Stacey Hume

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.

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.

Defining the Role of Laboratory Genetic Counselor

An increasing number of genetic counselors are moving into non-clinical roles, where their primary duties do not involve direct patient contact. According to the National Society of Genetic Counselors Professional Status Survey in 2010, 23% of counselors working in non-clinical roles identified laboratory or genetic testing as their primary area of work. Using a survey, we identified 43 genetic counselors who work predominately in laboratory settings. The two primary tasks performed by participants, include acting as a customer liaison (95%) and calling out test results (88%). Nineteen participants (44.2%) also reported spending a considerable amount of time signing reports. The most prevalent areas of job satisfaction were support from laboratory directors (76.8%), autonomy (76.7%), interactions with clinicians (69.7%) and interaction with other genetics counselors (67.5%). This is the first study specifically looking at the roles of laboratory genetic counselors, which is an expanding area of genetic counseling.

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

Abstract 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.

Toward optimal detection of the common prenatal aneuploidies by quantitative fluorescent-polymerase chain reaction: comparison of two commercial assays.

BACKGROUND/AIM To evaluate and compare the performance of the recently released Aneufast™ v2 (MolgentixSL) and QST*RplusV2 commercial assays (Gen-Probe), both designed for the quantitative fluorescent-polymerase chain reaction (PCR) detection of the common aneuploidies during pregnancy. METHODS A series of 160 consecutive fetal samples referred for rapid aneuploidy detection testing and an additional 25 samples enriched for the presence of an abnormality were selected for comparison. RESULTS To confidently rule out a chromosome abnormality, a second round of short tandem repeat typing was required for 14.1% (26) and 9.7% (18) of the specimens analyzed with Aneufast v2 and QST*RplusV2, respectively. Reflex testing was required for 7.6% (14) and 5.9% (11) of the specimens analyzed with respective assays to confidently rule out an autosomal trisomy. For the sex chromosomes, the difference in the amount of follow-up testing is greater between the assays, as a result of the inclusion in the initial PCR of the TAF9L paralogous marker in the QST*RplusV2 assay. CONCLUSIONS Overall, both assays performed similarly in the detection of aneuploidies. In this sample set, the QST*RplusV2 kit required less frequent reflex testing, which translates into shorter turnaround time and cost savings. The incorporation of the TAF9L paralogous sequence in the initial PCR is advantageous for diagnostic use.

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.

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

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.

Mitochondrial Replacement Therapy: The Road to the Clinic in Canada

Bartha Maria Knoppers, PhD; Arthur Leader, MD; Stacey Hume, PhD; Eric A. Shoubridge, PhD; Rosario Isasi, MPH; Forough Noohi, MSc; Ubaka Ogbogu, SJD; Vardit Ravitsky, PhD; Erika Kleiderman, LLB Centre of Genomics and Policy, Department of Human Genetics, McGill University, Montréal, QC Division of Reproductive Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON Department of Medical Genetics, University of Alberta, Edmonton, AB Department of Human Genetics, McGill University/Montréal Neurological Institute, Montréal, QC Department of Human Genetics, Institute for Bioethics and Health Policy, University of Miami Miller School of Medicine, Miami, FL Faculties of Law and Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB Bioethics Program, Department of Social and Preventive Medicine, School of Public Health, University of Montréal, Montréal, QC

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.