EMQN best practice guidelines for genetic testing in hereditary breast and ovarian cancer.

Hereditary Breast and Ovarian Cancer (HBOC) is a genetic condition associated with increased risk of cancers. The past decade has brought about significant changes to hereditary breast and ovarian cancer (HBOC) diagnostic testing with new treatments, testing methods and strategies, and evolving information on genetic associations. These best practice guidelines have been produced to assist clinical laboratories in effectively addressing the complexities of HBOC testing, while taking into account advancements since the last guidelines were published in 2007. These guidelines summarise cancer risk data from recent studies for the most commonly tested high and moderate risk HBOC genes for laboratories to refer to as a guide. Furthermore, recommendations are provided for somatic and germline testing services with regards to clinical referral, laboratory analyses, variant interpretation, and reporting. The guidelines present recommendations where 'must' is assigned to advocate that the recommendation is essential; and 'should' is assigned to advocate that the recommendation is highly advised but may not be universally applicable. Recommendations are presented in the form of shaded italicised statements throughout the document, and in the form of a table in supplementary materials (Table S4). Finally, for the purposes of encouraging standardisation and aiding implementation of recommendations, example report wording covering the essential points to be included is provided for the most common HBOC referral and reporting scenarios. These guidelines are aimed primarily at genomic scientists working in diagnostic testing laboratories.

Alberta Spinal Muscular Atrophy Newborn Screening-Results from Year 1 Pilot Project.

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by biallelic pathogenic/likely pathogenic variants of the survival motor neuron 1 (SMN1) gene. Early diagnosis via newborn screening (NBS) and pre-symptomatic treatment are essential to optimize health outcomes for affected individuals. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay using dried blood spot (DBS) samples for the detection of homozygous absence of exon 7 of the SMN1 gene. Newborns who screened positive were seen urgently for clinical evaluation. Confirmatory testing by multiplex ligation-dependent probe amplification (MLPA) revealed SMN1 and SMN2 gene copy numbers. Six newborns had abnormal screen results among 47,005 newborns screened during the first year and five were subsequently confirmed to have SMA. Four of the infants received SMN1 gene replacement therapy under 30 days of age. One infant received an SMN2 splicing modulator due to high maternally transferred AAV9 neutralizing antibodies (NAb), followed by gene therapy at 3 months of age when the NAb returned negative in the infant. Early data show that all five infants made excellent developmental progress. Based on one year of data, the incidence of SMA in Alberta was estimated to be 1 per 9401 live births.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Methodology for clinical genotyping of CYP2D6 and CYP2C19.

Many antidepressants, atomoxetine, and several antipsychotics are metabolized by the cytochrome P450 enzymes CYP2D6 and CYP2C19, and guidelines for prescribers based on genetic variants exist. Although some laboratories offer such testing, there is no consensus regarding validated methodology for clinical genotyping of CYP2D6 and CYP2C19. The aim of this paper was to cross-validate multiple technologies for genotyping CYP2D6 and CYP2C19 against each other, and to contribute to feasibility for clinical implementation by providing an enhanced range of assay options, customizable automated translation of data into haplotypes, and a workflow algorithm. AmpliChip CYP450 and some TaqMan single nucleotide variant (SNV) and copy number variant (CNV) data in the Genome-based therapeutic drugs for depression (GENDEP) study were used to select 95 samples (out of 853) to represent as broad a range of CYP2D6 and CYP2C19 genotypes as possible. These 95 included a larger range of CYP2D6 hybrid configurations than have previously been reported using inter-technology data. Genotyping techniques employed were: further TaqMan CNV and SNV assays, xTAGv3 Luminex CYP2D6 and CYP2C19, PharmacoScan, the Ion AmpliSeq Pharmacogenomics Panel, and, for samples with CYP2D6 hybrid configurations, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing and Luminex. Agena MassARRAY was also used for CYP2C19. This study has led to the development of a broader range of TaqMan SNV assays, haplotype phasing methodology with TaqMan adaptable for other technologies, a multiplex genotyping method for efficient identification of some hybrid haplotypes, a customizable automated translation of SNV and CNV data into haplotypes, and a clinical workflow algorithm.

Identification of high-impact gene-drug pairs for pharmacogenetic testing in Alberta, Canada.

