, and therapeutic decisions. Finally, the results can be considered in the review, evaluation, and optimization of individual State newborn testing algorithms.
Sensorineural hearing loss
Tremendous progress has been made in our understanding of the molecular basis of hearing and hearing loss. Through recent advances, the fascinating biology of the auditory system and new molecular mechanisms of hearing impairment have begun to be unveiled. Changes in the diagnostic impact of genetic testing have occurred, as well as exciting developments in therapeutic options (such as cochlear implants). Molecular diagnosis, which is already a reality for several hearing-associated genes, will continue to increase in the near future, both in terms of the number of mutations tested and the spectrum of genes. Inherited hearing loss, however, is characterized by impressive genetic heterogeneity. An abundance of genes carry a large number of mutations, but specific mutations in a single gene may lead to syndromic or nonsyndromic hearing loss. The Schrijver laboratory studies the genetics of hearing loss, with a focus on the DFNB1 locus, which contains the GJB2 and GJB6 genes. It is estimated that 50-70% of prelingual hearing loss is caused by genetic changes. Nonsyndromic (isolated) sensorineural hearing loss accounts for more than 70% of all hereditary hearing loss and up to half of that appears related to DFNB1. The Schrijver laboratory has shown that the GJB6 deletions del(GJB6-D13S1830) and del(GJB6-D13S1854), located upstream of the GJB2 gene, specifically inhibit the expression of GJB2 in cis with the deletions. We investigate the non-coding cis-regulatory sequence(s) responsible for controlling GJB2 expression and perform further genotyping in order to elucidate the genetic etiology underlying non-syndromic sensorineural hearing loss, and study genotype-phenotype correlations.
- Independent Studies (5)
Graduate and Fellowship Programs
Human Genetics and Genetic Counseling (Masters Program)
Medical Genetics (Fellowship Program)
Molecular and Genetic Medicine (Fellowship Program)
Molecular genetic testing for cystic fibrosis: laboratory performance on the College of American Pathologists external proficiency surveys
GENETICS IN MEDICINE
2015; 17 (3): 219-225
Molecular testing for cystic fibrosis mutations is widespread and routine in reproductive decision making and diagnosis. Our objective was to assess the level of performance of laboratories for this test.The College of American Pathologists administers external proficiency testing with multiple DNA samples distributed biannually. RESULTS are analyzed, reviewed, and graded by the joint College of American Pathologists/American College of Medical Genetics and Genomics Biochemical and Molecular Genetics Committee. Assessment is based on genotype and associated clinical interpretation.Overall, 357 clinical laboratories participated in the proficiency testing survey between 2003 and 2013 (322 in the United States and 35 international). In 2013, US participants reported performing nearly 120,000 tests monthly. Analytical sensitivity and specificity of US laboratories were 98.8% (95% confidence interval: 98.4-99.1%) and 99.6% (95% confidence interval: 99.4-99.7%), respectively. Analytical sensitivity improved between 2003 and 2008 (from 97.9 to 99.3%; P = 0.007) and remained steady thereafter. Clinical interpretation matched the intended response for 98.8, 86.0, and 91.0% of challenges with no, one, or two mutations, respectively. International laboratories performed similarly.Laboratory testing for cystic fibrosis in the United States has improved since 2003, and these data demonstrate a high level of quality. Neither the number of samples tested nor test methodology affected performance.
View details for DOI 10.1038/gim.2014.93
View details for Web of Science ID 000350733400007
View details for PubMedID 25077647
Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial
JOURNAL OF CLINICAL PATHOLOGY
2015; 68 (2): 111-118
Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation.13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation.Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8 ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation.In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation.
View details for DOI 10.1136/jclinpath-2014-202644
View details for Web of Science ID 000349532700003
View details for PubMedID 25430497
Results of the College of American Pathology/American College of Medical Genetics and Genomics external proficiency testing from 2006 to 2013 for three conditions prevalent in the Ashkenazi Jewish population
GENETICS IN MEDICINE
2014; 16 (9): 695-702
The purpose of this study was to determine analytic performance of laboratories offering molecular testing for conditions such as Tay-Sachs disease, Canavan disease, and familial dysautonomia, which are prevalent in the Ashkenazi Jewish population.The College of American Pathologists and the American College of Medical Genetics and Genomics cosponsor molecular proficiency testing for these disorders. Responses from 2006 to 2013 were analyzed for accuracy (genotyping and interpretations).Between 11 and 36 laboratories participated in each Tay-Sachs disease distribution. Samples tested per month were constant (2,900) from 2006 to 2011 but recently increased. Participants reporting <10 samples tested per month had longer turnaround times (42 vs. 7%, longer than 14 days; P = 0.03). Analytic sensitivity and specificity for US participants were 97.2% (95% confidence interval: 94.7-98.7%) and 99.8% (95% confidence interval: 99.1-99.9%), respectively. Of 11 genotyping errors, 2 were due to sample mix-up. Analytic interpretations were correct in 99.3% of challenges (956/963; 95% confidence interval: 98.5-99.7%). Better performance was found for Canavan disease and familial dysautonomia. International laboratories performed equally well.These results demonstrated high analytic sensitivity and specificity along with excellent analytic interpretation performance, confirming the genetics community impression that laboratories provide accurate test results in both diagnostic and screening settings. Proficiency testing can identify potential laboratory issues and helps document overall laboratory performance.
View details for DOI 10.1038/gim.2014.14
View details for Web of Science ID 000341830800007
View details for PubMedID 24577267
Methods-Based Proficiency Testing in Molecular Genetic Pathology
JOURNAL OF MOLECULAR DIAGNOSTICS
2014; 16 (3): 283-287
This Perspectives article describes methods-based proficiency testing (MBPT), the benefits and limitations of MBPT, why the time is right for MBPT in molecular diagnostics, and how MBPT for next-generation sequencing is being developed by the College of American Pathologists.
View details for DOI 10.1016/j.jmoldx.2014.02.002
View details for Web of Science ID 000335540200001
View details for PubMedID 24650895
A Balanced Look at the Implications of Genomic (and Other "Omics") Testing for Disease Diagnosis and Clinical Care.
2014; 5 (3): 748-766
The tremendous increase in DNA sequencing capacity arising from the commercialization of "next generation" instruments has opened the door to innumerable routes of investigation in basic and translational medical science. It enables very large data sets to be gathered, whose interpretation and conversion into useful knowledge is only beginning. A challenge for modern healthcare systems and academic medical centers is to apply these new methods for the diagnosis of disease and the management of patient care without unnecessary delay, but also with appropriate evaluation of the quality of data and interpretation, as well as the clinical value of the insights gained. Most critically, the standards applied for evaluating these new laboratory data and ensuring that the results and their significance are clearly communicated to patients and their caregivers should be at least as rigorous as those applied to other kinds of medical tests. Here, we present an overview of conceptual and practical issues to be considered in planning for the integration of genomic methods or, in principle, any other type of "omics" testing into clinical care.
View details for DOI 10.3390/genes5030748
View details for PubMedID 25257203
Mutation analysis of the SLC26A4, FOXI1 and KCNJ10 genes in individuals with congenital hearing loss.
Pendred syndrome (PDS) and DFNB4 comprise a phenotypic spectrum of sensorineural hearing loss disorders that typically result from biallelic mutations of the SLC26A4 gene. Although PDS and DFNB4 are recessively inherited, sequencing of the coding regions and splice sites of SLC26A4 in individuals suspected to be affected with these conditions often fails to identify two mutations. We investigated the potential contribution of large SLC26A4 deletions and duplications to sensorineural hearing loss (SNHL) by screening 107 probands with one known SLC26A4 mutation by Multiplex Ligation-dependent Probe Amplification (MLPA). A heterozygous deletion, spanning exons 4-6, was detected in only one individual, accounting for approximately 1% of the missing mutations in our cohort. This low frequency is consistent with previously published MLPA results. We also examined the potential involvement of digenic inheritance in PDS/DFNB4 by sequencing the coding regions of FOXI1 and KCNJ10. Of the 29 probands who were sequenced, three carried nonsynonymous variants including one novel sequence change in FOXI1 and two polymorphisms in KCNJ10. We performed a review of prior studies and, in conjunction with our current data, conclude that the frequency of FOXI1 (1.4%) and KCNJ10 (3.6%) variants in PDS/DFNB4 individuals is low. Our results, in combination with previously published reports, indicate that large SLC26A4 deletions and duplications as well as mutations of FOXI1 and KCNJ10 play limited roles in the pathogenesis of SNHL and suggest that other genetic factors likely contribute to the phenotype.
View details for DOI 10.7717/peerj.384
View details for PubMedID 24860705
- Feasibility of using microbeads with holographic barcodes to track DNA specimens in the clinical molecular laboratory PEERJ 2013; 1
Current Landscape and New Paradigms of Proficiency Testing and External Quality Assessment for Molecular Genetics
ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE
2013; 137 (7): 983-998
Context.-Participation in proficiency testing (PT) or external quality assessment (EQA) programs allows the assessment and comparison of test performance among different clinical laboratories and technologies. In addition to the approximately 2300 tests for individual genetic disorders, recent advances in technology have enabled the development of clinical tests that quickly and economically analyze the entire human genome. New PT/EQA approaches are needed to ensure the continued quality of these complex tests. Objectives.-To review the availability and scope of PT/EQA for molecular genetic testing for inherited conditions in Europe, Australasia, and the United States; to evaluate the successes and demonstrated value of available PT/EQA programs; and to examine the challenges to the provision of comprehensive PT/EQA posed by new laboratory practices and methodologies. Data Sources.-The available literature on this topic was reviewed and supplemented with personal experiences of several PT/EQA providers. Conclusions.-Proficiency testing/EQA schemes are available for common genetic disorders tested in many clinical laboratories but are not available for most genetic tests offered by only one or a few laboratories. Provision of broad, method-based PT schemes, such as DNA sequencing, would allow assessment of many tests for which formal PT is not currently available. Participation in PT/EQA improves the quality of testing by identifying inaccuracies that laboratories can trace to errors in their testing processes. Areas of research and development to ensure that PT/EQA programs can meet the needs of new and evolving genetic tests and technologies are identified and discussed.
View details for DOI 10.5858/arpa.2012-0311-RA
View details for Web of Science ID 000328257700018
View details for PubMedID 23808472
- Identification of the CFTR p.Phe508Del founder mutation in the absence of a polythymidine 9T allele in a Hispanic patient CLINICAL GENETICS 2013; 83 (6): 598-599
Integration of Genomic Medicine into Pathology Residency Training The Stanford Open Curriculum
JOURNAL OF MOLECULAR DIAGNOSTICS
2013; 15 (2): 141-148
Next-generation sequencing methods provide an opportunity for molecular pathology laboratories to perform genomic testing that is far more comprehensive than single-gene analyses. Genome-based test results are expected to develop into an integral component of diagnostic clinical medicine and to provide the basis for individually tailored health care. To achieve these goals, rigorous interpretation of high-quality data must be informed by the medical history and the phenotype of the patient. The discipline of pathology is well positioned to implement genome-based testing and to interpret its results, but new knowledge and skills must be included in the training of pathologists to develop expertise in this area. Pathology residents should be trained in emerging technologies to integrate genomic test results appropriately with more traditional testing, to accelerate clinical studies using genomic data, and to help develop appropriate standards of data quality and evidence-based interpretation of these test results. We have created a genomic pathology curriculum as a first step in helping pathology residents build a foundation for the understanding of genomic medicine and its implications for clinical practice. This curriculum is freely accessible online.
