Case Report: Two pregnancies in a patient with maple syrup urine disease

Dr. Shailly Jain

Maple syrup urine disease (MSUD) is an autosomal recessive disorder with deficiency of branched-chain alpha-ketoacid dehydrogenase. Mainstay of treatment revolves around dietary restriction of branch chain amino acids. With improved dietary management, female patients are surviving into reproductive years. Management of pregnant patients with inborn errors of metabolism is being reported more frequently but remains challenging. Here, we describe the course of one patient with MSUD through two pregnancies.

The patient was diagnosed with MSUD at 7 days of age with leucine (Leu) greater than 3500 umol/L.  Although her MSUD control was stable for many years, pre-conception control was poor with Leu between 600-700 umol/L. During her first trimester of first pregnancy, she required multiple hospitalizations due to metabolic decompensations precipitated by nausea, vomiting as well as food insecurity. With strict dietary management which included increasing the metabolic protein along with gradual increases in natural protein, Leu levels stabilized. Specialized metabolic protocols were created for labour and delivery and nutrition post-partum. Patient did not have metabolic decompensations during or after delivery. Similar management was followed during her second pregnancy. During the second post-partum period, patient presented with a severe decompensation with encephalopathy. This was in conjunction with a urinary tract infection.  Leu reached a peak of 1140 umol/L

In summary, we present two pregnancies in one MSUD patient. We outline dietary management through different trimesters, labour and delivery, and post-partum. The case highlights potential high-risk periods for metabolic decompensation during pregnancy and post-partum in MSUD patients. Close monitoring remains crucial. Both children, oldest one at 3 years of age, are showing normal early development and growth.

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Putative association of a germline mutation in HYOU1 with nephrosclerosis and end-stage renal disease.

Dr. Karen Bedard

We have ascertained a large pedigree from an isolated area of Maritime Canada displaying an apparently autosomal dominant inheritance pattern of renal failure. Affected individuals display biochemical features of kidney dysfunction in early adulthood, progressing to end-stage renal disease (ESRD) in later decades. Genetic linkage analysis identified shared regions on chromosomes 1 and 11.  Whole exome sequencing identified a variant of uncertain significance in the gene encoding the stress response protein HYOU1.  The single nucleotide alteration that leads to a lysine to arginine substitution (K398R) is at a highly conserved, putative ubiquitination site. The variant was present in 14/14 individuals with both severe (clinical score of 3/5 or higher) and early onset (diagnosed before age 65) disease.  The clinical severity was significantly greater in the 5 individuals homozygous for this variant. Overexpression of either the wild-type or mutant HYOU1 in HEK293 tetracycline-inducible stable cell lines led to increased susceptibility to cell death induced by peroxide, but not by sodium chloride, glucose or urea.  The HYOU1 K398R variant protein degraded more slowly than wild-type HYOU1 protein.  Together, these results suggest that the K398R variant in HYOU1 may be involved in risk for chronic kidney disease culminating in ESRD. However, uncertainty remains in part because the variant is present in the general population with a frequency of 0.6%.  Further studies to be conducted include: additional fine mapping with stricter inclusion criteria; whole genome sequencing; and outreach to identify other familial cases in which to validate the findings.

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Profile of genetic variants identified in patients with connective tissue disorders using a targeted next generation sequencing platform

Dr. Catherine Fen Li

Catherine F. Li, Calgary Laboratory Services, University of Calgary, Calgary, AB, Canada; Elizabeth Wei, Center for Human Genetics, Cambridge, MA, USA; Clinton Baldwin, Milunsky, Center for Human Genetics, Cambridge, MA, USA; Jeff M. Milunsky, Center for Human Genetics, Cambridge, MA, USA

Genetic connective tissue disorders are a spectrum of systemic diseases with a high degree of clinical variability. Significant clinical overlap exists between many of these disorders. Therefore, differential diagnosis is difficult clinically, however, definitive diagnosis impacts the clinical management of these patients. We have implemented a cost-effective targeted next generation sequencing platform to simultaneously analyse 22 genes whose mutations result in connective tissue disorders. MLPA assays are also available for those genes that have known deletions reported as pathogenic variants. All discovered pathogenic variants were confirmed by Sanger sequencing. Uniform coverage exceeds 30X, with supplemental Sanger sequencing improving overall coverage to greater than 99% of targeted regions. In patients with a suspected dominant undiagnosed hereditary connective tissue disorder or those with a dominant family history of aneurysms, pathogenic variants were found in ACTA2, COL2A1, COL3A1, COL5A1, FBN2, SMAD3, TGFB2 and TGFBR1 genes; likely pathogenic variants were found in COL11A1, COL1A1, COL2A1, COL3A1, COL5A1, COL5A2, FBN2, NOTCH1, TGFB2 and TGFBR1 genes; variants of uncertain significance were found in 20 genes tested, but not in NTM and TGFB2 genes; likely benign variants were found in all 22 genes. MLPA assay did not identify any deletions. These findings support the notion that targeted next generation sequencing is the optimal cost-effective first line test for these patients.

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Experience with a targeted next generation sequencing platform in patients with autism spectrum disorders

Dr. Catherine Fen Li

Catherine F. Li, Calgary Laboratory Services, University of Calgary, Calgary, AB, Canada; Elizabeth Wei, Center for Human Genetics, Cambridge, MA, USA; Clinton Baldwin,Center for Human Genetics, Cambridge, MA, USA; Jeff M. Milunsky, Center for Human Genetics, Cambridge, MA, USA

Autism is a global neurodevelopmental disorder with a range of symptomatic variations. Epidemiological studies show an important role of genetic factors in autism and several susceptible regions and genes have been identified. Targeted next generation sequencing was performed on the 53 genes known to be causally associated with primarily non-syndromic autism spectrum disorders, using the Ion Torrent platform. Approximately 5% of the gene regions were sequenced individually using a fluorescent Sanger Sequencing technology. The variant detection rate of this panel exceeds 95%. In patients with a suspected autism spectrum disorder and having previous unrevealing genetic testing (normal SNP DNA microarray; negative Fragile X; negative single gene testing), variants of uncertain significance were found in DLGAP2, DPP6, MBD3, MBD4, NLGN3, NRXN1, NRXN2 and SLC9A9 genes, while likely benign variants were found in 49 genes tested, but not in DPP6, MBD3, NLGN3 and NRXN2 genes. However, neither pathogenic nor likely pathogenic variants were found in a small cohort. These findings again point to the low percentage detection found in non-syndromic single genes contributing to autism, even when assayed by a next generation sequencing panel.

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HER2 amplification status in breast cancer: assessing the impacts caused by implantation of ASCO/CAP (2013) guidelines in clinical setting.

Dr. Flechere Fortin

Background. Her2/neu protein is overexpressed in about 20% invasive breast carcinoma, which can be treated with Her2-targeted therapies. Assessing Her2 amplification status is a critical laboratory investigation, involving both classical (immunohistochemistry (IHC)) and molecular pathology tests (fluorescence in situ hybridization (FISH)] on paraffin-embedded tissues). In 2013, the American Society of Clinical Oncology (ASCO)/ College of American Pathologist (CAP) updated their guidelines for Her2 testing.

Objectives. Determine impact of these guidelines on IHC and FISH Her2 testing, and results reported to clinicians.

Design/Method. Literature review was done to retrieve papers presenting retrospective and prospective data. They were examined to determine changes in HER2 positive, equivocal, and negative rates, for both Her2 IHC and FISH tests, as well as changes in overall Her2 status. Results. Most studies report an increased percentage of cases having either positive or equivocal Her2 amplification status (difference range: 0.9 - 6.8% positive cases; 0.3 – 5.9% equivocal cases). Conversely, less negative cases are reported (difference: 1.9 – 7.6%).

Conclusions. Increasing the number of cases reporting positive and equivocal results will lead to an increased use of Her2-targeted therapies. As these are costly therapies, economic cost for the society will increase. Actually, clinical impact is still unknown for “Her2 equivocal” patients.

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PGxome: Leveraging the power of many for whole exome sequencing

Dr. Diane Allingham-Hawkins

PGxome is PreventionGenetics' whole exome sequencing (WES) test. PGxome is intended for health care providers searching for a diagnosis when the clinical phenotype is unclear or previous test results have been uninformative. PGxome utilizes the Agilent Clinical Research Exome v1 platform. Patient’s DNA is sequenced using Illumina’s NextSeq 500 paired end technology. Quality metrics include >97% of target bases covered at >20x, and mean coverage of >120x. The analysis pipeline uses BWA for alignment and GATK for variant calling. Variants are filtered and annotated using VarSeq ( Variant interpretations are distributed among 20 PhD geneticists with specific expertise, thereby leveraging the collective knowledge of our PhD team.  Clinical indications for the PGxome WES assay included neurological disorders, including developmental delay and intellectual disability (51%), multiple congenital anomalies (23%), and neuromuscular disorders (16%). A definite molecular diagnosis (i.e. pathogenic/likely pathogenic variant) was obtained in 37% of cases, 44% had a variant(s) of uncertain significance which may explain the phenotype, and 19% were negative. Patients have the option to opt-out of secondary findings in the ACMG list of genes (Kalia et al., 2016. PMID: 27854360) and opt-in for other secondary findings and carrier status. No ACMG secondary findings were observed, but other secondary findings were observed in 46% of cases that opted in. Carrier status for ≥ 1 recessive disorders was reported in 100% of cases that opted in. In summary, PGxome leverages the power of many to deliver a definite or potential diagnosis in 81% of WES cases reported to date.