OBJECTIVES: To facilitate decision-making and priority-setting related to Alberta's Pharmacogenomics (PGx) testing implementation strategy by identifying gene-drug pairs with the highest potential impact on prescribing practices in Alberta. PATIENTS AND METHODS: Annual drug dispensing data for Alberta from 2012 to 2016 for 57 medications with PGx-based prescribing guidelines were obtained, along with population estimates and demographics (age and ethnicity). Frequencies of actionable PGx genotypes by ethnicity were obtained from the Pharmacogenomics Knowledgebase (PharmGKB). Annual dispensing activity for each of the 57 medications was calculated for the full population (all ages) and children/youth (0-19 years). Alberta ethnicity data were cross-referenced with genetic frequency data for each of the main ethnic groups from PharmGKB to estimate the proportion of individuals with actionable genotypes. Actionable genotype proportions and drug dispensing frequencies were collectively used to identify high impact gene-drug pairs. RESULTS: We found (a) half of the drugs with PGx-based prescribing guidelines, namely, analgesics, proton pump inhibitors, psychotropics, and cardiovascular drugs, were dispensed at high frequencies (>1% of the entire population), (b) the dispensing rate for about one-third of these drugs increased over the 5-year study period, (c) between 1.1 and 45% of recipients of these drugs carried actionable genotypes, and (d) the gene-drug pairs with greatest impact in Alberta predominatly included CYP2C19 or CYP2D6. CONCLUSIONS: We uncovered specific patterns in drug dispensing and identified important gene-drug pairs that will inform the planning and development of an evidenced-based PGx testing service in Alberta, Canada. Adaptation of our approach may facilitate the process of evidence-based PGx testing implementation in other jurisdictions.

Retrospective testing of respiratory specimens for COVID-19 to assess for earlier SARS-CoV-2 infections in Alberta, Canada.

Background: The first case of coronavirus disease 2019 (COVID-19) in Alberta, Canada, was confirmed on March 5, 2020. Because the virus testing criteria had changed significantly over this time period, we wanted to ascertain whether previous cases of COVID-19 had been missed in the province. Methods: Our aim was to retrospectively evaluate specimens submitted for respiratory virus testing from December 1, 2019, through March 7, 2020, for undetected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections before the first confirmed case. Results: Testing of 23,517 samples (representing 23,394 patients) identified 1 patient positive for SARS-CoV-2. This specimen was collected on February 24, 2020, from a patient with symptoms consistent with COVID-19 who had recently returned from the western United States. Phylogenetic analysis confirmed this viral isolate belonged to lineage B.1. The epidemiology of this case is consistent with those of other early cases before sustained community transmission, which included a travel history outside of Canada. Conclusion: This exercise provides support that local public health pandemic planning was satisfactory and timely.

Historique: Le premier cas de maladie à coronavirus 2019 (COVID-19) en Alberta, au Canada, a été confirmé le 15 mars 2020. Puisque les critères de dépistage ont beaucoup évolué pendant cette période, les chercheurs voulaient vérifier si des cas antérieurs de COVID-19 avaient été omis dans la province. Méthodologie: Les chercheurs ont procédé à l'évaluation rétrospective d'échantillons soumis en vue du dépistage d'un virus respiratoire entre le 1er décembre 2019 et le 7 mars 2020, afin de retracer les infections par le coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2) non décelées avant le premier cas confirmé. Résultats: Le dépistage de 23 517 échantillons (représentant 23 394 patients) a fait ressortir un patient positif au SARS-CoV-2. Le prélèvement avait été effectué le 24 février 2020 chez un patient éprouvant des symptômes correspondant à la COVID-19 revenu récemment de l'ouest des États-Unis. L'analyse phylogénétique a confirmé que l'isolat viral appartenait à la lignée B.1. L'épidémiologie de ce cas est compatible avec celle des autres premiers cas précédant une transmission communautaire soutenue, qui incluait un voyage à l'extérieur du Canada. Conclusion: Cet exercice appuie la pertinence et la rapidité de la planification sanitaire locale de la pandémie.

MITO-FIND: A study in 390 patients to determine a diagnostic strategy for mitochondrial disease.