View details for DOI 10.1016/j.jmoldx.2012.11.003
View details for Web of Science ID 000315841600001
View details for PubMedID 23313248
Feasibility of using microbeads with holographic barcodes to track DNA specimens in the clinical molecular laboratory.
We demonstrate the feasibility of using glass microbeads with a holographic barcode identifier to track DNA specimens in the molecular pathology laboratory. These beads can be added to peripheral blood specimens and are carried through automated DNA extraction protocols that use magnetic glass particles. We found that an adequate number of microbeads are consistently carried over during genomic DNA extraction to allow specimen identification, that the beads do not interfere with the performance of several different molecular assays, and that the beads and genomic DNA remain stable when stored together under regular storage conditions in the molecular pathology laboratory. The beads function as an internal, easily readable specimen barcode. This approach may be useful for identifying DNA specimens and reducing errors associated with molecular laboratory testing.
View details for DOI 10.7717/peerj.91
View details for PubMedID 23862106
Opportunities and Challenges Associated with Clinical Diagnostic Genome Sequencing A Report of the Association for Molecular Pathology
JOURNAL OF MOLECULAR DIAGNOSTICS
2012; 14 (6): 525-540
This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications.
View details for DOI 10.1016/j.jmoldx.2012.04.006
View details for Web of Science ID 000310178600001
View details for PubMedID 22918138
College of American Pathologists' Laboratory Standards for Next Generation Sequence Clinical Testing
ELSEVIER SCIENCE INC. 2012: 742-742
View details for Web of Science ID 000310178600432
Feasibility of Using Microbeads with Holographic Barcodes to Track DNA Specimens in the Molecular Pathology Laboratory
ELSEVIER SCIENCE INC. 2012: 735-735
View details for Web of Science ID 000310178600404
Increased incidence of profound biotinidase deficiency among Hispanic newborns in California
MOLECULAR GENETICS AND METABOLISM
2012; 106 (4): 485-487
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
Molecular genetic testing for fraglie X syndrome: laboratory performance on the College of American Pathologists proficiency surveys (2001-2009)
GENETICS IN MEDICINE
2012; 14 (3): 306-312
The College of American Pathologists offers biannual proficiency testing for molecular analysis of fragile X syndrome. The purpose of this study was to analyze laboratory performance on the fragile X proficiency surveys from 2001 to 2009.Individual laboratory responses were analyzed for accuracy of genotype determination (normal, gray zone, premutation, or full mutation) and size analysis of the FMR1 trinucleotide repeat region. The analytical sensitivity and specificity of testing for fragile X were calculated, and laboratory performance for trinucleotide repeat sizing was evaluated.Overall, laboratories demonstrated analytical sensitivity of 99% and 96% for detection of full mutations associated with fragile X syndrome in males and females, respectively; analytical sensitivity of 98% for detection of premutations; and analytical specificity of 99.9%. Size measurements of the CGG repeat region were acceptable from most laboratories, with an increase in the range of reported sizes observed for larger repeat expansions.Molecular genetic testing for fragile X syndrome demonstrated excellent sensitivity and specificity by laboratories participating in the College of American Pathologists (CAP) surveys. Allele sizing demonstrated good performance overall with improved accuracy over the study period. Participation in proficiency testing can aid laboratories in assessing individual performance and need for calibration of assays.
View details for DOI 10.1038/gim.2011.11
View details for Web of Science ID 000301475100004
View details for PubMedID 22241100
Analysis of the Alternative Splicing of an FGFR2 Transcript Due to a Novel 5 ' Splice Site Mutation (1084+1G > A): Case Report
CLEFT PALATE-CRANIOFACIAL JOURNAL
2012; 49 (1): 104-108
Craniosynostosis is characterized by premature fusion of one or more cranial sutures and is associated with mutations in fibroblast growth factor receptor (FGFR) genes. Here we describe a novel mutation (1084+1G>A) in the FGFR2 gene of a patient with isolated bicoronal synostosis. We detected two isoforms that result from the mutation and are characterized, respectively, by exon skipping and the use of a cryptic splice site. Interestingly, the alternatively spliced forms of FGFR2 appear to induce fusion of the cranial sutures suggesting that the mutation acts via a gain-of-function mechanism rather than a loss of protein functionality.
View details for DOI 10.1597/10-217
View details for Web of Science ID 000300352600014
View details for PubMedID 21524234
Verification of Performance Specifications of a Molecular Test Cystic Fibrosis Carrier Testing Using the Luminex Liquid Bead Array
ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE
2012; 136 (1): 14-19
The number of clinical laboratories introducing various molecular tests to their existing test menu is continuously increasing. Prior to offering a US Food and Drug Administration-approved test, it is necessary that performance characteristics of the test, as claimed by the company, are verified before the assay is implemented in a clinical laboratory.To provide an example of the verification of a specific qualitative in vitro diagnostic test: cystic fibrosis carrier testing using the Luminex liquid bead array (Luminex Molecular Diagnostics, Inc, Toronto, Ontario).The approach used by an individual laboratory for verification of a US Food and Drug Administration-approved assay is described.Specific verification data are provided to highlight the stepwise verification approach undertaken by a clinical diagnostic laboratory.Protocols for verification of in vitro diagnostic assays may vary between laboratories. However, all laboratories must verify several specific performance specifications prior to implementation of such assays for clinical use. We provide an example of an approach used for verifying performance of an assay for cystic fibrosis carrier screening.
View details for DOI 10.5858/arpa.2010-0536-OA
View details for Web of Science ID 000299045800002
View details for PubMedID 22208482
Design and Analytical Validation of Clinical DNA Sequencing Assays
ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE
2012; 136 (1): 41-46
DNA sequencing is the method of choice for mutation detection in many genes.To demonstrate the analytical accuracy and reliability of DNA sequencing assays developed in clinical laboratories. Only general guidelines exist for the validation of these tests. We provide examples of assay validation strategies for DNA sequencing tests.We discuss important design and validation considerations.The validation examples include an accuracy study to evaluate concordance between results obtained by the newly designed assay and analyzed by another method or laboratory. Precision (reproducibility) studies are performed to determine the robustness of the assay. To assess the quality of sequencing assays, several sequence quality measures are available. In addition, assessing the ability of primers to specifically and robustly amplify target regions before sequencing is important.Protocols for validation of laboratory-developed sequencing assays may vary between laboratories. An example summary of a validation is provided.
View details for DOI 10.5858/arpa.2010-0623-OA
View details for Web of Science ID 000299045800006
View details for PubMedID 22208486
Allelic discrimination of cis-trans relationships by digital polymerase chain reaction: GJB2 (p.V27I/p.E114G) and CFTR (p.R117H/5T)
GENETICS IN MEDICINE
2011; 13 (12): 1025-1031
: To distinguish the cis-trans relationship of two sequence changes and to arrive at an accurate molecular diagnosis for autosomal recessive disorders, methods such as Sanger sequencing cannot differentiate whether sequence changes are in cis or trans. In addition, most techniques theoretically appropriate for allelic discrimination depend on the specific identified sequence changes for assay design, need extensive optimization, or may not be suitable. We developed a method that does not fully depend on the specific nucleotide changes. It enables efficient assay design and practical implementation of allelic discrimination.: Digital polymerase chain reaction (PCR) was used to separate and amplify alleles. Sanger sequencing was subsequently used to identify sequence changes.: We developed a cost-effective digital PCR method for allelic discrimination of short amplicons and demonstrated it with p.Val27Ile and p.Glu114Gly in GJB2 as an example. We also successfully developed a long-range digital PCR approach to determine the cis-trans relationship of p.Arg117His and 5T in the CFTR gene.: Digital PCR for allelic discrimination can be clinically implemented to determine the allelic configuration of relatively common sequence changes which frequently appear together and have clinical ramifications, such as the combination of p.Val27Ile and p.Glu114Gly in the GJB2 gene and p.Arg117His and 5T in CFTR.
View details for DOI 10.1097/GIM.0b013e3182272e0b
View details for Web of Science ID 000298138700007
View details for PubMedID 21836520
A two-antibody mismatch repair protein immunohistochemistry screening approach for colorectal carcinomas, skin sebaceous tumors, and gynecologic tract carcinomas
2011; 24 (7): 1004-1014
Mismatch repair protein immunohistochemistry is a widely used method for detecting patients at risk for Lynch syndrome. Recent data suggest that a two-antibody panel approach using PMS2 and MSH6 is an effective screening protocol for colorectal carcinoma, but there are limited data concerning this approach for extraintestinal tumors. The purpose of this study was to review the utility of a two-antibody panel approach in colorectal carcinoma and extraintestinal tumors. We evaluated mismatch repair protein expression in two cohorts: (1) a retrospective analysis of intestinal and extraintestinal tumors (n=334) tested for mismatch repair protein immunohistochemistry and (2) a prospectively accrued series of intestinal, gynecologic tract, and skin sebaceous neoplasms (n=98). A total of 432 cases were analyzed, including 323 colorectal, 50 gynecologic tract, 49 skin sebaceous, and 10 other neoplasms. Overall, 102/432 tumors (24%) demonstrated loss of at least one mismatch repair protein. Concurrent loss of MLH1 and PMS2 was the most common pattern of abnormal expression (50/432, 12%) followed by concurrent loss of MSH2 and MSH6 (33/432, 8%). Of 55 cases with abnormal PMS2 expression, 5 (9%) demonstrated isolated loss of PMS2 expression. Of 47 cases with abnormal MSH6 expression, 14 (30%) demonstrated isolated loss of MSH6 expression. Isolated loss of MLH1 or MSH2 was not observed. Colorectal carcinomas more frequently demonstrated abnormal expression of PMS2 (39/59, 66%). Skin sebaceous neoplasms more frequently demonstrated abnormal expression of MSH6 (18/24, 75%, respectively). A total of 65 tumors with abnormal mismatch repair protein expression were tested for microsatellite instability (MSI): 47 (72%) MSI high, 9 (14%) MSI low, and 9 (14%) microsatellite stable (MSS). Abnormal MSH6 expression accounted for 14/18 (78%) cases that were MSS or MSI low. Our findings confirm the utility of a two-antibody approach using PMS2 and MSH6 in colorectal carcinoma and indicate that this approach is effective in extraintestinal neoplasms associated with Lynch syndrome.
View details for DOI 10.1038/modpathol.2011.55
View details for Web of Science ID 000292319700014
View details for PubMedID 21499234
Cystic Fibrosis Carrier Screening in Obstetric Clinical Practice: Knowledge, Practices, and Barriers, a Decade After Publication of Screening Guidelines
GENETIC TESTING AND MOLECULAR BIOMARKERS
2011; 15 (7-8): 517-523
Cystic fibrosis (CF) carrier screening guidelines have been in place for almost a decade. The purpose of this study was to determine the current awareness by obstetricians of the existence and content of practice guidelines, the variety in practice regarding CF carrier screening, and the level of knowledge regarding CF genetics and screening result interpretation. We also explored potential barriers to offering screening and whether academic affiliation or type of practice influenced outcome.An online survey program was used to deliver a questionnaire to obstetricians throughout the United States. One hundred fifty-six respondents participated, with 143 answering all questions in the survey.Although most obstetricians are aware of screening guidelines and have accurate knowledge about CF carrier screening, 12.3% were not aware of carrier screening guidelines, 17.7% were unable to interpret basic results, 16.5% experienced barriers to offering screening, and 43% lacked information regarding carrier rates, screening sensitivity, and residual risk.Most obstetricians offer CF carrier screening and will refer to genetic counseling services at times. However, we identified a deficiency of information in a concerning percentage of practitioners. This deficiency could be improved by targeted and readily accessible educational efforts, especially for obstetricians not affiliated with academia.