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Sexual dimorphism in SMAD3 mutation carriers: implications for management derived from a large family.

Dr. Julie Richer

Sharan Goobie, Maritime Medical Genetics, IWK Health Centre, Dalhousie University, NS; Hussein Al-Amodi, Division of Cardiac Surgery, Western University, London, ON; Michael W. Chu, Division of Cardiac Surgery, Western University, London, ON; Bart Loeys, Center of Medical Genetics, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium

SMAD3 mutation carriers are at-risk for aortic dissection and for arterial aneurysms/dissections from brain to pelvis.  Based on prior suggestions in the literature on gender differences in thoracic aortic aneurysm/dissection, we investigated whether there is a sexual dimorphism regarding risk of vascular events in SMAD3 mutation carriers.  In a large SMAD3 pedigree, we included mutation carrying individuals who were either (1) >40 years or (2) had aneurysm/vascular dissection/intervention.  We identified 22 mutation carriers and categorized them as unaffected or affected.  We subcategorized affected individuals, between (1) aneurysm/dissection excluding the aortic root/ascending aorta versus (2) aneurysm/dissection with disease including the aortic root/ascending aorta (TAAD).  In females, we identified 7 as unaffected whereas 6 were affected. Of these 6 affected females, 5 had arterial aneurysms without TAAD.  All 9 males were affected.  Eight of the males had TAAD and one had arterial aneurysm without TAAD.  In addition, four other males in the family presented with sudden death and/or aortic dissection, but were not included because the precise nature and location of their aneurysm was undocumented.  Our findings are in line with previous human SMAD3 family observations (VandeLaar et al, JMG, 2012; VanderLinde et al, JVS, 2013) and mouse data (van der Pluijm I et al, EBioMedicine, 2016).

In summary, clinicians caring for individuals with a personal/family history of SMAD3 mutations need to know that (1) the absolute risk of aneurysms is higher in males and (2) the risk of vascular events in the presence of a normal echocardiogram is higher in females.

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Formation of the Medical Genetics recommendations for Choosing Wisely Canada

Dr. Elaine Goh

Andrea Guerin, Kingston General Hospital, Department of Pediatrics, Division of Medical Genetics, Kingston, ON; Joanna Lazier, Department of Medical Genetics, University of Alberta, Edmonton, AB; Sharan Goobie, Medical Genetics, Department of Pediatrics, IWK Health Centre, Halifax, NS; Tanya N. Nelson, Department of Pathology and Laboratory Medicine, University of British Columbia, and Department of Pathology and Laboratory Medicine, BC Children's and BC Women's Hospitals, Vancouver, BC; Ron Agatep, Diagnostic Services Manitoba, Clinical Biochemistry and Genetics, Winnipeg, MB, Canada; and Victoria Siu, Division of Medical Genetics, Department of Pediatrics, London Health Sciences Centre, Western University, London, ON; Karen Y. Niederhoffer, Department of Medical Genetics, University of Alberta, Edmonton, AB; Julie Richer, Children’s Hospital of Eastern Ontario, Department of Medical Genetics, Ottawa, ON

Choosing Wisely Canada is a national campaign organized to promote a discussion about unnecessary use of tests, procedures and treatments in various specialties. Through the support and engagement of the Canadian College of Medical Geneticists (CCMG) membership, the Ethics, Education, and Public Policy Committee (E2P2) undertook an iterative process to help formulate the five things physicians and patients should question in the field of Medical Genetics. The E2P2 committee generated a potential list of statements in the summer of 2015 and a pilot survey was distributed during the CCMG annual conference general meeting in September 2015. Based on the feedback, the E2P2 modified the statements and added to the list. This list was then distributed to the entire membership via an electronic survey in March of 2016 asking for members to rank their top 5 statements. The answers were weighted and the top 5 statements were selected. Members of the E2P2 committee then reviewed the literature and generated the rationale for the statements which were then presented at the CCMG annual meeting in 2016 and subsequently made available for comments by the membership. Feedback and revisions were considered by the E2P2 committee prior to the submission of the final version of the statements focusing on non-invasive prenatal testing, direct to consumer genetic testing, chromosome analysis, whole exome sequencing and carrier testing in children. These statements will be released to the Choosing Wisely Canada organization. The goal is to provide greater public awareness of these issues related to Medical Genetics.  

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Dystrophin deletions: two cases of incidental, medically actionable results on microarray.

Dr. Victoria Siu

Background: Chromosome microarray(CMA) is a first-tier investigation for developmental delay, yielding a definitive etiology in about 10% of cases. The finding of clinically significant genomic imbalance may also be an incidental finding. Deletions in the dystrophin gene may result in Duchenne or Becker muscular dystrophy, or dilated cardiomyopathy. We present two unrelated families with the incidental finding of a dystrophin deletion.

Case 1. A young woman with Williams syndrome presented with gestational diabetes and hypertension at 33 weeks gestation. Her partner had a history of learning difficulties. CMA on their infant son unexpectedly revealed a deletion of exons 49 to 51 in the dystrophin gene. Microarray in mother confirmed a 7q11.2 microdeletion and heterozygous state for the dystrophin deletion which was inherited from her unaffected father.

Case 2. Two brothers with developmental delay had a maternally inherited 11p14.3 deletion on CMA, which did not explain their presentation. Mother was incidentally also found to be a carrier for a deletion of exons 49 to 51 in the dystrophin gene, and was a previously known carrier for X-linked ornithine transcarbamylase (OTC) deficiency. The grandfather had both OTC deficiency and the dystrophin deletion with normal echocardiogram and muscle function. A maternal half-brother to the proband had only the dystrophin deletion, as a result of a recombination event.

Genetic counselling issues: In both families, the finding of the dystrophin deletion raised the issue as to appropriate investigation and surveillance of the children.

Conclusion: "Clinically actionable" incidental findings can present management dilemmas in asymptomatic individuals.

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H3.1 K36 mutation in a congenital-onset soft tissue neoplasm

Dr. Sarah Sawyer

Kristin D. Kernohan, Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa; David Grynspan, Department of Pathology, Children’s Hospital of Eastern Ontario;  Raveena Ramphal, Division of Hematology/Oncology, Children’s Hospital of Eastern Ontario; Elizabeth Nizalik, Department of Pathology, Children’s Hospital of Eastern Ontario; Jiannis Ragoussis, McGill University and Genome Quebec Innovation Centre, Montreal; Care4Rare Canada Consortium, Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa; Nada Jabado, Department of Pediatrics, McGill University and McGill University Health Centre Research Institute, Department of Human Genetics, McGill University;  Kym M. Boycott, Children’s Hospital of Eastern Ontario Research Institute, Department of Genetics; Jacek Majewski, Department of Human Genetics, McGill University, Genome Quebec Innovation Centre; Sarah L. Sawyer, Children’s Hospital of Eastern Ontario Research Institute, Department of Genetics

Background: We describe a patient who presented with a congenital soft tissue lesion at birth, initially diagnosed as infantile fibromatosis at 15 days of age based on pathology from a biopsy. Unusually, the mass demonstrated malignant progression leading to death at 20 months of age. Biological progression to malignancy is not known to occur in fibromatosis, and fibrosarcoma is not known to progress from a benign lesion.

Objective: Given the unclear diagnosis based on pathology and history, we attempted to reach a diagnosis using molecular tecniques.

Methods: Whole-exome sequencing of the tumor and blood in our patient was undertaken.

Results: Whole-exome sequencing of the tumor identified a driver mutation in histone 3.1 (H3.1) at lysine (K)36.

Conclusion: Our findings support the link between oncohistones and infantile soft tissue tumours and provide additional evidence for the oncogenic effects of p.K36M in H3 variants. Whole exome sequencing of rare pediatric tumors provides an avenue for diagnosis when traditional pathology techniques are unable to provide a diagnosis to patients.