Mitochondrial diseases, due to nuclear or mitochondrial genome mutations causing mitochondrial dysfunction, have a wide range of clinical features involving neurologic, muscular, cardiac, hepatic, visual, and auditory symptoms. Making a diagnosis of a mitochondrial disease is often challenging since there is no gold standard and traditional testing methods have required tissue biopsy which presents technical challenges and most patients prefer a non-invasive approach. Since a diagnosis invariably involves finding a disease-causing DNA variant, new approaches such as next generation sequencing (NGS) have the potential to make it easier to make a diagnosis. We evaluated the ability of our traditional diagnostic pathway (metabolite analysis, tissue neuropathology and respiratory chain enzyme activity) in 390 patients. The traditional diagnostic pathway provided a diagnosis of mitochondrial disease in 115 patients (29.50%). Analysis of mtDNA, tissue neuropathology, skin electron microscopy, respiratory chain enzyme analysis using inhibitor assays, blue native polyacrylamide gel electrophoresis were all statistically significant in distinguishing patients between a mitochondrial and non-mitochondrial diagnosis. From these 390 patients who underwent traditional analysis, we recruited 116 patients for the NGS part of the study (36 patients who had a mitochondrial diagnosis (MITO) and 80 patients who had no diagnosis (No-Dx)). In the group of 36 MITO patients, nuclear whole exome sequencing (nWES) provided a second diagnosis in 2 cases who already had a pathogenic variant in mtDNA, and a revised diagnosis (GLUL) in one case that had abnormal pathology but no pathogenic mtDNA variant. In the 80 NO-Dx patients, nWES found non-mitochondrial diagnosis in 26 patients and a mitochondrial diagnosis in 1 patient. A genetic diagnosis was obtained in 53/116 (45.70%) cases that were recruited for NGS, but not in 11/116 (9.48%) of cases with abnormal mitochondrial neuropathology. Our results show that a non-invasive, bigenomic sequencing (BGS) approach (using both a nWES and optimized mtDNA analysis to include large deletions) should be the first step in investigating for mitochondrial diseases. There may still be a role for tissue biopsy in unsolved cases or when the diagnosis is still not clear after NGS studies.

The novel p.Ser263Phe mutation in the human high-affinity choline transporter 1 (CHT1/SLC5A7) causes a lethal form of fetal akinesia syndrome.

A subset of a larger and heterogeneous class of disorders, the congenital myasthenic syndromes (CMS) are caused by pathogenic variants in genes encoding proteins that support the integrity and function of the neuromuscular junction (NMJ). A central component of the NMJ is the sodium-dependent high-affinity choline transporter 1 (CHT1), a solute carrier protein (gene symbol SLC5A7), responsible for the reuptake of choline into nerve termini has recently been implicated as one of several autosomal recessive causes of CMS. We report the identification and functional characterization of a novel pathogenic variant in SLC5A7, c.788C>T (p.Ser263Phe) in an El Salvadorian family with a lethal form of a congenital myasthenic syndrome characterized by fetal akinesia. This study expands the clinical phenotype and insight into a form of fetal akinesia related to CHT1 defects and proposes a genotype-phenotype correlation for the lethal form of SLC5A7-related disorder with potential implications for genetic counseling.

CCMG practice guideline: laboratory guidelines for next-generation sequencing.

PurposeThe purpose of this document is to provide guidance for the use of next-generation sequencing (NGS, also known as massively parallel sequencing or MPS) in Canadian clinical genetic laboratories for detection of genetic variants in genomic DNA and mitochondrial DNA for inherited disorders, as well as somatic variants in tumour DNA for acquired cancers. They are intended for Canadian clinical laboratories engaged in developing, validating and using NGS methods. METHODS OF STATEMENT DEVELOPMENT: The document was drafted by the Canadian College of Medical Geneticists (CCMG) Ad Hoc Working Group on NGS Guidelines to make recommendations relevant to NGS. The statement was circulated for comment to the CCMG Laboratory Practice and Clinical Practice committees, and to the CCMG membership. Following incorporation of feedback, the document was approved by the CCMG Board of Directors. DISCLAIMER: The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada and should not be considered to be inclusive of all information laboratories should consider in the validation and use of NGS for a clinical laboratory service.

Hybrid gel electrophoresis using skin fibroblasts to aid in diagnosing mitochondrial disease.

OBJECTIVE: We developed a novel, hybrid method combining both blue-native (BN-PAGE) and clear-native (CN-PAGE) polyacrylamide gel electrophoresis, termed BCN-PAGE, to perform in-gel activity stains on the mitochondrial electron transport chain (ETC) complexes in skin fibroblasts. METHODS: Four patients aged 46-65 years were seen in the Metabolic Clinic at Alberta Children's Hospital and investigated for mitochondrial disease and had BN-PAGE or CN-PAGE on skeletal muscle that showed incomplete assembly of complex V (CV) in each patient. Long-range PCR performed on muscle-extracted DNA identified 4 unique mitochondrial DNA (mtDNA) deletions spanning the ATP6 gene of CV. We developed a BCN-PAGE method in skin fibroblasts taken from the patients at the same time and compared the findings with those in skeletal muscle. RESULTS: In all 4 cases, BCN-PAGE in skin fibroblasts confirmed the abnormal CV activity found from muscle biopsy, suggesting that the mtDNA deletions involving ATP6 were most likely germline mutations that are associated with a clinical phenotype of mitochondrial disease. CONCLUSIONS: The BCN-PAGE method in skin fibroblasts has a potential to be a less-invasive tool compared with muscle biopsy to screen patients for abnormalities in CV and other mitochondrial ETC complexes.