View details for DOI 10.1089/gtmb.2010.0228
View details for Web of Science ID 000292773700011
View details for PubMedID 21453058
Mutation analysis of SLC26A4 for Pendred syndrome and nonsyndromic hearing loss by high-resolution melting.
journal of molecular diagnostics
2011; 13 (4): 416-426
Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection.
View details for DOI 10.1016/j.jmoldx.2011.03.003
View details for PubMedID 21704276
Evaluation of a Gene Expression Microarray-based Assay to Determine Tissue Type of Origin on a Diverse Set of 49 Malignancies
AMERICAN JOURNAL OF SURGICAL PATHOLOGY
2011; 35 (7): 1030-1037
The Tissue of Origin Frozen (TOO-FRZ) assay from Pathwork Diagnostics has been cleared by the Food and Drug Administration as a diagnostic study for malignancies of unknown primary. The goal of this study was to evaluate the performance of TOO-FRZ on a diverse collection of malignancies. We collected a diverse set of 49 malignancies. We classified each case into 1 of 4 groups: common morphology from a tissue type included in the TOO-FRZ assay (n=29), uncommon morphology from a tissue type included in the TOO-FRZ assay (n=10), tumor from a tissue type not included in the TOO-FRZ assay (n=3), and malignancies of unknown primary (n=7). We found strong diagnostic performance for common morphologies from tissue types on the TOO-FRZ [overall accuracy=26 of 29 (90%, 95% CI, 73% to 97%)], with perfect performance in all tissue types except gastric (0 of 2) and pancreatic (1 of 2) tissues. There was a significant decline in performance for uncommon morphologies from tissue types included in the TOO-FRZ assay [6 of 10 (60%) cases with an indeterminate result, 1 of 10 (10%) cases with an incorrect prediction, and 3 of 10 (30%) with a correct prediction] and for tumors from tissue types not included in the assay (incorrect prediction in 2 of 3 cases). For the 7 malignancies of unknown primary in our study set, the TOO-FRZ provided a likely clinically useful result in only 2 of 7 cases. These results provide an insight into the strengths and limitations of this molecular assay for the surgical pathologist, and our findings suggest future directions for research in this area.
View details for DOI 10.1097/PAS.0b013e3182178b59
View details for Web of Science ID 000291676200011
View details for PubMedID 21602661
Ultrasensitive Detection of Drug-Resistant Pandemic 2009 (H1N1) Influenza A Virus by Rare-Variant-Sensitive High-Resolution Melting-Curve Analysis
JOURNAL OF CLINICAL MICROBIOLOGY
2011; 49 (7): 2602-2609
Oseltamivir (Tamiflu), an oral neuraminidase inhibitor, has been widely used to treat pandemic 2009 (H1N1) influenza A. Although a majority of 2009 (H1N1) influenza A virus remains oseltamivir susceptible, the threat of resistance due to the His275Tyr mutation is highlighted by the limitations of alternative therapies and the potential for rapid, global fixation of this mutation in the circulating influenza A virus population. In order to better understand the emergence of resistance, we developed a rare-variant-sensitive high-resolution melting-curve analysis method (RVS-HRM) that is able to detect the His275Tyr oseltamivir resistance mutation to 0.5% in a background of susceptible virus. We applied RVS-HRM to clinical specimens from patients who developed oseltamivir resistance and demonstrated the ultrasensitive detection of influenza A virus N1 neuraminidase quasispecies. Interestingly, we were unable to detect the oseltamivir resistance mutation in pretreatment samples, suggesting that resistant virus does not reach even this very low detection threshold until exposed to selective drug pressure. Thus, patients naive to oseltamivir are most likely to be susceptible when this drug is used as a first-line treatment modality.
View details for DOI 10.1128/JCM.00277-11
View details for Web of Science ID 000292276200035
View details for PubMedID 21543559
Allele-Specific Impairment of GJB2 Expression by GJB6 Deletion del(GJB6-D13S1854)
2011; 6 (6)
Mutations in the GJB2 gene, which encodes connexin 26, are a frequent cause of congenital non-syndromic sensorineural hearing loss. Two large deletions, del(GJB6-D13S1830) and del(GJB6-D13S1854), which truncate GJB6 (connexin 30), cause hearing loss in individuals homozygous, or compound heterozygous for these deletions or one such deletion and a mutation in GJB2. Recently, we have demonstrated that the del(GJB6-D13S1830) deletion contributes to hearing loss due to an allele-specific lack of GJB2 mRNA expression and not as a result of digenic inheritance, as was postulated earlier. In the current study we investigated the smaller del(GJB6-D13S1854) deletion, which disrupts the expression of GJB2 at the transcriptional level in a manner similar to the more common del(GJB6-D13S1830) deletion. Interestingly, in the presence of this deletion, GJB2 expression remains minimally but reproducibly present. The relative allele-specific expression of GJB2 was assessed by reverse-transcriptase PCR and restriction digestions in three probands who were compound heterozygous for a GJB2 mutation and del(GJB6-D13S1854). Each individual carried a different sequence variant in GJB2. All three individuals expressed the mutated GJB2 allele in trans with del(GJB6-D13S1854), but expression of the GJB2 allele in cis with the deletion was almost absent. Our study clearly corroborates the hypothesis that the del(GJB6-D13S1854), similar to the larger and more common del(GJB6-D13S1830), removes (a) putative cis-regulatory element(s) upstream of GJB6 and narrows down the region of location.
View details for DOI 10.1371/journal.pone.0021665
View details for Web of Science ID 000292290100054
View details for PubMedID 21738759
- Mutation Distribution in Expanded Screening for Cystic Fibrosis: Making Up the Balance in a Context of Ethnic Diversity CLINICAL CHEMISTRY 2011; 57 (6): 799-801
Quality Assurance for Duchenne and Becker Muscular Dystrophy Genetic Testing Development of a Genomic DNA Reference Material Panel
JOURNAL OF MOLECULAR DIAGNOSTICS
2011; 13 (2): 167-174
Duchenne and Becker muscular dystrophies (DMD/BMD) are allelic X-linked recessive disorders that affect approximately 1 in 3500 and 1 in 20,000 male individuals, respectively. Approximately 65% of patients with DMD have deletions, 7% to 10% have duplications, and 25% to 30% have point mutations in one or more of the 79 exons of the dystrophin gene. Most clinical genetics laboratories test for deletions, and some use technologies that can detect smaller mutations and duplications. Reference and quality control materials for DMD/BMD diagnostic and carrier genetic testing are not commercially available. To help address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing and the DMD/BMD patient communities and the Coriell Cell Repositories, have characterized new and existing cell lines to create a comprehensive DMD/BMD reference material panel. Samples from 31 Coriell DMD cell lines from male probands and female carriers were analyzed using the Affymetrix SNP Array 6.0 and Multiplex Ligation-Dependent Probe Amplification (MRC-Holland BV, Amsterdam, the Netherlands), a multiplex PCR assay, and DNA sequence analysis. Identified were 16 cell lines with deletions, 9 with duplications, and 4 with point mutations distributed throughout the dystrophin gene. There were no discordant results within assay limitations. These samples are publicly available from Coriell Institute for Medical Research (Camden, NJ) and can be used for quality assurance, proficiency testing, test development, and research, and should help improve the accuracy of DMD testing.
View details for DOI 10.1016/j.jmoldx.2010.11.018
View details for Web of Science ID 000298306300008
View details for PubMedID 21354051
Population Carrier Screening for Spinal Muscular Atrophy A Position Statement of the Association for Molecular Pathology
JOURNAL OF MOLECULAR DIAGNOSTICS
2011; 13 (1): 3-6
Spinal muscular atrophy is a common and often fatal autosomal recessive disorder for which carrier screening is available. The Association for Molecular Pathology has evaluated recent opinions regarding population carrier screening, reviewed the current literature, and developed a position statement that includes specific recommendations addressing both diagnostic and practical issues that affect implementation.
View details for DOI 10.1016/j.jmoldx.2010.11.012
View details for Web of Science ID 000286287200002
View details for PubMedID 21227388
Identification of rare DNA variants in mitochondrial disorders with improved array-based sequencing
NUCLEIC ACIDS RESEARCH
2011; 39 (1): 44-58
A common goal in the discovery of rare functional DNA variants via medical resequencing is to incur a relatively lower proportion of false positive base-calls. We developed a novel statistical method for resequencing arrays (SRMA, sequence robust multi-array analysis) to increase the accuracy of detecting rare variants and reduce the costs in subsequent sequence verifications required in medical applications. SRMA includes single and multi-array analysis and accounts for technical variables as well as the possibility of both low- and high-frequency genomic variation. The confidence of each base-call was ranked using two quality measures. In comparison to Sanger capillary sequencing, we achieved a false discovery rate of 2% (false positive rate 1.2 × 10??, false negative rate 5%), which is similar to automated second-generation sequencing technologies. Applied to the analysis of 39 nuclear candidate genes in disorders of mitochondrial DNA (mtDNA) maintenance, we confirmed mutations in the DNA polymerase gamma POLG in positive control cases, and identified novel rare variants in previously undiagnosed cases in the mitochondrial topoisomerase TOP1MT, the mismatch repair enzyme MUTYH, and the apurinic-apyrimidinic endonuclease APEX2. Some patients carried rare heterozygous variants in several functionally interacting genes, which could indicate synergistic genetic effects in these clinically similar disorders.
View details for DOI 10.1093/nar/gkq750
View details for Web of Science ID 000286008500009
View details for PubMedID 20843780
Diagnostic Yield in the Workup of Congenital Sensorineural Hearing Loss Is Dependent on Patient Ethnicity
OTOLOGY & NEUROTOLOGY
2011; 32 (1): 81-87
Diagnostic yield on GJB2 sequencing and computed tomography in the workup for idiopathic congenital sensorineural hearing loss is related to patient ethnicity.GJB2 sequencing and computed tomography of the temporal bones are important initial diagnostic tests in the workup of idiopathic congenital sensorineural hearing loss. Previous studies showed an association between mild or unilateral hearing loss and positive imaging findings and between severe or bilateral deafness and GJB2 mutations. Recent studies on connexin 26-associated deafness demonstrate a wide range of phenotypes that vary with ethnicity.We present a retrospective case series of 271 consecutive ethnically diverse patients evaluated for idiopathic congenital sensorineural hearing loss. Results of genetic testing and imaging were correlated with audiologic findings and ethnicity.All patients with asymmetric hearing loss had more positive findings on imaging. With respect to the severity of hearing loss, however, differences were noted between ethnic groups. Whereas white patients conformed to previous findings, Hispanics with severe hearing loss had similar rates of positive imaging and genetic testing results. Asians with mild hearing loss had significantly greater yield on genetic testing rather than on imaging. This reflects the high prevalence of the p.V37I mutation in GJB2 among Asians, which gives rise to a mild, frequently progressive phenotype.Ethnicity should be considered when determining the optimal sequence of diagnostic testing for idiopathic congenital sensorineural hearing loss. Asian patients, in particular, should all be screened for mutations in GJB2, especially in the case of mild hearing loss.