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Clinical validation of PhenoVar as a diagnostic tool in the interpretation of exome sequencing for genetic disorders

Mrs. Fanny Thuriot

Fanny Thuriot, Université de Sherbrooke, Department of Pediatrics; Caroline Buote, Université de Sherbrooke, Department of Pediatrics; Laura-Dempsey-Nunez, Université de Sherbrooke, Department of Pediatrics; Marie Edmont, Université de Sherbrooke, Department of Pediatrics; Elaine Gravel, Université de Sherbrooke, Department of Pediatrics; Yannis Trakadis, Université McGill, Department of Medical Genetics; Bruno Marand, Université de Sherbrooke, Department of Pediatrics; Valérie Désilets, Université de Sherbrooke, Department of Pediatrics; Sébastien Chénier, Université de Sherbrooke, Department of Pediatrics; Pierre-Étienne Jacques, Université de Sherbrooke, Department of Biology; Sébastien Lévesque, Université de Sherbrooke, Department of Pediatrics

Background. Although phenotypic heterogeneity of genetic diseases limits the identification of the causal gene by conventional approaches, whole-exome sequencing (WES) has been instrumental in reaching a diagnosis in several cases recently.  However, the number of variants identified by WES poses a significant challenge for clinical interpretation. To address this, we previously developed PhenoVar, a phenotype-driven diagnostic tool. Here, we report its clinical validation.

Objectives. We sought to determine the yield of WES in patients with suspected genetic disorders and compare PhenoVar with a manual analysis and Exomiser, another phenotype-driven tool.

Design/Methods. WES was performed on a cohort of 51 patients presenting dysmorphisms with or without neurodevelopmental disorders of undetermined etiology. For each patient, we used PhenoVar, which analysed filtered variants and phenotypic traits, and returned a prioritized list of potential diagnoses. This list was reviewed by a clinical geneticist, who selected canadidate variants to be confirmed by segregation analysis. Conventional bioinformatics analysis was performed in parallel and candidate variations were reviewed afterwards by the same clinical geneticist to identify any potential diagnosis missed by PhenoVar.

Results. A molecular diagnosis was identified in 19 patients with both PhenoVar and the conventional bioinformatics analysis; a yield of 37%. PhenoVar reduced by half the number of potential diagnoses to be reviewed and outperformed Exomiser, with 17 diagnoses ranking in the top 10 compared to 11 diagnoses for the latter.

Conclusions. PhenoVar prioritizes variants obtained from WES and provides a reliable and efficient diagnostic aid to clinical geneticists.

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Naxos disease in French-Canadian families with a novel founder mutation

Dr. Tania Cruz Marino

Tania Cruz Marino, Department of Medical Biology, CIUSSS; Bruno Maranda, Department of Pediatrics, division of Medical Genetics, Centre Hospitalier Universitaire de Sherbrooke and Université de Sherbrooke, Sherbrooke; Josianne Leblanc, Department of Medical Biology, CIUSSS; Annabelle Pratte, Department of Medical Biology, CIUSSS; Melinda Barabas, Department of Cardiology, CIUSSS; Audrey Dupéré, Department of Dermatology, CIUSSS;  Sébastien Lévesque, Department of Pediatrics, division of Medical Genetics, Centre Hospitalier Universitaire de Sherbrooke and Université de Sherbrooke

Background. Naxos disease (ND) is an autosomal recessive condition characterized by arrhythmogenic right ventricular cardiomyopathy (ARVC), palmoplantar keratoderma (PPK) and peculiar woolly hair. Objective. To describe the phenotype associated with a novel JUP gene variant identified in French-Canadian individuals. Method. Through exome sequencing we identified the JUP homozygous variant c.902A>G (p.Glu301Gly) in one patient presenting features of ND. Further potential ND cases were ascertained through clinics of dermatology and cardiology and subsequently referred to the medical genetics clinics in Chicoutimi, Quebec City, and Sherbrooke. Index cases were tested independently at different clinical laboratories. The demographic and clinical data were obtained from the medical records upon the identification of a JUP mutation. Results. Seven unrelated cases ranging in age at last follow-up from 12 to 77 years, shared the same homozygous mutation c.902A>G (p.Glu301Gly), pointing to a founder effect in Caucasians of French-Canadian descent. The youngest patients were ascertained through dermatology clinics due to the woolly hair and the PPK, features that were present since childhood in all the individuals but had not gained medical attention (PPK was mild in most of them). Four out of the five adults were diagnosed with ARVC at the age of 28 or above, suggesting an ARVC phenotype not completely penetrant by adolescence. Conclusions. ND should be considered in those cases with cutaneous and hair findings even if they do not fulfill the criteria for ARVC by adolescence, and/or in those cases with an ARVC without prominent cutaneous findings, especially if of French-Canadian descent.

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17q25.3 duplication in a patient with psychosis and autism spectrum disorder, further support for this novel psychiatric illness susceptibility locus

Ms Kellie MacDonald

Background. Prior linkage evidence and co-segregation of translocation t(9;17)(q33.2;q25.3) with psychiatric disease suggest that 17q25.3 is a psychiatric illness susceptibility locus. A recent publication using data from prior GWAS studies revealed NPTX1 and ENDOV, in this region, to be associated with bipolar disease and schizophrenia, respectively (Bipolar Disord 2015;17:205-211).

Objectives. We present a patient investigated for psychosis and autism spectrum disorder (ASD) carrying a copy number variant (CNV) at this locus.

Results. aCGH revealed a 500kb duplication at 17q25.3, encompassing NPTX1, RPTOR and containing a portion of ENDOV. This CNV was inherited from the proband’s mother who appears to have mild cognitive issues and is awaiting formal neuropsychiatric evaluation.

Discussion. We hypothesize that this CNV alters the expression of NPTX1, a gene implicated in synaptic plasticity, and ENDOV, an endoribonuclease involved in DNA replication and RNA transcription, contributing to our patient’s phenotype. The proband’s father is unaffected. However, given the significant paternal family history of psychiatric illness, we suspect a paternally inherited second deleterious genetic hit, which would explain the more severe phenotype of the proband. Ongoing investigations, including sequencing the paternal allele of the above genes for point mutations, functional assays, and linkage analysis on the father’s family will likely shed more light in the genetic contributors of psychiatric disease in this family.

Conclusions. This case supports 17q25.3 as a candidate region for neuropsychiatric illness and illustrates the complexity of genetic evaluation in patients with psychiatric disease, exemplifying how they can be approached at a clinical level.

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Inherited Tatton-Brown-Rahman syndrome due to novel DNMT3A germline mutations in French Canadian and Old Order Amish families

Dr. Tania Cruz Marino

Tania Cruz Marino, Department of Medical Biology, CIUSSS; Baozhong Xin, DDC Clinic Center for Special Needs Children, Middlefield, OH; Julia Szekely, DDC Clinic Center for Special Needs Children, Middlefield, OH; Josianne Leblanc, Department of Medical Biology, CIUSSS; Karen Cechner, DDC Clinic Center for Special Needs Children, Middlefield, OH; Valerie Sency, DDC Clinic Center for Special Needs Children, Middlefield, OH; Chris Wensel, DDC Clinic Center for Special Needs Children, Middlefield, OH; Melinda Barabas, Department of Cardiology, CIUSSS; Valérie Therriault, Manicouagan Familial Medicine Unit, CISSS; Heng Wang, Department of Pediatrics, Rainbow Babies & Children’s Hospital, Department of Molecular Cardiology, DDC Clinic Center for Special Needs Children, Middlefield, OH

Background. Tatton-Brown-Rahman syndrome (TBRS) is an autosomal dominant condition characterized by tall stature, intellectual disability and distinctive dysmorphic features. It was recently described in 13 isolated cases with de novo mutations in the DNMT3A gene. Objective. To describe the phenotype associated with two novel DNMT3A germline mutations identified in two sib-ships: a French-Canadian family in Canada and an Old Older Amish family in America. Method. We performed clinical exome sequencing and identified two mutations in the DNMT3A gene, a c.2296_2297delAA (p.Lys766Glufs*15) small deletion in the French-Canadian family and a c.2312G>A (p.Arg771Gln) missense mutation in the Amish family. Sanger sequencing was performed to confirm the DNMT3A mutations and to analyse the targeted mutations sites for family members. The demographic and clinical data were obtained from the medical records upon the identification of a DNMT3A mutation. Results. We identified six cases of inherited TBRS caused by two novel DNMT3A germline mutations. Despite their different ethnic background, affected individuals (ranging in age from 11 to 54 years) shared the characteristic features of TBRS, including distinctive dysmorphic features, increased height, and intellectual disability. Variable additional features were identified as well. Parental DNA analysis revealed that the French-Canadian mutation was absent in three maternal tissues (the father was deceased) and the Amish mutation was inherited from the healthy mosaic father. Conclusions. This study reflects the first North American families with inherited TBRS and expands its phenotypic spectrum. We recommend considering the TBRS among the differential diagnosis of individuals with tall stature and intellectual deficiency.