Newborn Screening: Current Status in Alberta, Canada.

Newborn screening (NBS) in Alberta is delivered by a number of government and health service entities who work together to provide newborn screening to infants born in Alberta, the Northwest Territories, and the Kitikmeot region of the Nunavut territory. The Alberta panel screens for 21 disorders (16 metabolic, two endocrine, cystic fibrosis, severe combined immunodeficiency, and sickle cell disease). NBS is a standard of care, but is not mandatory. NBS performance is monitored by the Alberta Newborn Metabolic Screening (NMS) Program and NMS Laboratory, who strive for continuous quality improvement. Performance analysis found that over 99% of registered infants in Alberta received a newborn screen and over 98% of these infants received a screen result within 10 days of age.

Plasma-derived cell-free mitochondrial DNA: A novel non-invasive methodology to identify mitochondrial DNA haplogroups in humans.

BACKGROUND: Mitochondrial diseases are a clinically heterogeneous group of diseases caused by mutations in either nuclear or mitochondrial DNA (mtDNA). The diagnosis is challenging and has frequently required a tissue biopsy to obtain a sufficient quantity of mtDNA. Less-invasive sources mtDNA, such as peripheral blood leukocytes, urine sediment, or buccal swab, contain a lower quantity of mtDNA compared to tissue sources which may reduce sensitivity. Cellular apoptosis of tissues and hematopoetic cells releases fragments of DNA and mtDNA into the circulation and these molecules can be extracted from plasma as cell-free DNA (cfDNA). However, entire mtDNA has not been successfully identified from the cell free fraction previously. We hypothesized that the circular nature of mtDNA would prevent its degradation and a higher sensitivity method, such as next generation sequencing, could identify intact cf-mtDNA from human plasma. METHODS: Plasma was obtained from patients with mitochondrial disease diagnosed from skeletal muscle biopsy (n = 7) and healthy controls (n = 7) using a specially cfDNA collection tube (Streck Inc.; La Vista, NE). To demonstrate the presence of mtDNA within these samples, we amplified the isolated DNA using custom PCR primers specific to overlapping fragments of mtDNA. cfDNA samples were then sequenced using the Illumina MiSeq sequencing platform. RESULTS: We confirmed the presence of mtDNA, demonstrating that the full mitochondrial genome is in fact present within the cell-free plasma fraction of human blood. Sequencing identified the mitochondrial haplogroup matching with the tissue specimen for all patients. CONCLUSION: We report the existence of full length mtDNA in cell-free human plasma that was successfully used to perform haplogroup matching. Clinical applications for this work include patient monitoring for heteroplasmy status after mitochondrially-targeted therapies or haplogroup monitoring as a measure of stem cell transplantation.

Data sharing as a national quality improvement program: reporting on BRCA1 and BRCA2 variant-interpretation comparisons through the Canadian Open Genetics Repository (COGR).

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.

Assessing the cost of implementing the 2011 Society of Obstetricians and Gynecologists of Canada and Canadian College of Medical Genetics practice guidelines on the detection of fetal aneuploidies.

BACKGROUND: The Society of Obstetricians and Gynecologists of Canada and the Canadian College of Medical Genetics published guidelines, in 2011, recommending replacement of karyotype with quantitative fluorescent polymerase chain reaction when prenatal testing is performed because of an increased risk of a common aneuploidy. STUDY OBJECTIVE: This study's objective is to perform a cost analysis following the implementation of quantitative fluorescent polymerase chain reaction as a stand-alone test. RESULTS: A total of 658 samples were received between 1 April 2014 and 31 August 2015: 576 amniocentesis samples and 82 chorionic villi sampling. A chromosome abnormality was identified in 14% (93/658) of the prenatal samples tested. The implementation of the 2011 Society of Obstetricians and Gynecologists of Canada and the Canadian College of Medical Genetics guidelines in Edmonton and Northern Alberta resulted in a cost savings of $46 295.80. The replacement of karyotype with chromosomal microarray for some indications would be associated with additional costs. CONCLUSION: The implementation of new test methods may provide cost savings or added costs. Cost analysis is important to consider during the implementation of new guidelines or technologies. © 2017 John Wiley & Sons, Ltd.

The clinical application of genome-wide sequencing for monogenic diseases in Canada: Position Statement of the Canadian College of Medical Geneticists.