View details for DOI 10.1097/MAO.0b013e3181fc786f
View details for Web of Science ID 000285334400019
View details for PubMedID 21042228
- Laboratory Guidelines for Detection, Interpretation, and Reporting of Maternal Cell Contamination in Prenatal Analyses A Report of the Association for Molecular Pathology JOURNAL OF MOLECULAR DIAGNOSTICS 2011; 13 (1): 7-11
Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples
AMER SOC HEMATOLOGY. 2010: 1130-1131
View details for Web of Science ID 000289662203074
Rare Variant Sensitive High Resolution Melting Demonstrates that the His275Tyr Substitution in Pandemic H1N1 Influenza A Virus Appears after Oseltamivir Treatment in Infected Patients
ELSEVIER SCIENCE INC. 2010: 875-875
View details for Web of Science ID 000283973700096
ACANTHOSIS NIGRICANS AND HYPOCHONDROPLASIA IN A CHILD WITH A K650Q MUTATION IN FGFR3
2010; 27 (6): 664-666
View details for Web of Science ID 000285111600024
Hereditary diffuse gastric cancer due to a previously undescribed CDH1 splice site mutation
2010; 41 (8): 1200-1203
Our patient was a 52-year-old man who was diagnosed with signet ring cell gastric adenocarcinoma. An extensive family history of gastric cancer raised suspicion for hereditary diffuse gastric cancer. Sequencing of the patient's CDH1 gene revealed a novel point mutation in a strictly conserved splice site within intron 6, c.833-2 A > G. This mutation was predicted to result in loss of function due to defective RNA splicing. To characterize the pathogenic mechanism of this mutation, we amplified the patient's CDH1 gene products by reverse transcriptase polymerase chain reaction. Primers flanking the region of the mutation detected 3 distinct transcripts. In addition to the wild-type product, a larger product consistent with activation of a cryptic splice site within intron 6 and a smaller product shown to result from exon 7 skipping were detected. In summary, we have identified a novel CDH1 mutation in a large hereditary diffuse gastric cancer kindred and identified its pathogenic mechanism.
View details for DOI 10.1016/j.humpath.2010.01.022
View details for Web of Science ID 000280128300019
View details for PubMedID 20624523
Genotyping with a 198 Mutation Arrayed Primer Extension Array for Hereditary Hearing Loss: Assessment of Its Diagnostic Value for Medical Practice
2010; 5 (7)
Molecular diagnostic testing of individuals with congenital sensorineural hearing loss typically begins with DNA sequencing of the GJB2 gene. If the cause of the hearing loss is not identified in GJB2, additional testing can be ordered. However, the step-wise analysis of several genes often results in a protracted diagnostic process. The more comprehensive Hereditary Hearing Loss Arrayed Primer Extension microarray enables analysis of 198 mutations across eight genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5, MTRNR1 and MTTS1) in a single test. To evaluate the added diagnostic value of this microarray for our ethnically diverse patient population, we tested 144 individuals with congenital sensorineural hearing loss who were negative for biallelic GJB2 or GJB6 mutations. The array successfully detected all GJB2 changes previously identified in the study group, confirming excellent assay performance. Additional mutations were identified in the SLC26A4, SLC26A5 and MTRNR1 genes of 12/144 individuals (8.3%), four of whom (2.8%) had genotypes consistent with pathogenicity. These results suggest that the current format of this microarray falls short of adding diagnostic value beyond the customary testing of GJB2, perhaps reflecting the array's limitations on the number of mutations included for each gene, but more likely resulting from unknown genetic contributors to this phenotype. We conclude that mutations in other hearing loss associated genes should be incorporated in the array as knowledge of the etiology of hearing loss evolves. Such future modification of the flexible configuration of the Hereditary Hearing Loss Arrayed Primer Extension microarray would improve its impact as a diagnostic tool.
View details for DOI 10.1371/journal.pone.0011804
View details for Web of Science ID 000280297300027
View details for PubMedID 20668687
A 30-month-old Child With Acute Renal Failure Due to Primary Renal Cytotoxic T-cell Lymphoma
AMERICAN JOURNAL OF SURGICAL PATHOLOGY
2010; 34 (7): 1066-1070
We present a case of a 30-month-old child who presented with anemia and acute renal failure, and was found to have bilateral renal involvement by primary cytotoxic T-cell lymphoma. This was characterized by a monotonous interstitial lymphoid infiltrate with extensive necrosis. The tumor cells showed a CD8, granzyme, and TIA1-positive phenotype with no evidence of Epstein-Barr virus by in situ hybridization. The differential diagnosis based on the biopsy findings included a reactive interstitial nephritis; however, molecular studies confirmed T-cell clonality. She was started on induction chemotherapy and subsequently received maintenance therapy with methotrexate and 6-mercaptopurine. The patient had a complete response after chemotherapy and at 21 months of follow-up, she has no evidence of residual lymphoma; however, she has developed a dilated cardiomyopathy and she remains in renal failure. We discuss the morphologic, immunophenotypic, and molecular features of our case and describe the clinical course of our patient. We review the literature on primary renal lymphoma with an emphasis on T-lineage lymphomas and those that occur in children.
View details for DOI 10.1097/PAS.0b013e3181de693c
View details for Web of Science ID 000279167400021
View details for PubMedID 20495447
- Next Generation DNA Sequencing and the Future of Genomic Medicine GENES 2010; 1 (1): 38-69
Combined Use of PCR-Based TCRG and TCRB Clonality Tests on Paraffin-Embedded Skin Tissue in the Differential Diagnosis of Mycosis Fungoides and Inflammatory Dermatoses
JOURNAL OF MOLECULAR DIAGNOSTICS
2010; 12 (3): 320-327
The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility.
View details for DOI 10.2353/jmoldx.2010.090123
View details for Web of Science ID 000277531700009
View details for PubMedID 20203005
Rare Sequence Variation in the Genome Flanking a Short Tandem Repeat Locus Can Lead to a Question of "Nonmaternity"
JOURNAL OF MOLECULAR DIAGNOSTICS
2010; 12 (3): 384-389
Typing of STR (short tandem repeat) alleles is used in a variety of applications in clinical molecular pathology, including evaluations for maternal cell contamination. Using a commercially available STR typing assay for maternal cell contamination performed in conjunction with prenatal diagnostic testing, we were posed with apparent nonmaternity when the two fetal samples did not demonstrate the expected maternal allele at one locus. By designing primers external to the region amplified by the primers from the commercial assay and by performing direct sequencing of the resulting amplicon, we were able to determine that a guanine to adenine sequence variation led to primer mismatch and allele dropout. This explained the apparent null allele shared between the maternal and fetal samples. Therefore, although rare, allele dropout must be considered whenever unexplained homozygosity at an STR locus is observed.
View details for DOI 10.2353/jmoldx.2010.090201
View details for Web of Science ID 000277531700018
View details for PubMedID 20203001
Comprehensive and Efficient HBB Mutation Analysis for Detection of beta-Hemoglobinopathies in a Pan-Ethnic Population
AMERICAN JOURNAL OF CLINICAL PATHOLOGY
2010; 133 (5): 700-707
Current methods that assay hemoglobin beta-globin chain variants can have limited clinical sensitivity when applied techniques identify only a predefined panel of mutations. Even sequence-based assays may be limited depending on which gene regions are investigated. We sought to develop a clinically practical yet inclusive molecular assay to identify beta-globin mutations in multicultural populations. We highlight the beta-globin mutation detection assay (beta-GMDA), an extensive gene sequencing assay. The polymerase chain reaction (PCR) primers are located to encompass virtually all hemoglobin beta locus (HBB) mutations. In addition, this assay is able to detect, by gap PCR, a common large deletion (Delta619 base pair), which would be missed by sequencing alone. We describe our 5-year experience with the beta-GMDA and indicate its capability for detecting homozygous, heterozygous, and compound heterozygous sequence changes, including previously unknown HBB variants. The beta-GMDA offers superior sensitivity and ease of use with comprehensive detection of HBB mutations that result in beta-globin chain variants.
View details for DOI 10.1309/AJCP7HQ2KWGHECIO
View details for Web of Science ID 000277476500004
View details for PubMedID 20395516
Connexin-26-associated deafness: Phenotypic variability and progression of hearing loss
GENETICS IN MEDICINE
2010; 12 (3): 174-181
To evaluate genotype-phenotype correlation over time for a cohort of children with connexin-26 (GJB2)-associated autosomal recessive hearing loss.Fifty-two children were identified from a database of individuals with homozygous or compound heterozygous mutations in GJB2 and subjected to chart review of their otolaryngologic and serial audiometric evaluations. Genotype-phenotype correlations were identified among the members of this group by appropriate statistical analyses.Hearing loss was most severe in individuals with two truncating mutations in GJB2 and mildest in those with two nontruncating mutations. Progressive hearing loss was seen directly by serial audiometry in 24% of all subjects, and suggested in a total of 28% when those with normal newborn hearing screens and subsequent hearing loss were included. Progression was particularly common among carriers of the p.V37I allele either in homozygosity or in compound heterozygosity with a truncating allele; these children are primarily of Asian descent and demonstrate mild, slowly progressive hearing loss.Phenotype in GJB2-associated hearing loss is correlated with genotype, with truncating mutations giving rise to more severe hearing loss. Progression of hearing loss is not uncommon, especially in association with the p.V37I allele. These results suggest that close audiometric follow-up is warranted for patients with GJB2-associated recessive hearing loss.
View details for DOI 10.1097/GIM.0b013e3181d0d42b
View details for Web of Science ID 000276023100007
View details for PubMedID 20154630
Design and Evaluation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-Type JAK2 Transcript Levels in the Clinical Laboratory
JOURNAL OF MOLECULAR DIAGNOSTICS
2010; 12 (1): 58-64
The somatic mutation JAK2 V617F is associated with BCR-ABL1-negative myeloproliferative neoplasms. Detection of this mutation aids diagnosis of these neoplasms, and quantification of JAK2 V617F may provide a method to monitor response to therapy. For these reasons, we designed a clinical assay that uses allele-specific PCR and real-time detection with hydrolysis probes for the quantification of JAK2 V617F, wild-type JAK2, and GAPDH transcripts. Mutant and wild-type JAK2 were quantified by using external plasmid standards that contain the relevant JAK2 V617F or JAK2 sequence, respectively. We tested 55 peripheral blood specimens from patients with suspected myeloproliferative neoplasms and 55 peripheral blood specimens from patients not known to have myeloproliferative neoplasms. Low-level, nonspecific amplification was detected in reactions containing a high copy number of plasmid standards and in specimens from patients not known to have myeloproliferative neoplasms, necessitating the use of a laboratory-established mutant to wild-type cutoff. The limit of detection established by using cell line dilutions is 0.1%, and this method identified three JAK2 V617F-positive patients who were not detected by a less sensitive method. The assay characteristics and our initial evaluation indicate this method can be used for the detection and quantification of JAK2 V617F, which should be useful for diagnosis of myeloproliferative neoplasms and potentially for monitoring minimal residual disease in future trials of therapies targeted to myeloproliferative neoplasms.