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WDR26 haploinsufficiency causes a recognizable syndrome of intellectual disability, seizures, abnormal gait, and distinctive facial features

Dr. Matt Deardorff

Background.  Using exome sequencing, we identified de novo heterozygous loss of function WDR26 gene variants in individuals with intellectual disability.

Objectives.  The purpose of this study was to better characterize the clinical spectrum and specific features that result from pathogenic WDR26 variants.

Design/Method. We utilized a network of collaborators and GeneMatcher to identify individuals with WDR26 variants, carefully assessed clinical information and correlated findings with cellular and molecular data.

Results. We identified eleven individuals with de novo pathogenic variants in WDR26. Eight carry loss-of-function mutations and three harbor missense substitutions. All have intellectual disability with delayed speech, a history of seizures and abnormal gaits. Common facial features include a prominent maxilla, widely spaced teeth, and a broad nasal tip that together comprise a recognizable facial phenotype. These features are consistent with those seen with heterozygous chromosome 1q41q42 microdeletions, suggesting that haploinsufficiency of WDR26 contributes to the pathology. To support this, WDR26 loss-of-function single nucleotide variants lead to nonsense-mediated decay with subsequent reduction of RNA expression and protein levels. Finally, structural modeling of missense mutations also supports haploinsufficiency as a mechanism of pathogenicity by disrupting conserved domains of the protein.

Conclusions. Haploinsufficiency of WDR26 causes an emerging clinical syndrome of intellectual disability, seizures, and distinctive facial features.

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Algorithmic analysis of all possible mitochondrial tRNA mutations

Dr. Neal Sondheimer

Novel or rare mutations in mitochondrial tRNA sequence are frequently observed after sequencing of mitochondrial DNA in patients where mitochondrial disease is suspected. Determining whether these mutations are potentially pathogenic is crucial, but biochemical confirmation can be challenging, requiring skin, muscle or other specimens and research lab capacity. We have used available databases of polymorphic and pathogenic mutations, alignment between diverse tRNAs, structural information and comparative genomics to predict the impact of all possible single-base mutations and deletions. A reanalysis of all confirmed pathogenic and polymorphic variants showed that this algorithmic approach had an optimal sensitivity and specificity of 70% at a single cutoff value. This exceeded the sensitivity and specificity of currently available systems based upon machine-learning or interspecific alignment alone. The algorithm also correctly predicted the impact of uncommon variants such as mutations within the anti-codon. The use of the system, in conjunction with studies of maternal relatives and level of heteroplasmy, will aid physicians in diagnosis of mitochondrial disorders.

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Identification of a DNA methylation signature of PTEN loss in prostate cancer

Dr. Erfan Aref-Eshghi

Erfan Aref-Eshghi, Department of Pathology and Laboratory Medicine, Western University; Laila C. Shcenkel, Department of Pathology and Laboratory Medicine, Western University; Peter Ainsworth, Department of Pathology and Laboratory Medicine, Western University Jean-Claude Cutz, Department of Pathology and Molecular Medicine, McMaster University; Bekim Sadikovic,Department of Pathology and Laboratory Medicine, Western University

Background: Loss of function mutations in Phosphatase and Tensin homolog (PTEN) are associated with a poor prognosis in prostate cancer; however, molecular consequences of loss of PTEN are not well understood.

Objectives: Using a genome-wide approach, we aimed to describe the DNA methylation profile of human prostate cancer tissues, and to identify the specific “epi-signature” associated with loss of PTEN gene expression.

Methods: The PTEN status of macrodisected FFPE tissues (30 tumors and 16 normal; prostatectomy and matched needle biopsy) was assessed by immunohistochemistry and FISH analysis. Genome-wide methylation analysis was conducted using Illumina infinium methylation 450k arrays. A moderated T-test and a multivariable linear model analysis compared PTEN loss with genome-wide DNA methylation. Hierarchical clustering and gene enrichment analysis were conducted, and validated algorithms were used to identify regional methylation changes.

Results: Comparison of the normal and tumor tissues identified over 250 abnormally methylated regions including promoter hypermethylation of tumor suppressor genes such as APC and the Protocadherin cluster. The level of PTEN loss was associated with 28 defectively methylated regions including hypomethylation of the PTK6 promoter (associated with cancer progression), and genes involved in the regulation of cell growth, differentiation and senescence including AGPAT4, FGFR2, ISL2, and DIP2C.

Conclusion: We identified a DNA methylation epi-signature of PTEN loss in human prostate cancer involving genes and pathways responsible for cancer progression. Follow up study of a larger clinically-defined prostate cancer cohort is warranted to confirm these findings and assess clinical prognostic utility of these findings.

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Proposed guidelines for utilization of the comprehensive assessment of sequence and copy number mutations in Epilepsy-related disorders in Ontario

Dr. Bekim Sadikovic

Bekim Sadikovic, Schulich school of Medicine and Dentistry, Western University, Molecular Diagnostic Laboratory, Victoria Hospital, London Health Science Centre; Narayan Prasad, Schulich school of Medicine and Dentistry, Western University;  Jennifer Kerkhof, Molecular Diagnostic Laboratory, Victoria Hospital, London Health Science Centre;  Alan Stuart, Molecular Diagnostic Laboratory, Victoria Hospital, London Health Science Centre; Erfan Aref-Eshghi, Schulich school of Medicine and Dentistry, Western University; Peter Ainsworth, Schulich school of Medicine and Dentistry, Western University; Hanxin Lin, Schulich school of Medicine and Dentistry, Western University; Sharan Goobie,Schulich school of Medicine and Dentistry, Western University

The overall prevalence of epilepsy is 5/1000 persons, with an estimated 200,000 Canadians affected by this condition. Approximately 55% of epilepsies have a genetic component, characterization of which can be used to improve optimization of medical management, use of targeted therapeutics, avoidance of adverse drug effects, prognosis, and genetic counseling. The Ontario Epilepsy Genetic Testing Criteria (OEGTC) Working Group has proposed Criteria for Genetic Testing Related to Epilepsy, which describes nine clinical criteria for genetic testing, and outlines a list of specific clinical and diagnostic prerequisites for genetic analysis. Genetic testing ranges from monogenic to gene panel and/or clinical exome sequencing. While most targeted monogenic testing approaches provide high sensitivity and specificity “rule-out” assays, large gene panels and exome sequencing generally result in reduced analytical sensitivity.

Based on the OEGTC criteria, and incorporating advice from clinical geneticists and epileptologists, we have developed a “rule-out” epilepsy panel for the accurate assessment of both sequence and copy number changes in 69 genes associated with major epilepsy syndromes. These have been grouped into four major clinical categories: 1. genes with therapeutic implications; 2. infantile and childhood epileptic encephalopathies; 3. progressive myoclonic epilepsy; and 4. other genetic syndromes with epilepsy as a major presentation.

Here we present an approach combining the utilization of OEGTC guidelines for assessment of epilepsy with a comprehensive clinical test for assessment of monogenic and polygenic epileptic conditions, including technical, validation, and early clinical testing data. This approach enables guidance and regulation of appropriate utilization of epilepsy genetic testing.

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Mutations in cellular mechanotransduction genes are associated with idiopathic scoliosis. A genetics and cellular based evidence study

Ms Niaz Oliazadeh

Background: Idiopathic Scoliosis (IS) is a complex pediatric disease of unknown cause, defined by abnormal spinal curvatures and a genetic component. Cilium is a membranous sensory organelle that senses the surrounding mechanical stimuli in osteoblasts and accordingly affects important cellular-molecular aspects of the bone tissue.

Objectives: To investigate cilia abnormalities in structural, functional and genetic levels in IS patients.

Design/Methods: The morphology of cilia was studied by confocal microscopy in stained osteoblasts from 4 patients and 4 controls. The same group of IS and control osteoblasts were then subjected to defined fluid flow shear stress. Changes in gene expression of several markers in response to mechanical stimulation were quantified to assess the functional capacity of cilia. We also performed a whole exome sequencing (WES) on 73 IS and 70 controls to investigate if the accumulation of rare variants in ciliary genes could be causal to IS.

Results: We found significant elongation in cilia of IS osteoblasts compared to controls. Our results also showed a different gene expression profile in IS cells with longer cilia in response to mechanical stimulation. This impaired gene expression response mostly affected bone formation factors and WNT pathway players. Our SKAT-O analysis of WES results showed enrichment for rare variants in genes that are involved in cellular mechanotransduction (p-value≤0.01).  

Conclusions: Together these data suggest that IS bone cells have a differential sensation of mechanical stimulation. This impairment has a genetic component and can affect important signalling pathways and bone formation process in response to mechanical loading.