PURPOSE AND SCOPE: The aim of this Position Statement is to provide recommendations for Canadian medical geneticists, clinical laboratory geneticists, genetic counsellors and other physicians regarding the use of genome-wide sequencing of germline DNA in the context of clinical genetic diagnosis. This statement has been developed to facilitate the clinical translation and development of best practices for clinical genome-wide sequencing for genetic diagnosis of monogenic diseases in Canada; it does not address the clinical application of this technology in other fields such as molecular investigation of cancer or for population screening of healthy individuals. METHODS OF STATEMENT DEVELOPMENT: Two multidisciplinary groups consisting of medical geneticists, clinical laboratory geneticists, genetic counsellors, ethicists, lawyers and genetic researchers were assembled to review existing literature and guidelines on genome-wide sequencing for clinical genetic diagnosis in the context of monogenic diseases, and to make recommendations relevant to the Canadian context. The statement was circulated for comment to the Canadian College of Medical Geneticists (CCMG) membership-at-large and, following incorporation of feedback, approved by the CCMG Board of Directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. RESULTS AND CONCLUSIONS: Recommendations include (1) clinical genome-wide sequencing is an appropriate approach in the diagnostic assessment of a patient for whom there is suspicion of a significant monogenic disease that is associated with a high degree of genetic heterogeneity, or where specific genetic tests have failed to provide a diagnosis; (2) until the benefits of reporting incidental findings are established, we do not endorse the intentional clinical analysis of disease-associated genes other than those linked to the primary indication; and (3) clinicians should provide genetic counselling and obtain informed consent prior to undertaking clinical genome-wide sequencing. Counselling should include discussion of the limitations of testing, likelihood and implications of diagnosis and incidental findings, and the potential need for further analysis to facilitate clinical interpretation, including studies performed in a research setting. These recommendations will be routinely re-evaluated as knowledge of diagnostic and clinical utility of clinical genome-wide sequencing improves. While the document was developed to direct practice in Canada, the applicability of the statement is broader and will be of interest to clinicians and health jurisdictions internationally.

Discrepant HIV results resolved by human DNA testing.

A high-risk patient was informed of a positive HIV antibody/antigen test. However, follow-up samples taken 2-3 months later for HIV RNA and anti-HIV antibodies were negative. Human DNA testing confirmed that all samples were from this patient, excluding a sample mix-up. Laboratory investigations revealed a likely splash-over contamination event.

Exome sequencing identifies a novel variant in ACTC1 associated with familial atrial septal defect.

BACKGROUND: The genetics of congenital heart disease (CHD) remain incompletely understood. Exome sequencing has been successfully used to identify disease-causing mutations in familial disorders in which candidate gene analyses and linkage mapping have failed. METHODS: We studied a large family characterized by autosomal dominant isolated secundum atrial septal defect (ASD) (MIM No. 612794). Candidate gene resequencing and linkage analysis were uninformative. RESULTS: Whole-exome sequencing of 2 affected family members identified 44 rare shared variants, including a nonsynonymous mutation (c.532A>T, p.M178L, NM_005159.4) in alpha-cardiac actin (ACTC1). This mutation was absent from 1834 internal controls as well as from the 1000 Genomes and the Exome Sequencing Project (ESP) databases, but predictions regarding its effect on protein function were divergent. However, p.M178L was the only rare mutation segregating with disease in our family. CONCLUSIONS: Our results provide further evidence supporting a causative role for ACTC1 mutations in ASD. Massively parallel sequencing of the exome allows for the detection of novel rare variants causing CHD without the limitations of a candidate gene approach. When mutation prediction algorithms are not helpful, studies of familial disease can help distinguish rare pathologic mutations from benign variants. Consideration of the family history can lead to genetic insights into CHD.

Copy number variant analysis in CHM to detect duplications underlying choroideremia.

PURPOSE: To investigate the possibility of duplications or deletions within the CHM gene as a cause of choroideremia (CHM). MATERIALS AND METHODS: Eight males and one female subject were identified in whom clinical features were consistent with a clinical diagnosis of CHM. In all cases, sequencing of the coding region and adjacent intronic splice sites did not identify a mutation. In some cases, supplemental immunoblot analysis of protein from peripheral blood leukocytes with anti-REP-1 antibody confirmed absence of Rab escort protein-1, REP-1. A multiplex ligation-dependent probe amplification assay (MLPA) for the CHM gene was developed to test for deletions and duplications within the CHM gene. RESULTS: A duplication of exons 3-8 of the CHM gene (NM_000390.2) was identified in one case. DISCUSSION: While likely an uncommon event, duplication within the CHM gene could be considered as an explanation for CHM cases in which no mutation is found by sequence analysis.