View details for DOI 10.2353/jmoldx.2010.090068
View details for Web of Science ID 000273664100009
View details for PubMedID 19959796
The digenic hypothesis unraveled: The GJB6 del(GJB6-D13S1830) mutation causes allele-specific loss of GJB2 expression in cis
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2009; 389 (2): 354-359
Connexin 26 and connexin 30 are the major connexins expressed in the cochlea, where they are co-localized and form heteromeric gap junctions. Mutations in the GJB2 gene, which encodes connexin 26, are the most common cause of prelingual non-syndromic sensorineural hearing loss. The large del(GJB6-D13S1830) mutation which involves GJB6 (connexin 30), causes hearing loss in homozygous individuals, or when compound heterozygous with a GJB2 mutation. Until now, it remained unresolved whether this phenomenon results from digenic inheritance or because of lack of GJB2 mRNA expression. After RNA extraction from buccal epithelium, a tissue known to express connexin 26 as well as connexin 30, allele-specific expression of GJB2 was investigated by reverse-transcriptase PCR and restriction digestions in three unrelated individuals compound heterozygous for a GJB2 mutation and del(GJB6-D13S1830). Each proband carried a different sequence change in GJB2. The mutated GJB2 allele in trans with del(GJB6-D13S1830) was expressed in all three individuals whereas the GJB2 allele located in cis with the deletion was not expressed at all. Thus, mutations in these two genes do not cause hearing loss through a digenic mechanism of inheritance alone, as was postulated previously, but instead GJB2 expression is abolished through an effect in cis with the deletion. Our study provides unequivocal support for the hypothesis that del(GJB6-D13S1830) eliminates a putative cis-regulatory element located within the deleted region.
View details for DOI 10.1016/j.bbrc.2009.08.152
View details for Web of Science ID 000270764400028
View details for PubMedID 19723508
Development and Characterization of Reference Materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 Genetic Testing
JOURNAL OF MOLECULAR DIAGNOSTICS
2009; 11 (6): 553-561
Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website.
View details for DOI 10.2353/jmoldx.2009.090078
View details for Web of Science ID 000271681400009
View details for PubMedID 19767587
The role of the cytoskeleton in the formation of gap junctions by Connexin 30
EXPERIMENTAL CELL RESEARCH
2009; 315 (10): 1683-1692
Mutations in the genes that encode Connexin 26 (GJB2) and Connexin 30 (GJB6) are the most common known cause of hereditary nonsyndromic sensorineural deafness. Cx26 and Cx30 share a similar protein structure, as well as the same expression distribution pattern in the cochlea. Cx26 has different intracellular trafficking properties compared to those of Cx43 and Cx32, whose trafficking manner is consistent with the classical membrane protein secretory pathway. Until now, however, the trafficking patterns of Cx30 have not been studied. By means of an immunofluorescence staining approach, we found that the targeting of Cx30 to gap junctions in transfected HeLa cells is not affected by brefeldin A, suggesting a Golgi-independent feature, similar to Cx26. Nocodazole had a minimal effect on assembly and distribution of Cx30 gap junctions. Cytochalasin B-induced actin filament depolymerization, however, affected both the pattern and the distribution of Cx30 gap junctions. Co-localization with and/or interaction between Cx30 and microtubules and cortical actin filaments, but not with the tight/adherens junction protein ZO-1, was confirmed by immunofluorescence and/or immunoprecipitation methods. The results suggest that the cytoskeleton, and especially actin filaments, are important components in the processes of assembly, trafficking and stabilization of Cx30 gap junctions.
View details for DOI 10.1016/j.yexcr.2009.03.001
View details for Web of Science ID 000266281000005
View details for PubMedID 19285977
Detection of Maternal Cell Contamination (MCC) using STR/microsatellite markers on a Capillary Electrophoresis System
AMER ASSOC CLINICAL CHEMISTRY. 2009: A45-A46
View details for Web of Science ID 000266895400136
Mitochondrial DNA analysis by multiplex denaturing high-performance liquid chromatography and selective sequencing in pediatric patients with cardiomyopathy
GENETICS IN MEDICINE
2009; 11 (2): 118-126
Mitochondrial DNA testing is typically performed by targeted mutation analysis only. We applied a more comprehensive approach to study the mitochondrial genome in 24 pediatric patients with idiopathic cardiomyopathy.Patients in the cohort did not show overt multisystemic disease and were previously tested for mutations in a subset of structural genes associated with cardiomyopathy. Mutation screening of the mitochondrial DNA by multiplex denaturing high-performance liquid chromatography was complemented by sequence analysis.We identified 130 individual (unique) sequence changes. Among several potentially pathogenic changes, a novel heteroplasmic mutation in nicotinamide adenine dinucleotide dehydrogenase subunit 4 (10677G>A) was identified in one fraternal twin with worse clinical symptoms than his sibling. Another proband carried homoplasmic mutation 13708G>A (in nicotinamide adenine dinucleotide dehydrogenase subunit 5) that has been associated with Leber's hereditary optic neuropathy.Changes in mitochondrial DNA may represent a relatively rare cause of idiopathic pediatric cardiomyopathies and/or influence their phenotypic expression. Interpretation of variants with uncertain pathogenicity, however, currently impedes clinical diagnostic use of comprehensive mitochondrial DNA testing. Whereas combined use of multiplex denaturing high-performance liquid chromatography and sequencing is more comprehensive than targeted mutation analysis, measurement of additional functional parameters, such as tissue respiratory chain activity, remains important to establishing a definitive diagnosis.
View details for DOI 10.1097/GIM.0b013e318190356b
View details for Web of Science ID 000263663300009
View details for PubMedID 19265752
Splice variant IVS2-2A > G in the SLC26A5 (Prestin) gene in five Estonian families with hearing loss
INTERNATIONAL JOURNAL OF PEDIATRIC OTORHINOLARYNGOLOGY
2009; 73 (1): 103-107
The aim of our study was to identify the IVS2-2A>G sequence change in the SLC26A5 (Prestin) gene in Estonian individuals with hearing loss and in their family members.In the years 2005-2007 we have screened 194 probands with early onset hearing loss and 68 family members with an arrayed primer extension (APEX) microarray, which covers 201 mutations in six nuclear genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5) and two mitochondrial genes encoding 12S rRNA and tRNA-Ser (UCN).In four probands with early onset hearing loss and in five unaffected family members from five families we identified the IVS2-2A>G change in one allele of the SLC26A5 gene. We did not find any homozygosity for this splice variant. IVS2-2A>G was identified in 2.1% of probands. One of these probands, however, is also homozygous for the 35delG mutation in the GJB2 gene and a second patient has Down syndrome, which is also associated with hearing impairment. Therefore, in those two cases the etiology of the hearing loss is probably not associated with the IVS2-2A>G sequence change in the SLC26A5 gene.Our data support the hypothesis that heterozygosity for the mutation IVS2-2A>G in SLC26A5 gene may not, by itself, be sufficient to cause hearing loss.
View details for DOI 10.1016/j.ijporl.2008.10.003
View details for Web of Science ID 000262554100012
View details for PubMedID 19027966
Diagnostic Value of a Primer Extension Microarray for Sensorineural Hearing Loss: A Study of Danish Individuals
ELSEVIER SCIENCE INC. 2008: 571-571
View details for Web of Science ID 000260533600039
Design and Validation of a Real-Time PCR Assay for Quantification of JAK2 V617F and Wild-type JAK2 Transcript Levels
ELSEVIER SCIENCE INC. 2008: 581-581
View details for Web of Science ID 000260533600084
Genetic Analysis of Presbycusis by Arrayed Primer Extension
ANNALS OF CLINICAL AND LABORATORY SCIENCE
2008; 38 (4): 352-360
Using the Hereditary Hearing Loss arrayed primer extension (APEX) array, which contains 198 mutations across 8 hearing loss-associated genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5, 12S-rRNA, and tRNA Ser), we compared the frequency of sequence variants in 94 individuals with early presbycusis to 50 unaffected controls and aimed to identify possible genetic contributors. This cross-sectional study was performed at Stanford University with presbycusis samples from the California Ear Institute. The patients were between ages 20 and 65 yr, with adult-onset sensorineural hearing loss of unknown etiology, and carried a clinical diagnosis of early presbycusis. Exclusion criteria comprised known causes of hearing loss such as significant noise exposure, trauma, ototoxic medication, neoplasm, and congenital infection or syndrome, as well as congenital or pediatric onset. Sequence changes were identified in 11.7% and 10% of presbycusis and control alleles, respectively. Among the presbycusis group, these solely occurred within the GJB2 and SLC26A4 genes. Homozygous and compound heterozygous pathogenic mutations were exclusively seen in affected individuals. We were unable to detect a statistically significant difference between our control and affected populations regarding the frequency of sequence variants detected with the APEX array. Individuals who carry two mild mutations in the GJB2 gene possibly have an increased risk of developing early presbycusis.
View details for Web of Science ID 000260722000005
View details for PubMedID 18988928
Microsatellite instability and mismatch repair protein defects in ovarian epithelial neoplasms in patients 50 years of age and younger
AMERICAN JOURNAL OF SURGICAL PATHOLOGY
2008; 32 (7): 1029-1037
Ovarian malignancies occurring in the setting of hereditary nonpolyposis colorectal carcinoma syndrome typically present in young women, often as the first or "sentinel" cancer, but the frequency of microsatellite instability (MSI) and mismatch repair (MMR) defects in ovarian surface epithelial malignancies in women
View details for Web of Science ID 000257298000010
View details for PubMedID 18469706
Multiplex ligation-dependent probe amplification identification of whole exon and single nucleotide deletions in the CFTR gene of Hispanic individuals with cystic fibrosis
JOURNAL OF MOLECULAR DIAGNOSTICS
2008; 10 (4): 368-375
A disparity between Caucasian and Hispanic mutation detection for cystic fibrosis continues to exist, although the carrier frequency is only moderately lower in Hispanics. We aimed to identify exonic rearrangements that remained undetected by conventional methods. In seven of 32 cystic fibrosis-affected self-identified Hispanics for whom only one or no mutations were identified by extensive molecular testing, exon deletions appeared to be present with a multiplex ligation-dependent probe amplification (MLPA) assay. Two recurrent deletions (of exons 2-3 and exons 22-23) were identified in one and three patients, respectively (12.5%, 11.1% of unidentified alleles). Two apparently novel deletions (exons 6b and 20) were identified in three additional patients. Subsequent sequencing to characterize deletion breakpoints, however, identified single nucleotide deletions at the probe binding sites close to the ligation point. All resulted in false positive MLPA deletion signals. Interestingly, these mutations were not common in Caucasians, and one (935delA) was common in U.S. Hispanics. On examination of all probe binding sites, we identified a total of 76 reported mutations and five silent variants that immediately surrounded the MLPA ligation sites, with 22 occurring in non-Caucasians. These mutations are not all rare. Thus, apparent exon deletions by MLPA may indicate the presence of both large deletions and point mutations, with important implications for pan-ethnic MLPA testing in cystic fibrosis and other genetic conditions.
View details for DOI 10.2353/jmoldx.2008.080004
View details for Web of Science ID 000257262400013
View details for PubMedID 18556774
Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (11): 4335-4339
Blood neutrophils recruited to cystic fibrosis (CF) airways are believed to be rapidly killed by resident bacteria and to passively release elastase and other toxic by-products that promote disease progression. By single-cell analysis, we demonstrate that profound functional and signaling changes readily occur within viable neutrophils recruited to CF airways, compared with their blood counterparts. Airway neutrophils have undergone conventional activation, as shown by decreased intracellular glutathione, increased lipid raft assembly, surface mobilization of CD11b+ and CD66b+ granules, and increased levels of the cytoskeleton-associated phospho-Syk kinase. Unexpectedly, they also mobilize to the surface CD63+ elastase-rich granules, usually confined intracellularly, and lose surface expression of CD16 and CD14, both key receptors in phagocytosis. Furthermore, they express CD80, major histocompatibility complex type II, and the prostaglandin D2 receptor CD294, all normally associated with other lineages, which reflects functional reprogramming. This notion is reinforced by their decreased total phosphotyrosine levels, mirroring a postactivated stage, and increased levels of the phospho-S6 ribosomal protein, a key anabolic switch. Thus, we identified a subset of neutrophils within CF airways with a viable but dysfunctional phenotype. This subset provides a possible therapeutic target and indicates a need to revisit current paradigms of CF airway disease.