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Discrepant genotype-phenotype correlation in two siblings with hypophosphatasia

Dr. Alan O'Brien

Background. Hypophosphatasia (HPP)  is a rare genetic disorder causing defective bone mineralization, due to a deficit of tissue-nonspecific alkaline phosphatase (TNSALP), encoded by the ALPL gene. The clinical severity and age of onset vary greatly and are thought to reflect residual enzyme activity. There is, however, a more limited intra-familial variation, and affected siblings sharing the same genotype tend to be similarly affected.

Objectives. We report on two siblings that are followed in our clinic. Both are compound heterozygotes for ALPL mutations: c.571G>A in exon 6 and c.1553_1568del16 in exon 12. However, they are both differentially affected, with the oldest having findings typical of childhood type HPP and the youngest having almost exclusively dental findings, more in keeping with odontohypophosphatasia.

Design/Method. We compare the phenotype of the two siblings by reviewing the clinical findings, imaging and laboratory results.

Results. Sibling 1, the oldest, presented between one and two years of age with loss of four primary teeth, an ankle fracture and x-ray anomalies including decreased bone density and rachitic changes. His alkaline phosphatase level was 20 U/L. Sibling 2 was diagnosed prenatally through molecular testing, and presented clinically after one year of age with tooth decay and hypoplastic, abnormally shaped teeth. Her ALP level was 23 U/L. She has not had any skeletal complications, including fractures.

Conclusion. Our report adds to the available descriptions of genotype-phenotype correlations in HPP and increases awareness to the phenomenon of intra-familial phenotypic discrepancy.

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Explaining the 'missing heritability' in schizophrenia: preliminary evidence using a large number of exomes

Ms Vanessa Fulginiti

Background: Schizophrenia (SCZ) is a highly heritable (80%) disorder. The multifactorial threshold hypothesis proposes that SCZ occurs when one’s exposure to deleterious genetic and/or environmental factors increases past a 'disease threshold'. Different whole exome sequencing (WES) studies have found an increased de novo mutation (DNM) rate in schizophrenic patients.

Objectives: Our "network hypothesis" is that affected individuals accumulate inherited and de novo genetic variants at random within a set of candidate genes, which cooperatively increase one's predisposition to SCZ. The probability to be affected is mainly influenced by the total number of mutations that each parent carries on this network.

Design/Method: SCZ candidate genes were selected if reported to bear DNM in schizophrenic patients in at least two original WES independent publications. Control genes associated with hypertrophic cardiomyopathy (HC) were also selected. WES data from 598 SCZ trios and an independent SCZ case control dataset (n=5090), accessed via dbGaP, were analyzed for rare functional variants in these genes.

Results: Fourteen SCZ candidate genes and fourteen HC genes were identified. Paired t-test analysis of the trios showed that the mutation load of probands was increased compared to the average mutation load of parents in the SCZ "network" (p = 0.041), but not in the HC genes (p = 0.212). All SCZ "network" variants in probands were inherited. Similar results were found when using the case control dataset (p = 0.012 and p = 0.389, respectively).

Conclusions: We validate fourteen SCZ candidate genes and provide preliminary evidence to support our “network hypothesis”.

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Chromosome 2q duplication: A case report on a recombinant chromosome 2 with pure 2q24.3q36.1 duplication.

Dr. Tchakarska Gany

Background. Approximately 30 cases of pure 2q duplication were described to date. Inherited cases resulted from the recombination or abnormal segregation of intra- and interchromosomal insertions in a balanced carrier parent. Most reported pure 2q duplications cluster within 2q33q37 or 2q11.1q21 regions and present distinct phenotypic features. A cleft lip/palate locus is present at band 2q13.

Objectives. To describe the cytogenetics findings in a female presenting with Pierre Robin sequence, broad, depressed nasal bridge, long philtrum, limb contractures and axial hypotonia and to summarize the literature on phenotype-genotype correlations in cases with pure 2q duplication.

Design/Method. Chromosomal microarray (CMA) analysis was performed on the proband using the CGXTM-HD 4x180K array (Agilent). G-banded karyotype analysis of the proband and her parents was performed following standard procedures.

Results. CMA analysis detected a 64 Mb duplication (274 OMIM genes) of the q24.3q36.1 segment. G-banded karyotype analysis revealed the presence of an intra-arm inverted insertion in 2q in the father (46,XY,ins(2)(q13q36.1q24.3)). The proband’s karyotype findings are consistent with a recombinant chromosome 2 with the duplicated segment inverted and inserted at band 2q13.<br style="mso-data-placement:same-cell;">

Conclusions. Most of the observed features in the proband are consistent with those described in association with the duplication of 2q33q37 material. Cleft lip/palate and Pierre Robin sequence are associated with the 2q13 region. Therefore, we suggest that the insertion breakpoint in 2q13 disrupted this critical region and accounts for the presence of Pierre Robin sequence in our patient. This is the largest 2q pure duplication reported to date in a liverborn.

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Cohen Syndrome: a mitochondrial masquerader?

Dr. Karen Niederhoffer

Karen Y. Niederhoffer, Department of Medical Genetics, University of Alberta; Gabriella Horvath, Biocehmical Diseases, Pediatrics, University of British Columbia, Adult Metabolic Disease Clinic, Vancouver General Hospital

Background: Cohen syndrome is caused by mutations in VPS13B. It can be challenging to diagnose given variable presentation.

Objectives: To appreciate the overlap of Cohen syndrome with mitochondrial disease. To appreciate the impact of diagnostic momentum.

Method: Case report of a patient with mutation confirmed Cohen syndrome.

Results: The proband presented with hypotonia and delay. She later developed retinitis pigmentosa, myopia, bilateral cataracts, decreased exercise tolerance, and type 2 diabetes.  Given multi-organ involvement in the context of hypotonia/weakness and delay, consideration was made for Kearns-Sayre syndrome or other mitochondrial disease.

Over the course of several decades, she was seen by multiple specialties including biochemical diseases, genetics, ophthalmology, cardiology, hematology, and endocrinology. Investigations, including muscle biopsy, ECHO, ECG, abdominal ultrasound, acylcarnitine profile, urine organic acids, lactate, CK, LFTs, and renal tests were normal or nonspecific. mtDNA sequencing and deletion testing were unremarkable. CPEO panels identified a heterozygous variant in PCK2 and several variants of unclear significance.

Given an unconfirmed diagnosis, microarray was performed in adulthood. This identified a 139.9 Kb loss at chr8:100,128,046 - 100,267,919 resulting in partial deletion of VPS13B. Sequencing identified a pathogenic mutation, c.11595del p.Arg3865Serfs*13, and confirmed deletion of exons 7-17.

Conclusions: Despite a well described phenotype, Cohen syndrome can demonstrate wide variability, resulting in delay to diagnosis. Mitochondrial disorders also display phenotypic heterogeneity.  Despite close medical surveillance, our patient went misdiagnosed for over 30 years. As new technologies become available, re-visiting unconfirmed diagnoses provides the potential for correct reclassification and change in patient management.

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Regulation of Matrix Gla Protein Gene Expression by an Intronic Sequence

Mr. Jobran Mohammed Moshi

Introduction: Matrix Gla protein (MGP) is a potent inhibitor of extracellular matrix (ECM) mineralization. MGP‐deficiency in humans leads to Keutel syndrome, a rare genetic disease, hallmarked by abnormal soft tissue calcification. How Mgp gene is regulated in various cell types is still not well-understood. In a recent study, SOX9, a chondrogenic transcription factor, has been shown to interact with several regions in the promoter as well as intronic sequences of Mgp gene. In the current study, we investigated the effect of a SOX9 binding intronic sequence on the Mgp promoter activity.

Methods: We amplified the rat intronic sequence earlier shown to bind SOX9. This sequence was inserted in front of the 2 kb mouse proximal promoter driving the firefly luciferase gene. This construct was used to transfect ATDC5 chondrogenic cells or primary vascular smooth muscle cells (VSMCs). The transfected cells were grown for 48 hrs and luciferase activity was measured.

Results: Transfection of ATDC5 cells by the pMgp-Luc construct induced luciferase activity in ATDC5 cells. Unexpectedly, when the SOX9 binding intronic sequence was added upstream of the promoter, there was a significant downregulation of the luciferase activity in the transfected cells. Co-transfection with a Sox9 expression vector failed to increase the luciferase activity. We next split the intronic sequence into 3 segments and cloned in front of the 2.0 kb Mgp proximal promoter driving the luciferase gene. Interestingly, we found that all 3 fragments induced the luciferase activity. We identified two CCCTC sequences within the intronic sequence, which are known to bind a DNA interacting protein CTCF with promoter silencing activity. Deletion of one CTCF binding site in the intronic sequence was sufficient to inhibit the repressor activity.