View details for DOI 10.1073/pnas.0712386105
View details for Web of Science ID 000254263300048
View details for PubMedID 18334635
Inherited hearing loss: molecular genetics and diagnostic testing.
Expert opinion on medical diagnostics
2008; 2 (3): 231-248
Background: Hearing loss is a clinically and genetically heterogeneous condition with major medical and social consequences. It affects up to 8% of the general population. Objective: This review recapitulates the principles of auditory physiology and the molecular basis of hearing loss, outlines the main types of non-syndromic and syndromic deafness by mode of inheritance, and provides an overview of current clinically available genetic testing. Methods: This paper reviews the literature on auditory physiology and on genes, associated with hearing loss, for which genetic testing is presently offered. Results/conclusion: The advent of molecular diagnostic assays for hereditary hearing loss permits earlier detection of the underlying causes, facilitates appropriate interventions, and is expected to generate the data necessary for more specific genotype-phenotype correlations.
View details for DOI 10.1517/17530059.2.3.231
View details for PubMedID 23495655
Progressive cerebral vascular degeneration with mitochondrial encephalopathy
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
2008; 146A (3): 361-367
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
Interlaboratory performance of a microarray-based gene expression test to determine tissue of origin in poorly differentiated and undifferentiated cancers
JOURNAL OF MOLECULAR DIAGNOSTICS
2008; 10 (1): 67-77
Clinical workup of metastatic malignancies of unknown origin is often arduous and expensive and is reported to be unsuccessful in 30 to 60% of cases. Accurate classification of uncertain primary cancers may improve with microarray-based gene expression testing. We evaluated the analytical performance characteristics of the Pathwork tissue of origin test, which uses expression signals from 1668 probe sets in a gene expression microarray, to quantify the similarity of tumor specimens to 15 known tissues of origin. Sixty archived tissue specimens from poorly and undifferentiated tumors (metastatic and primary) were analyzed at four laboratories representing a wide range of preanalytical conditions (eg, personnel, reagents, instrumentation, and protocols). Cross-laboratory comparisons showed highly reproducible results between laboratories, with correlation coefficients between 0.95 to 0.97 for measurements of similarity scores, and an average 93.8% overall concordance between laboratories in terms of final tissue calls. Bland-Altman plots (mean coefficients of reproducibility of 32.48+/-3.97) and kappa statistics (kappa >0.86) also indicated a high level of agreement between laboratories. We conclude that the Pathwork tissue of origin test is a robust assay that produces consistent results in diverse laboratory conditions reflecting the preanalytical variations found in the everyday clinical practice of molecular diagnostics laboratories.
View details for DOI 10.2353/jmoldx.2008.070099
View details for Web of Science ID 000252521200009
View details for PubMedID 18083688
Identification of an intronic single nucleotide polymorphism leading to allele dropout during validation of a CDH1 sequencing assay: implications for designing polymerase chain reaction-based assays
GENETICS IN MEDICINE
2007; 9 (11): 752-760
The CDH1 gene encodes the cell adhesion protein E-cadherin, and CDH1 germline mutations are associated with hereditary diffuse gastric cancer. Identification of individuals at high risk of developing diffuse gastric cancer affords the opportunity for endoscopic screening or elective prophylactic gastrectomy. We set out to develop a CDH1 sequencing assay for clinical use.All exons of the CDH1 gene were amplified and sequenced with published and modified primers.While validating the assay, we encountered a case in which a single nucleotide polymorphism located in intron 15 led to allele dropout and therefore to a false-negative result. The polymorphism leading to allele dropout was located within a primer-binding sequence, five bases away from the 3' end of the primer. A frameshift mutation in exon 15 was detected by an alternative primer that binds away from the polymorphic site. A search of the University of California Santa Cruz single nucleotide polymorphism database revealed other polymorphisms located within primer-binding sites. A total of 12 primers in nine primer sets were modified to minimize allele dropout risk.The approach of designing primers to avoid known single nucleotide polymorphisms can be generalized to the design of any polymerase chain reaction-based assay and should be employed whenever possible.
View details for DOI 10.1097/GIM.0b013e318159a369
View details for Web of Science ID 000251233500004
View details for PubMedID 18007144
T-cell clonality analysis in biopsy specimens from two different skin sites shows high specificity in the diagnosis of patients with suggested mycosis fungoides
JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY
2007; 57 (5): 782-790
The diagnosis of mycosis fungoides (MF) is often difficult because of significant clinical and histopathologic overlap with inflammatory dermatoses. T-cell receptor (TCR)gamma chain rearrangement by polymerase chain reaction (PCR) (TCR-PCR) is a helpful adjuvant tool in this setting, but several of the inflammatory dermatoses in the differential diagnosis of MF may contain a clonal T-cell proliferation.We examined whether analysis for T-cell clonality and comparison of the clones with the standardized BIOMED-2 PCR multiplex primers for the TCRgamma chain from two anatomically distinct skin sites improves diagnostic accuracy.We examined two biopsy specimens each from 10 patients with unequivocal MF, from 18 patients with inflammatory dermatoses, and from 18 patients who could initially not be definitively given a diagnosis based on clinical and histopathologic criteria.Eight of 10 patients with unequivocal MF had an identical clone in both biopsy specimens. Two of 18 patients with inflammatory dermatoses were found to have a clone in one of the biopsy specimens. On further follow-up of the 18 patients with morphologically nondiagnostic biopsy specimens, 13 of 18 were later confirmed to have MF and 5 of 18 had inflammatory dermatoses. Eleven of 13 patients with MF had an identical clone in both biopsy specimens; two of 13 had a polyclonal amplification pattern in both biopsy specimens. Four of 5 patients with inflammatory dermatoses had no clone in either biopsy specimen. One patient with an inflammatory dermatosis had an identical clone in both specimens. The sensitivity of TCR-PCR analysis to evaluate for an identical clone at different anatomic skin sites (dual TCR-PCR) is 82.6% and the specificity is 95.7%.The number of patients in the study group was limited.These data suggest that dual TCR-PCR is a very promising technique with high specificity in distinguishing MF from inflammatory dermatoses.
View details for DOI 10.1016/j.jaad.2007.06.004
View details for Web of Science ID 000250387100004
View details for PubMedID 17646032
Testing for maternal cell contamination in prenatal samples - A comprehensive survey of current diagnostic practices in 35 molecular diagnostic laboratories
JOURNAL OF MOLECULAR DIAGNOSTICS
2007; 9 (3): 394-400
The potential presence of maternal cell contamination (MCC) in chorionic villus or amniotic fluid samples poses a serious preanalytical risk for prenatal misdiagnosis. The aim of this study was to identify current diagnostic practices in the absence of comprehensive practice guidelines. Thirty-five clinical molecular laboratories that conduct prenatal testing agreed to participate in a clinical practice survey. The survey included questions about sample requirements, test indications, assay type, test performance and limitations, criteria and management of uninformative test results, reporting, and billing. Sixty percent of participating laboratories performed testing on direct and cultured amniotic fluid, whereas forty percent tested cultured cells only. Most also accepted chorionic villus samples. Although MCC testing of fetal samples is recommended in guidelines by the American College of Medical Genetics, only 60% of surveyed laboratories performed it without exception. Commercially available assays were used by 75% of participating laboratories, and at least five identity markers were evaluated at 87% of the laboratories. The reported lower limit of MCC detection ranged from 1 to 20% but was not determined in all laboratories. MCC testing was performed in the majority of molecular diagnostic laboratories, but guidelines for standardization are needed to ensure optimal and accurate prenatal patient care.
View details for DOI 10.2353/jmoldx.2007.070017
View details for Web of Science ID 000247691200015
View details for PubMedID 17591939
A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort
GENETICS IN MEDICINE
2007; 9 (7): 413-426
The aim of the study was to determine the actual GJB2 and GJB6 mutation frequencies in North America after several years of generalized testing for autosomal recessive nonsyndromic sensorineural hearing loss to help guide diagnostic testing algorithms, especially in light of molecular diagnostic follow-up to universal newborn hearing screening.Mutation types, frequencies, ethnic distributions, and genotype-phenotype correlations for GJB2 and GJB6 were assessed in a very large North American cohort.GJB2 variants were identified in 1796 (24.3%) of the 7401 individuals examined, with 399 (5.4%) homozygous and 429 (5.8%) compound heterozygous. GJB6 deletion testing was performed in 12.0% (888/7401) of all cases. The >300-kb deletion was identified in only nine individuals (1.0%), all of whom were compound heterozygous for mutations in GJB2 and GJB6. Among a total of 139 GJB2 variants identified, 53 (38.1%) were previously unreported, presumably representing novel pathogenic or benign variants.The frequency and distribution of sequence changes in GJB2 and GJB6 in North America differ from those previously reported, suggesting a considerable role for loci other than GJB2 and GJB6 in the etiology of autosomal recessive nonsyndromic sensorineural hearing loss, with minimal prevalence of the GJB6 deletion.
View details for DOI 10.1097/GIM.0b013e3180a03276
View details for Web of Science ID 000248370700003
View details for PubMedID 17666888
Comprehensive arrayed primer extension array for the detection of 59 sequence variants in 15 conditions prevalent among the (Ashkenazi) Jewish population
JOURNAL OF MOLECULAR DIAGNOSTICS
2007; 9 (2): 228-236
In the Ashkenazi Jewish population, serious and lethal genetic conditions occur with relatively high frequency. A single test that encompasses the majority of population-specific mutations is not currently available. For comprehensive carrier screening and molecular diagnostic purposes, we developed a population-specific and inclusive microarray. The arrayed primer extension genotyping microarray carries 59 sequence variant detection sites, of which 53 are detectable bi-directionally. These sites represent the most common variants in Tay-Sachs disease, Bloom syndrome, Canavan disease, Niemann-Pick A, familial dysautonomia, torsion dystonia, mucolipidosis type IV, Fanconi anemia, Gaucher disease, factor XI deficiency, glycogen storage disease type 1a, maple syrup urine disease, nonsyndromic sensorineural hearing loss, familial Mediterranean fever, and glycogen storage disease type III. Several mutations in the selected disorders that are not prevalent per se in the Ashkenazi Jewish populations, as well pseudodeficiency alleles, are also included in the array. The initial technical evaluation of this microarray demonstrates that it is comprehensive, robust, sensitive, specific, and easily modifiable. This cost-effective array is based on a diversely applied platform technology and is suitable for both carrier screening and disease detection in Ashkenazi and Sephardic Jewish populations.