Conclusion: Our study shows the presence of a regulatory element in the intron 1 of Mgp gene. We identified the presence of two CCCTC binding sites within this intronic sequence, which has been shown to bind SOX9. Selective ablation of the upstream CCCTC sequence leads to an upregulation promoter activity suggesting that the intronic sequence confers important epigenetic regulation of the Mgp promoter.

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Mutations In MYBPC1 Gene Are Associated With Myogenic Tremor

Dr. Baiba Lace

Myosin Binding Protein-C comprises a family of accessory proteins in striated muscles, and includes three isoforms: cardiac, fast skeletal, and slow skeletal. Reports have identified mutations in the MYBPC1 gene underlying the development of severe and lethal forms of distal arthrogryposis. We describe the presence of a novel missense mutation in MYBPC1 in three generations of a family presenting with muscle weakness, skeletal deformities, tremor and spinal rigidity. Exome sequencing analysis on three of the affected members, as well as one unaffected member, followed by a custom data analysis pipeline was performed. Our data indicated the presence of a novel heterozygous variation in the MYBPC1 gene (NM_002465.3, c.742G>A, p.E248K), which segregated with the disease. The E248K mutation is located in the unique M-motif of sMyBP-C, which is constitutively expressed in all splice variants. To assess the effects of the E248K mutation on the ability of the NH2-terminus of sMyBP-C to directly bind actin or myosin, we performed blot overlay assays and observed that mutant protein carrying the E248K substitution exhibited ~3.5 fold increased binding to heavy meromyosin compared to wild type protein. We hypothesize that the specific tremor seen in this family could be a “myogenic” tremor related to the increased heavy meromyosin binding of the mutant sMyBP-C, and that this increased binding could lead to cycling force production as the basis of the tremor. 

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Detection of microdeletion/microduplication using next generation sequencing; three case studies.

Dr. Jeanne Morin-Leisk

Jeanne Morin-Leisk, Fulgent Genetics, Temple City, CA USA; Régen Drouin, CHUQ, Dep Paediatrics, Faculty Medicine, Laval University; and, Harry Gao, Fulgent Genetics, Temple City, CA USA

Background. Next generation sequencing (NGS) data is primarily used for detection of SNPs and small duplications/deletions. However, NGS is also a powerful method that can be leveraged using data processing to detect copy number variants (CNVs).

Objectives. The purpose of the case studies described herein is to demonstrate the clinical utility of CNV analysis using NGS data. Specifically, the presentation of cases with either a multi-exon deletion or multi-exon duplication, detected by NGS and confirmed by an orthogonal method, that can be reasonably expected to explain the clinical phenotype of the individuals tested.

Cases. Here we describe three children in whom a CNV, detected by NGS, was identified which is likely diagnostic. The first was found to have a duplication of all coding exons of the UBE3A gene. This duplication was inherited from the proband’s very slightly affected father. The second was found to have a de novo deletion of all coding exons of the PRRT2 gene. And the third was found to have a 2-exon duplication of the GRIA3 gene, which is located on the X-chromosome and transmitted by the proband’s mother to him and not to his unaffected brother.

Conclusions. These findings support the notion that there is clinical utility in using NGS data to detect CNVs.

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Thiemann disease and Familial digital arthropathy – brachydactyly: two sides of the same coin?

Dr. Samseh Nadirah

Click for image

Nadirah S. Damseh, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto; Jennifer Stimec, Department of Diagnostic Imaging, The Hospital for Sick Children; Alan O'Brien, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto; Christian R. Marshall, Department of Paediatric Laboratory Medicine and Laboratory Medicine and Pathobiology, The Hospital for Sick Children and University of Toronto; Ravi Savarirayan, Division of Rheumatology, Departments of Pediatrics and Medicine, The Hospital for Sick Children; Ali S. Jawad, Department of Rheumatology, The Royal London Hospital, The University of London; Laxer M. Ronald, Division of Rheumatology, Departments of Pediatrics and Medicine, The Hospital for Sick Children; Peter Kannu, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto, Developmental and Stem Cell Biology


Thiemann disease is a rare familial autosomal dominant deforming interphalangeal joint arthropathy of currently unknown genetic cause. Familial digital arthropathy-brachydactyly (FDAB) is distinctive form of autosomal dominant digital arthropathy with overlapping features. Transient receptor potential vanilloid 4 (TRPV4) mutations cause FDAB. To date, three families affected by FDAB have been reported and thirty- two cases of Thiemann disease identified.

Method and results:

We describe a father and son referred with a diagnosis of Thiemann disease and subsequently identified with a heterozygous mutation (c.809G>T) in TRPV4. The identical genetic mutation was previously reported to cause FDAB.

PUBMED database searches were conducted in to retrieve articles related to Thiemann disease and FDAB. We review the clinical and radiological features of nineteen individuals with a diagnosis of Thiemann disease and compare them with the three reported families affected by FDAB. We compare and contrast the clinical and radiological features of the two conditions.


FDAB and Thiemann disease are non-inflammatory digital arthropathies with phenotypic overlap.Although more extensive joint involvement, a distal hand joint preponderance and bracydactyly are expected in FDAB, there are striking radiological similarities between the two entities. Our report describing a TRPV4 mutation in a father and son referred with a diagnosis of Thiemann disease suggests the historical accounts of these two different phenotypes may be imprecise in some instances and that they may represent phenotypic variabilitiy of the same disease.

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Molecular diagnosis of PIK3CA-related overgrowth spectrum (PROS) in 162 patients and recommendations for genetic testing

Dr. Jean-Baptiste Rivière

Purpose: Postzygotic activating mutations of PIK3CA cause a wide range of mosaic disorders collectively referred to as PIK3CA-related overgrowth spectrum (PROS). Here, we describe the diagnostic yield and characteristics of PIK3CA sequencing in PROS.

Methods: We performed ultra-deep next-generation sequencing (NGS) of PIK3CA in various tissues from 162 patients referred to our clinical laboratory, and assessed diagnostic yield by phenotype and tissue tested.

Results: We identified disease-causing mutations in 66.7% (108/162) of patients, with mutant allele levels down to 1%. The diagnostic rate was higher (74%) in syndromic than in isolated cases (35.5%; P = 9.03 × 10−5). We identified 40 different mutations, and found strong oncogenic mutations more frequently in patients without (50.6%) than in those with brain overgrowth (15.2%; P = 0.00055). Mutant allele levels were higher in skin and overgrown tissues than in blood and buccal samples (P = 3.9 × 10−25), regardless of the phenotype.

Conclusion: Our data demonstrate the value of ultra-deep NGS for molecular diagnosis of PROS, highlight its substantial allelic heterogeneity, and confirm that optimal diagnosis requires fresh skin or surgical samples from affected regions. Our findings may be of value in guiding future recommendations for genetic testing in PROS and other mosaic conditions.

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Severe rhizomelic shortening in a child with a complex duplication/deletion rearrangement of chromosome X.

Dr. Ashish Deshwar

Ashish R. Deshwar, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto; Lucie Dupuis, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto; Carsten Bergmann, Institute for Medical Diagnostics GmbH, Center for Human Genetics, Ingelheim, Germany; James Stavropoulos, Genome Diagnostics, The Hospital for Sick Children and University of Toronto; and Roberto Mendoza-Londono, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children and University of Toronto


In this study, we present a patient who exhibits mesomelic involvement and severe rhizomelic shortening associated with developmental delay. This patient presents with traits characteristic of multiple genetic syndromes and no clear unifying diagnosis.

Design and Methods:

Karyotyping was performed followed by fluorescence in situ hybridization and multiplex ligation dependent probe amplification to characterize a re-arrangement of the X chromosome.  Sequencing across the SHOX locus was completed given the skeletal presentation. A panel of 43 genes implicated in connective tissue disorders with bone involvement were sequenced. 


Karyotyping revealed a recombinant X chromosome in which the region distal to Xp22.33 (where SHOX is found) was replaced with material from Xq28 (including MECP2). Sequencing across the SHOX locus on the Y chromosome revealed a single base pair mutation upstream of the coding sequence of unknown significance. The connective tissue gene panel revealed a predicted pathogenic mutation in HSPG2.


This patient has features of both Langer Mesomelic dysplasia and MECP2 duplication syndrome and unique features not typically present in either. Further delineation of this rearrangement may allow for the identification of genes critical for the development of the limb’s proximal segment.

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Characterization of VHL promoter variants in patients suspected of Von Hippel Lindau

Dr. Saleh Albanyan

Click to download Powerpoint


  • Von Hippel-Lindau disease (VHL) is a hereditary cancer syndrome in which carriers are at increased risk of developing a variety of tumors in multiple organ systems.
  • A clinical diagnosis of VHL is determined by the presence of specific clinical manifestations while molecular genetic diagnosis results from a pathogenic variant in the VHL gene.
  • The majority of mutations occur in VHL coding exons and DNA analysis of these regions has a sensitivity of nearly 100%. 

Patients & Results:

Family 1:

  • 53 year old male presenting with a renal cyst and pancreatic nodule found to have a Variant of uncertain significance (VUS)  in the 5’ UTR of VHL (c-61_-51 dup11).
  • Variant was examined in his 3 children, and was found in his daughter and who had negative VHL phenotyping (Figure 1)
  • Transcript analysis by RT-PCR ( daughter’s peripheral blood lymphocytes)  revealed 61% RNA expression VHL transcripts compared to healthy control.• Protein analysis with western blotting of the VHL protein in peripheral blood lymphocytes also showed decreased protein expression.

Family 2:

  • 22 year old female presenting with 2 right sided renal masses on abdominal ultrasound and multiple pancreatic cysts found to have a VUS in the 5’ UTR of VHL (c.-77_-32del)
  • This variant segregated with 2 affected family members (Figure 1)
  • Transcript analysis by RT-PCR was performed and revealed that our patient expressed 45% of normal VHL transcripts decreased VHL protein gene expression(figure2). 
  • Protein analysis with western blotting of the VHL protein using the patient’s lymphocytes showed marked decreased expression  


  • RNA and protein studies in these families were taken in context of the clinical manifestations (More marked decrease in Patient 2)
  • Both promoter variants may play a role in the clinical phenotype in both families, it is likely more contributory in the second family.
  • Promoter regions could be of importance for the molecular characterization of VHL patients that have no variations in the coding region.
  • we propose extending the molecular analysis of highly suspicious VHL cases to include the regulatory regions of the gene along with RNA and protein studies

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Analysis of exome filtering techniques for the prioritization of clinically relevant genes

Dr. Kristin Kernohan

Whole exome sequencing is a next-generation sequencing based technique which selectively sequences DNA from the ~1% of the genome that codes for proteins. This cost efficient and effective technology has revolutionized rare disease research as a tool for novel disease gene identification, and has already begun to transition into the clinical realm for diagnostics. However, one of the challenges for clinical exome sequencing is variant interpretation; each exome contains ~20,000 variants, ~300-500 of which are rare (seen in  < 1 % of the population). Clinical labs must interrogate this data to identify the variants in genes which may contribute to an individual’s phenotype, a task which is difficult given the constantly evolving rare disease literature. Efficient exome filtering also allows for control over the identification of incidental findings. We conducted a strategic assessment of 50 exomes with known disease variants using a number of available rare disease datasets as filters: Online Mendelian Inheritance in Man (OMIM), Orphanet, Human Phenotype Ontology (HPO) annotation files and the gene panels curated at Radboud University Medical Center. We found that personalized panels produced using the HPO terms for each participant’s PhenomeCentral profile and the HPO annotation files had the same success rate as the OMIM and Orphanet panels, while providing a significantly smaller pool of variants to individually assess. We also find that this method is successful for the identification of patients with multiple diagnoses. We conclude that HPO term derived gene panels are an efficient and effective way to prioritize and identify clinically relevant exome variants.

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Increased nuchal translucency and Noonan syndrome spectrum – A Mount Sinai hospital experience

Dr. Pierre Sinajon

Objective: To examine a cohort of patients seen at the PNDMG program - Mount Sinai Hospital between 2013 – 2015, with fetal ultrasound findings of an increased nuchal translucency (NT ≥ 3.5mm), and to:

  • Correlate between NT measurements and a diagnosis of Noonan Syndrome Spectrum (NSS) 
  • Conduct a systematic literature review to determine the correlation between NT measurement and a diagnosis of NSS 
  • To create a clinical protocol for the investigation of an increased fetal NT

Methods: Patients presenting between 2013 – 2015 with fetal ultrasound findings of increased NT. All were offered amniocentesis/CVS for QF-PCR as an initial test. If negative, patients proceeded to karyotype/microarray analysis and NSS panel testing. Patients offered fetal ultrasounds at 16 weeks and 18 – 22 weeks gestation (GA) along with fetal echocardiogram.

A systematic review was conducted in accordance to PRISMA criteria. Pubmed, Embase, Ovid MEDLINE and Web of Science were searched from January 2005 – August 2016 for articles involving NSS and increased NT. Seventeen papers were included for analysis.

Results: 226 patients were seen. In 116/226 patients, chromosomal aneuploidy was detected through QF-PCR.  The remaining 110/226 patients had further testing. 8 had karyotype abnormalities, 13 had abnormal microarray findings and 5 had NSS findings through DNA analysis.

Discussion: Given the small numbers found to have positive result for NSS, a systematic review of literature was performed. The systematic review was used to address small numbers from the original data set and to help create a clinical protocol for investigating an increased NT.

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Characterization of 5 adult patients with m.15152G>A, a novel mutation in mitochondrial cytochrome b

Dr. Natascia Anastasio

Objectives: Mutations in mitochondrially-encoded cytochrome b, MT-CYB, are one of the most common causes of CIII deficiency and this disorder has huge phenotypic variability (FEBS J 2005;272:3583-3592.). We present a case of familial CIII deficiency and characterize the variability of symptoms in adult patients.

Method: Clinical phenotyping, mitochondrial DNA familial variant testing and heteroplasmy analysis, muscle biopsy. 

Results: The proband and his sister presented in childhood with recurrent episodes of ketotic hypoglycemia, decreased endurance, leg cramps and gastrointestinal dysmotility. Both were found to be homoplasmic for an m.15152G>A (p.G136S) mutation in MT-CYB. They have had good response to coenzyme Q10. Their mother was also found to be homoplasmic and showed findings in different organ systems, as is typical of a mitochondrial disorder. These include neurological, muscular, psychiatric, gastrointestinal, cardiovascular, auditory, and visual symptoms. These findings were similar to those described by 4 other adult family members; all heteroplasmic for the familial MT-CYB mutation. Three of five patients had abnormal muscle biopsies which showed ragged red fibers (1/3), myopathic changes (2/3) and mitochondrial proliferation (1/3). Two of five adult patients were treated with coQ10 and showed subjective improvement.

Conclusions: This is the first report of the m.15152G>A mutation in MT-CYB causing mitochondrial disease. Our case highlights the phenotypic variability in symptoms, even amongst patients with the same mutation. Our case also raises the possibility of response to treatment with coenzyme Q10. Future directions include muscle mtDNA heteroplasmy testing, blue native stain and respiratory chain enzyme analysis to further characterize the phenotype.

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Second family with Mabry Syndrome caused by PIGL mutation

Dr. Ruqaiah Altassan

Keywords: Hyperphosphatasia mental retardation syndrome (HPMRS), Mabry syndrome, PIGL gene, CHIME syndrome.


To describe the second family with Mabry syndrome (Hyperphosphatasia mental retardation syndrome-HPRMS) caused by PIGL mutation.

Design and Methods:

Clinical evaluation and molecular testing of two siblings presented to our institution with distinctive facial features, developmental delay, mental retardation, seizure disorder, skeletal deformities, and high alkaline phosphatase.


Molecular testing for the index case showed two heterozygous mutations in PIGL gene (C.60G>A; p.TRP20*, and c.262C>T; p.ARG88Cys). Segregation analysis confirmed compound heterozygous inheritance of the two alleles. Same changes found in the other affected sibling.

To date, six mutations in PIGL gene have been identified in seven patients presenting with CHIME syndrome ( coloboma, heart defect, early-onset ichthyosiform dermatosis, mental retardation and ear anomalies /hearing loss alternate with epilepsy) and only one  patient with Mabry phenotype ( Fujiwara I et al, 2015).

In our patients, some of the clinical features overlap between CHIME and Mabry syndrome especially the CNS involvement; however, they do not have coloboma, heart defect or hearing impairment. The distinctive skeletal phenotype of short terminal phalanges and the high alkaline phosphatase diverged our diagnosis to Mabry syndrome.


Our clinical, biochemical and molecular findings support the previous report of Mabry syndrome caused by PIGL mutation, which is another PhosphatidylInositol Glycan (PIG) anchor biosynthesis class that should be considered in the differential diagnosis of the known PIG classes (PIGV, PIGO, and PIGW) and the Post GPI Attachment to Proteins genes (PGAP2, PGAP3) that are currently linked to Mabry syndrome.


Fujiwara I, Murakami Y, Niihori T, Kanno J, Hakoda A, Sakamoto O, Okamoto N, Funayama R, Nagashima T, Nakayama K, Kinoshita T, Kure S, Matsubara Y, Aoki Y. Mutations in PIGL in a patient with Mabry syndrome. Am J Med Genet A. 2015 Apr;167A(4):777-85.