View details for DOI 10.2353/jmoldx.2007.060100
View details for Web of Science ID 000245427600013
View details for PubMedID 17384215
Cystic fibrosis detection in high-risk Egyptian children and CFTR mutation analysis
JOURNAL OF CYSTIC FIBROSIS
2007; 6 (2): 111-116
Knowledge about Cystic Fibrosis (CF) in Egypt is very limited. The objective of this study was to screen for CF in Egyptian children with suggestive clinical features and to identify causative genetic mutations.Sixty-one patients from the Chest Unit, Cairo University Children's Hospital, Egypt, were included. Subjects presented with persistent or recurrent respiratory symptoms, failure to thrive, diarrhea and/or steatorrhea and unexplained persistent jaundice. Patients were screened using the CF Indicatortrade mark sweat test system (PolyChrome Medical, Inc., Brooklyn Center, MN). A quantitative sweat testing was conducted on 10 of the 12 positive patients. Seven probands and one sibling underwent molecular analysis by direct DNA sequencing of the coding region and of the intronic sequences adjacent to the 27 exons of the CFTR gene.Of 61 patients, 12 (20%) had positive sweat chloride screening. Ten of the 12 patients underwent quantitative sweat testing and were positive. Eight CFTR sequence changes were identified in seven affected probands and two were confirmed in one sibling by direct DNA sequencing.The study results suggest that CF is more common in Egypt than previously anticipated. Larger studies are warranted to identify the incidence, molecular basis and clinical pattern of CF in the Egyptian population.
View details for DOI 10.1016/j.jcf.2006.04.004
View details for Web of Science ID 000245778900003
View details for PubMedID 16837250
Clinical evaluation of a novel oncologic tissue of origin assay based on gene expression microarray.
ASSOC CLINICAL SCIENTISTS. 2007: 197-197
View details for Web of Science ID 000246264700023
Two patients with the V371/235delC genotype: Are radiographic cochlear anomalies part of the phenotype?
INTERNATIONAL JOURNAL OF PEDIATRIC OTORHINOLARYNGOLOGY
2006; 70 (12): 2109-2113
We present two East Asian patients with sensorineural hearing loss (SNHL) and compound heterozygosity for the 235delC and V37I mutations in the GJB2 gene. One patient has a unilaterally enlarged vestibular aqueduct, which underscores the importance of routine CT examination in children with SNHL, even if GJB2 (connexin 26) mutations have been identified. The second patient was not available for evaluation by CT. The pathogenic role of the V37I mutation has been controversial. We review the literature and present evidence in support of pathogenicity. Larger studies in compound heterozygous individuals and co-transfection studies will allow better genotype-phenotype correlations and prognostication.
View details for DOI 10.1016/j.ijporl.2006.07.015
View details for Web of Science ID 000242707900015
View details for PubMedID 16952406
Simultaneous multigene mutation detection in patients with sensorineural hearing loss through a novel diagnostic microarray: A new approach for newborn screening follow-up
2006; 118 (3): 985-994
The advent of universal newborn hearing screening in the United States and other countries, together with the identification of genes involved in the process of hearing, have led to an increase in both the need and opportunity for accurate molecular diagnosis of patients with hearing loss. Deafness and hearing impairment have a genetic cause in at least half the cases. The molecular genetic basis for the majority of these patients remains obscure, however, because of the absence of associated clinical features in approximately 70% (ie, nonsyndromic hearing loss) of patients, genetic heterogeneity, and the lack of molecular genetic tests that can evaluate a large number of mutations across multiple genes.We report on the development of a diagnostic panel with 198 mutations underlying sensorineural (mostly nonsyndromic) hearing loss. This panel, developed on a microarray, is capable of simultaneous evaluation of multiple mutations in 8 genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser[UCN]).The arrayed primer extension array for sensorineural hearing loss is based on a versatile platform technology and is a robust, cost-effective, and easily modifiable assay. Because hearing loss is a major public health concern and common at all ages, this test is suitable for follow-up after newborn hearing screening and for the detection of a genetic etiology in older children and adults.Comprehensive and relatively inexpensive genetic testing for sensorineural hearing loss will improve medical management for affected individuals and genetic counseling for their families.
View details for DOI 10.1542/peds.2005-2519
View details for Web of Science ID 000240959100016
View details for PubMedID 16950989
Detection of the JAK2 V617F mutation by LightCycler PCR and probe dissociation analysis
JOURNAL OF MOLECULAR DIAGNOSTICS
2006; 8 (3): 330-334
A point mutation in the JAK2 gene, a member of the tyrosine kinase family, was recently identified and shown to be associated with several myeloproliferative disorders. Several studies identified the same JAK2 point mutation (1,849G>T), resulting in the substitution of a valine to phenylalanine at codon 617 (V617F). We developed a simple and sensitive method to detect this mutation via polymerase chain reaction and probe dissociation analysis using the LightCycler platform, and we compared this method to existing restriction fragment-length polymorphism, direct sequencing, and amplification refractory mutation system methods. We found that the LightCycler method offered advantages of speed, reliability, and more straightforward interpretation over the restriction fragment-length polymorphism and sequencing approaches.
View details for DOI 10.2353/jmoldx.2006.050130
View details for Web of Science ID 000239106800006
View details for PubMedID 16825505
Hereditary sensorineural hearing loss: advances in molecular genetics and mutation analysis
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
2006; 6 (3): 375-386
Hearing loss has a genetic etiology in the majority of cases and is very common. The universal newborn hearing screening program, together with remarkable recent progress in the characterization of genes associated with the function of hearing, have resulted in increased demand and exciting possibilities of detecting the molecular basis of hereditary hearing loss through DNA testing. Future molecular diagnostic assays are expected to offer a greater variety of gene-specific tests, as well as combined mutation panels, which will aid in the management of the impressive genetic heterogeneity observed in hereditary hearing loss, especially in individuals with nonsyndromic forms. This review addresses the genetics of hearing loss, discusses the most commonly offered genetic assays for nonsyndromic hearing loss, with advantages and limitations, proposes a practical testing algorithm, and highlights current developments.
View details for DOI 10.1586/1473718.104.22.1685
View details for Web of Science ID 000237916000010
View details for PubMedID 16706740
Advanced mutation detection for hereditary sensorineural hearing loss through a comprehensive microarray.
ASSOC CLINICAL SCIENTISTS. 2006: 227-227
View details for Web of Science ID 000237603400026
GJB2 mutations and degree of hearing loss: A multicenter study
AMERICAN JOURNAL OF HUMAN GENETICS
2005; 77 (6): 945-957
Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.
View details for Web of Science ID 000233241200005
View details for PubMedID 16380907
Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations
JOURNAL OF MOLECULAR DIAGNOSTICS
2005; 7 (3): 375-387
Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.
View details for Web of Science ID 000231054600008
View details for PubMedID 16049310
Identification of mislabeled specimen by molecular methods: Case report and review
INTERNATIONAL JOURNAL OF SURGICAL PATHOLOGY
2005; 13 (3): 253-258
Specimen misidentification is a common cause of errors in surgical pathology. We report a case where bone-marrow biopsies from patients of different genders were mislabeled and molecular methods were applied to resolve the identity. A short tandem repeat (STR)-polymerase chain reaction-based assay, commonly used in paternity testing, was employed in an attempt to assign the correct identity to the specimens. However, the specimens had been processed by decalcification and the DNA yield was poor. One of the markers in the assay is the non-STR amelogenin locus that distinguishes the X and Y chromosomes. This amelogenin marker results in a product of low molecular weight, enabling unequivocal resolution of identity despite a poor DNA yield. The prevalence of errors in pathology due to specimen misidentifications is reviewed.
View details for Web of Science ID 000231185700004
View details for PubMedID 16086080
Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics novel mutations and assessment of a population-specific mutation spectrum
JOURNAL OF MOLECULAR DIAGNOSTICS
2005; 7 (2): 289-299
Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.
View details for Web of Science ID 000228736900018
View details for PubMedID 15858154
- High frequency of premature termination mutations in the factor V gene: Three factor V deficiency case reports and a mutation review THROMBOSIS AND HAEMOSTASIS 2005; 93 (3): 610-611
- Gender differences and performance in science SCIENCE 2005; 307 (5712): 1043-1043
- Novel Contributions to the Asian CFTR mutation spectrum: genotype and phenotype in Thai patients with cystic fibrosis. Am J Med Genet. 2005; 133A: 103-5
Hereditary non-syndromic sensorineural hearing loss - Transforming silence to sound
JOURNAL OF MOLECULAR DIAGNOSTICS
2004; 6 (4): 275-284
Tremendous progress has been made in our understanding of the molecular basis of hearing and hearing loss. Through recent advances, we have begun to understand the fascinating biology of the auditory system and unveiled new molecular mechanisms of hearing impairment. Changes in the diagnostic impact of genetic testing have occurred, as well as exciting developments in therapeutic options. Molecular diagnosis, which is already a reality for several hearing-associated genes, will doubtlessly continue to increase in the near future, both in terms of the number of mutations tested and the spectrum of genes. Genetic analysis for hearing loss is mostly used for diagnosis and treatment, and relatively rarely for reproductive decisions, in contrast to other inherited disorders. Inherited hearing loss, however, is characterized by impressive genetic heterogeneity. An abundance of genes carry a large number of mutations, but specific mutations in a single gene may lead to syndromic or non-syndromic hearing loss. Some mutations predominate in individual ethnic groups. For clinical and laboratory diagnosticians, it is challenging to keep abreast of the unfolding discoveries. This review aims to provide the framework pertinent to diagnosticians and a practical approach to mutation analysis in the hearing impaired.
View details for Web of Science ID 000226190000001
View details for PubMedID 15507665
Rapid combined genotyping assay for four achondroplasia and hypochondroplasia mutations by real-time PCR with multiple detection probes
2004; 8 (2): 185-189
Achondroplasia (ACH) and hypochondroplasia (HYCH) are the most prevalent genetic short-stature syndromes. Whereas the diagnosis of ACH can be established on clinical and radiologic grounds alone in the majority of cases, HYCH is more difficult to confirm. Molecular genetic analysis of both skeletal dysplasias can be especially helpful for the purpose of prenatal diagnosis, in early childhood to differentiate definitively between the largely overlapping phenotypes, and in atypical presentations. The two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide. These mutations can be identified by a variety of molecular methods, including PCR with restriction enzyme digestion or direct DNA sequencing. We have developed a single-step, real-time PCR assay in which two detection probes are applied in combination with a single anchor probe at each mutation position. Because the two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide, this approach guarantees optimal differentiation during probe dissociation analysis after amplification. This assay, which is performed on the LightCycler thermocycler, enables the rapid and reliable detection of the two most common FGFR3 mutations associated with ACH (1138G --> A and 1138G --> C; G380R) and HYCH (1620C --> A and 1620 C --> G; N540K) in a single test.
View details for Web of Science ID 000223513900019
View details for PubMedID 15345118
Molecular diagnosis of hypercoagulable states
2004; 35 (4): 214-221
View details for Web of Science ID 000220469400016
Prothrombin gene variants in non-Caucasians with fetal loss and intrauterine growth retardation
JOURNAL OF MOLECULAR DIAGNOSTICS
2003; 5 (4): 250-253
Thrombotic predisposition may affect pregnancy outcome, but in non-Caucasians the contributing genetic factors are poorly characterized. Two recently identified prothrombin gene mutations (20209C>T and 20221C>T) have been observed in non-Caucasian patients with thrombosis. The mutations are located near the commonly identified variant 20210G>A and have not been reported in Caucasian patients. The authors report a novel connection with pregnancy complications. The identification of sequence variants other than 20210G>A in the 3'-untranslated region of the prothrombin gene suggests that additional nucleotide substitutions may contribute to the development of thrombotic events and adverse pregnancy outcomes, especially in less well-characterized populations.
View details for Web of Science ID 000186292700009
View details for PubMedID 14573785
Diagnostic single nucleotide polymorphism analysis of factor V Leiden and prothrombin 20210G > A - A comparison of the nanogen electronic microarray with restriction enzyme digestion and the Roche LightCycler
AMERICAN JOURNAL OF CLINICAL PATHOLOGY
2003; 119 (4): 490-496
Genetic thrombosis risk factors include a sequence variant in the prothrombin gene (20210G > A) and factor V Leiden (1691G > A). These single nucleotide polymorphisms can be diagnosed with restriction fragment length polymorphism (RFLP) analysis, fluorescent genotyping on the LightCycler (Roche Diagnostics, Indianapolis, IN), and microarray-based testing on the novel NanoChip electronic microarray (NanoChip Molecular Biology Workstation, Nanogen, San Diego, CA). We compared these methods for accuracy, time to results, throughput, and interpretation. Results from 789 of 800 individual amplicons analyzed on the NanoChip were in complete agreement with the other assays. Eleven were "no calls" (uninterpreted by the NanoChip system) resulting from failed polymerase chain reaction amplifications. Although the NanoChip System, when used in a low-throughput setting, requires more overall time than the LightCycler, it is nearly equivalent per genotyping call. Owing to minimal sample handling, assay results are more reliable on the NanoChip platform and on the LightCycler than with RFLP. The NanoChip assay is reliable and may be especially valuable to laboratories with a large volume of thrombophilia test requests.
View details for DOI 10.1309/3VTR7TL2X7TXL0XY
View details for Web of Science ID 000181872500002
View details for PubMedID 12710121
Labor and cost requirements of two commercial assays for qualitative molecular detection of hepatitis C virus
JOURNAL OF CLINICAL MICROBIOLOGY
2002; 40 (9): 3476-3477
The Bayer transcription-mediated amplification (TMA) and the Roche PCR Amplicor version 2.0 molecular assays for the qualitative detection of hepatitis C virus were compared for cost, hands-on time, assay duration, and complexity. The TMA assay compares well to PCR and may be especially useful for laboratories with large numbers of test requests.
View details for DOI 10.1128/JCM.40.9.3476-3477.2002
View details for Web of Science ID 000177829900057
View details for PubMedID 12202596
Premature termination mutations in FBN1: Distinct effects on differential allelic expression and on protein and clinical phenotypes
AMERICAN JOURNAL OF HUMAN GENETICS
2002; 71 (2): 223-237
Marfan syndrome (MFS) and other type 1 fibrillinopathies result from mutations in the FBN1 gene, which encodes the connective-tissue microfibrillar protein fibrillin 1. Attempts at correlating genotype with phenotype have suggested considerable heterogeneity. To define the subtype of fibrillinopathy caused by premature termination codon (PTC) mutations, we integrate genotype information and mRNA expression levels with clinical and biochemical phenotypes. By screening the entire FBN1 gene for mutations, we identified 34 probands with PTC mutations. With the exception of two recurrent mutations, these nonsense and frameshift mutations are unique and span the entire FBN1 gene, from IVS2 to IVS63. Allele-specific reverse-transcriptase polymerase chain reaction analyses revealed differential allelic expression in all studied samples, with variable reduction of the mutant transcript. Fibrillin protein synthesis and deposition into the extracellular matrix were studied by pulse-chase analysis of cultured fibroblasts. In the majority of PTC samples, synthesis of normal-sized fibrillin protein was approximately 50% of control levels, but matrix deposition was disproportionately decreased. Probands and mutation-positive relatives were clinically evaluated by means of a standardized protocol. Only 71% (22/31) of probands and 58% (14/24) of the mutation-positive family members met current clinical diagnostic criteria for MFS. When compared with our previously reported study group of 44 individuals with FBN1 cysteine substitutions, the PTC group showed statistically significant differences in the frequency of individual signs, especially in the ocular manifestations. Whereas large-joint hypermobility was more common, lens dislocation and retinal detachment were distinctly less common in the PTC group. We conclude that PTC mutations have a major impact on the pathogenesis of type 1 fibrillinopathies and convey a distinct biochemical, clinical, and prognostic profile.
View details for Web of Science ID 000176977700002
View details for PubMedID 12068374
Homozygous factor V splice site mutation associated with severe factor V deficiency
2002; 99 (8): 3063-3065
We investigated a family whose proband has a severe bleeding disorder and factor V antigenic and functional levels of 8% and less than 1% of control values, respectively. Molecular analysis of the factor V gene revealed a novel homozygous mutation in the last nucleotide of exon 10. 1701G>T causes activation of a cryptic exonic splice site in exon 10, which encodes part of the factor V heavy chain (A2 domain). This leads to the deletion of 35 nucleotides and results in a frameshift with a premature stop codon at amino acid position 498. The G1701 and corresponding Gln509 are conserved in murine, bovine, and porcine factor V and in human factor VIII. Few factor V deficiency mutations have been identified as yet. Several are present in the heterozygous form in combination with factor V Leiden (Arg506Gln). This is the first reported homozygous splice site mutation in a patient with factor V deficiency.
View details for Web of Science ID 000174866500055
View details for PubMedID 11929802
Spontaneous spinal cerebrospinal fluid leaks and minor skeletal features of Marfan syndrome: a microfibrillopathy
JOURNAL OF NEUROSURGERY
2002; 96 (3): 483-489
Spontaneous spinal cerebrospinal fluid (CSF) leaks are increasingly recognized as a cause of postural headaches. The authors examined a group of patients suffering from spontaneous spinal CSF leaks who also had minor skeletal features of Marfan syndrome for abnormalities of fibrillin-containing microfibrils.Patients with spontaneous CSF leaks were evaluated for the clinical characteristics of connective tissue disorders. Skin biopsies were obtained in three patients with skeletal manifestations that constitute part of the Marfan syndrome phenotype. Cultured fibroblasts were studied for fibrillin-1 synthesis and incorporation into the extracellular matrix (ECM) by performing quantitative metabolic labeling and immunohistochemical analysis. Among 20 consecutive patients found to have spinal CSF leaks, four (20%) exhibited minor skeletal features of Marfan syndrome, but lacked any ocular or cardiovascular abnormalities. The mean age of these patients (30 years) was lower than that of the 16 patients without skeletal abnormalities (44 years; p = 0.01). Abnormalities in fibrillin-1 metabolism and immunostaining were detected in all three patients with the skeletal abnormalities who underwent examination, but not in a control patient without these skeletal manifestations.Twenty percent of patients who experience spontaneous spinal CSF leaks have minor skeletal features of Marfan syndrome. The authors demonstrated abnormalities in fibrillin-1 protein deposition in all patients examined, but only one person was found to have a fibrillin-1 abnormality typically found in classic Marfan syndrome. The results indicate that there is a heterogeneous involvement of other components of ECM microfibrils at the basis of this cerebrospinal manifestation. In addition, the authors identified a connective-tissue etiological factor in a group of disorders not previously classified as such.
View details for Web of Science ID 000174104100004
View details for PubMedID 11883832
Novel factor VC2-domain mutation (R2074H) in two families with factor V deficiency and bleeding
THROMBOSIS AND HAEMOSTASIS
2002; 87 (2): 294-299
The molecular basis of Factor V deficiency has been defined in few patients only. We report a homozygous nucleotide change (G6395A) in two Tunisian probands with Factor V deficiency and bleeding episodes. This substitution results in the replacement of an arginine (R) by a histidine (H) in amino acid position 2074, located in the Factor V C2-domain. Mutations in this protein domain have not previously been described. Several lines of evidence support that this sequence variant is indeed disease causing: 1) Crystal structures of Factor V and molecular C2-domain modeling studies of H2074 suggest that the conserved R2074 is required for correct folding; 2) Structure-function studies of selective Factor V mutants (R2074A) demonstrate the importance of R2074 for structural stability of the Factor V C2-domain and for cofactor activity (1); 3) In Factor VIII, point mutations in codon 2209, which corresponds to position 2074 in Factor V, cause hemophilia A.
View details for Web of Science ID 000173869300020
View details for PubMedID 11858490
Multi-exon deletions of the FBN1 gene in Marfan syndrome.
BMC medical genetics
2001; 2: 11-?
Mutations in the fibrillin -1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant multi-system connective tissue disorder. The 200 different mutations reported in the 235 kb, 65 exon-containing gene include only one family with a genomic multi-exon deletion.We used long-range RT-PCR for mutation detection and long-range genomic PCR and DNA sequencing for identification of deletion breakpoints, allele-specific transcript analyses to determine stability of the mutant RNA, and pulse-chase studies to quantitate fibrillin synthesis and extracellular matrix deposition in cultured fibroblasts. Southern blots of genomic DNA were probed with three overlapping fragments covering the FBN1 coding exonsTwo novel multi-exon FBN1 deletions were discovered. Identical nucleotide pentamers were found at or near the intronic breakpoints. In a Case with classic MFS, an in-frame deletion of exons 42 and 43 removed the C-terminal 24 amino acids of the 5th LTBP (8-cysteine) domain and the adjacent 25th calcium-binding EGF-like (6-cysteine) domain. The mutant mRNA was stable, but fibrillin synthesis and matrix deposition were significantly reduced. A Case with severe childhood-onset MFS has a de novo deletion of exons 44-46 that removed three EGF-like domains. Fibrillin protein synthesis was normal, but matrix deposition was strikingly reduced. No genomic rearrangements were detected by Southern analysis of 18 unrelated MFS samples negative for FBN1 mutation screening.Two novel deletion cases expand knowledge of mutational mechanisms and genotype/phenotype correlations of fibrillinopathies. Deletions or mutations affecting an LTBP domain may result in unstable mutant protein cleavage products that interfere with microfibril assembly.
View details for PubMedID 11710961
Cysteine substitutions in epidermal growth factor-like domains of fibrillin-1: Distinct effects on biochemical and clinical phenotypes
AMERICAN JOURNAL OF HUMAN GENETICS
1999; 65 (4): 1007-1020
Fibrillin-1 (FBN1) contains 47 epidermal growth factor (EGF)-like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified 19 new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant fibroblast strains with substitutions of 22 different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in 20/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and 19 mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26-32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.
View details for Web of Science ID 000082910000009
View details for PubMedID 10486319
The pathogenicity of the Pro1148Ala substitution in the FBN1 gene: Causing or predisposing to Marfan syndrome and aortic aneurysm, or clinically innocent?
1997; 99 (5): 607-611
In individuals with the Marfan syndrome (MFS), mutations have been identified in the fibrillin-1 gene (FBN1) at 15q21.1. A proline-to-alanine change at position 1148 in exon 27 (Pro1148Ala) has been reported in probands with MFS, aortic aneurysm or Marfanoid-craniosynostosis. It was suggested that this mutation could be a risk factor for aortic dilatation, since it was rarely observed in control populations. To investigate further the pathogenicity of this substitution, we screened 416 unrelated control individuals by allele-specific oligonucleotide (ASO) hybridization. We found 16 individuals who carried the alanine allele (3.8%), 3 of whom were homozygous. Five were of Latin American and eight were of Asian extraction. We also screened 133 probands with MFS, aortic aneurysm or related connective tissue disorders and found 4 (3%) that were heterozygous for the 1148Ala allele. All positive results were confirmed by DNA sequencing. In 20 individuals with 1148Ala, we confirmed the association with the rarer A allele at the IVS27-5G-->A polymorphism. Our results suggest that the Pro1148Ala change is a polymorphism of ancient evolutionary origin that is more prevalent in Asian and Latin American than in Caucasian or African populations.
View details for Web of Science ID A1997WW19000009
View details for PubMedID 9150726
- Retinal dystrophy in long-chain 3-hydroxy-acyl-CoA dehydrogenase deficiency Br J Opthalmol 1997; 81: 291-4