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Diagnosing multiple genetic diseases in families by whole exome sequencing debunks Occam’s razor

Dr. Tugce Balci

Background. Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown.

Objectives. We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada, examine the factors that might contribute to this finding, and put this in context of the existing literature.

Design / Methods. We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past five years in either the FORGE or Care4Rare Canada WES sequencing initiatives.

Results. 226 of the 802 probands (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the eight probands had family members with one or more of the molecularly diagnosed diseases. There were no multiple genetic diagnoses identified in sporadic trios. Consanguinity and the presence of multisystem disease both appeared to increase the likelihood of multiple genetic diagnoses in a single family.

Conclusions. Our findings highlight that in the setting of complete family ascertainment, multiple genetic diagnoses in a proband is almost akin to diagnosing one or more of them in their family and speaks to the importance of comprehensive clinical phenotyping alongside WES analysis of multiple family members.

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Delineating the impact of rare copy number variation on IQ in schizophrenia

Ms Chelsea Lowther

Background: Schizophrenia (SCZ) is associated with significant impairments in IQ. Yet, most studies investigating the burden of rare copy number variation (CNV) in SCZ have excluded individuals with low IQs (SCZ-LIQ).

Objectives: To determine the yield of clinically relevant CNVs and compare the functional impact of all genome-wide rare CNVs between SCZ-LIQ (FSIQ < 8 5) and SCZ-no ID (FSIQ≥85).

Methods: All genic CNVs >100 kb detected by high-resolution microarrays were annotated for pathogenicity following standard clinical guidelines. We also performed a gene-set enrichment analysis using all genome-wide CNVs to determine if there are pathways specifically related to SCZ-LIQ.

Results: The burden of clinically relevant CNVs was higher in the SCZ-LIQ compared to the SCZ-no ID group (OR=2.08 [1.31-3.30], p=0.001); this finding was primarily driven by SCZ subjects with comorbid intellectual disability (ID; n=23/59; 38.9%). We also identified a high proportion of clinically relevant CNVs in SCZ subjects with a non-verbal learning disorder (n=9/28; 32.1%), which did not significantly differ from SCZ-ID (p=0.537). Of the 31 gene-sets tested, only the GO Nervous System Development gene-set was significantly enriched for duplications in the SCZ-LIQ compared to SCZ-no ID group (p=0.002). These duplications implicated known and potentially novel genes related to SCZ-LIQ. 

Conclusions: To our knowledge, this is the largest study to examine the genome-wide impact of rare structural variation on IQ in SCZ to date. These data have important clinical and research implications, including demonstrating that individual with SCZ and ID or a non-verbal learning disorder should be prioritized for clinical microarray testing.

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Improved diagnostic yield and comparable coverage to targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test in paediatric medicine

Dr. Christian R. Marshall

Background: Genetic testing is an integral component of paediatric medicine, but can be costly, time-consuming, and inconclusive. Standard of care is often a step-wise approach involving targeted gene sequencing panels and chromosomal microarray analysis. Whole-genome sequencing (WGS) is a comprehensive approach that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.

Objectives: The purpose of the study was to compare the diagnostic yield of WGS with current clinical testing methods.

Methods: We prospectively recruited 103 patients from paediatric subspecialty clinics with clinical symptoms suggestive of an underlying single gene genetic disorder, and compared the diagnostic yield and coverage of WGS with conventional genetic testing.

Results: WGS identified diagnostic variants in 41% of the cohort, representing a significant increase over conventional testing by targeted panels +/- microarray (24%; p=0.01). Genes that were clinically sequenced in this cohort (n=1,226) were well covered by WGS with median exonic coverage of 32X +/- 8.3 (mean+/-SD) across all cases. All diagnostic nuclear and mitochondrial DNA variants, and all reportable copy number variations, discovered with conventional testing were identified with WGS. New molecular diagnoses made via WGS included disease associations with the PIGG, RNU4ATAC, TRIO, and UNC13A genes, as well as variants not detectable with whole-exome sequencing.

Conclusion: WGS as a primary clinical genetic test can provide a higher diagnostic yield than conventional testing, with comparable sensitivity.

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Exome Reanalysis: Integration of New Knowledge in Genomic Reports

Dr. Val Lapin

Val Lapin, EGL Genetics, Eurofins Clinical Diagnostics, 2460 Mountain Industrial Boulevard, Tucker, GA, USA;  Lindsey Mighion, EGL Genetics, Eurofins Clinical Diagnostics, 2460 Mountain Industrial Boulevard, Tucker, GA, USA;  Cristina da Silva, EGL Genetics, Eurofins Clinical Diagnostics, 2460 Mountain Industrial Boulevard, Tucker, GA, USA;  Lora J. H. Bean, EGL Genetics, Eurofins Clinical Diagnostics, 2460 Mountain Industrial Boulevard, Tucker, GA, USA, Dept. of Human Genetics, Emory University School of Medicine

Background. Whole exome sequencing (WES) has become an increasingly common and useful tool for identifying the underlying genetic cause in individuals with rare or complex etiologies. However, even with diagnostic yield of WES close to 30%, the majority of cases do not have a definitive answer.

Objectives. WES reanalysis offers the opportunity to review previous data with improved bioinformatic tools, recent publications and an ever-growing list of genes associated with diseases. Clinical laboratories must have a system to curate emerging genetic knowledge to reanalyze reported cases either through physician driven requests or retrospectively due to new gene disease-association.

Method. Of 37 cases reanalyzed, 31 were by physician request, while the remaining 6 were reanalyzed as part of routine laboratory procedures. The majority of the samples were reanalyzed only 1 time after the initial analysis (25/37); however, some cases have been reanalyzed as many as 4 times to date.

Results. Of 37 cases reanalyzed, 12 (32%) led to the finding of additional variants that were reported as phenotype-related, and 4 of these resulted in the identification of a genetic diagnosis for the proband.

Conclusions. The successes of reanalysis highlight the importance of providing the most up-to-date clinical data to the laboratory for analysis and of the need to continually evaluate genes as they move from genes of unknown significance to genes associated with disease. The ability to reanalyze and re-evaluate sequencing data as described will become an increasingly valuable tool in finding an end-point for some patients on a diagnosis odyssey.

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Clinical diagnosis of Duchenne Muscular Dystrophy in a case with an as-yet unidentified genetic cause

Dr. Michelle Axford

Michelle M Axford, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Jennifer Orr, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Marianne Eliou, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Carol Ann Ryan, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Christian R Marshall, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children, Laboratory Medicine and Pathobiology, University of Toronto; Raveen Basran, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children, Laboratory Medicine and Pathobiology, University of Toronto; Lauren Brady, Department of Pediatrics, McMaster Children’s Hospital; Mark A Tarnopolsky, Department of Pediatrics, McMaster Children’s Hospital

Background. Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene. Out-of-frame exonic deletions and duplications account for ~70% of DMD diagnoses. Nonsense variants, small indels, and splice site changes make up ~30%. Approximately 1% of cases have other genetic etiologies, such as deep intronic mutations or translocations.

Objectives. To identify the genetic cause of DMD in a 6 year old male with a family history of clinically but not molecularly diagnosed DMD.

Design/Methods. MLPA and Sanger sequencing of the DMD gene was performed on the proband, a maternal uncle clinically diagnosed with unusually severe DMD who died at age 10, and the proband’s grandmother.  cDNA studies on muscle tissue from the proband were carried out.  SMN del/dup analysis and Sanger sequencing of genes involved in congenital muscular dystrophy (CDM) were also carried out.

Results. SMN1 and CDM testing were both negative for pathogenic variants. Copy number and sequencing of DMD did not identify a causative variant; however cDNA analysis on the proband revealed an abnormal RNA transcript in which exons 1 through 45 did not amplify.  The underlying genetic cause of this mutant RNA was not identified, therefore FISH analysis is planned to attempt to detect potential genomic rearrangements.

Conclusions. A fraction ( < 1 %) of DMD cases are caused by pathogenic variants that are not detectable by typical DMD testing strategies. We report here a multigenerational family in which such a mutation may be causative of the DMD clinical phenotype.

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Discrepant Sanger and NGS sequencing results for an Ehlers-Danlos syndrome (EDS) III pathogenic variant in the high homology gene TNXB

Dr. Michelle Axford

Michelle M Axford, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children;   Jennifer Orr, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children;  Marianne Eliou, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Cameron Ellahi, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Liping Han, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Lynette Lau, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children; Christian R Marshall, Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children, Laboratory Medicine and Pathobiology, University of Toronto; Lianna Kyriakopoulou,Genome Diagnostics, Department of Pediatric and Laboratory Medicine, The Hospital for Sick Children

Background. Pathogenic variants causing the dominantly inherited EDS III occur in the TNXB gene, for which the truncated pseudogene TNXA shares ~96% sequence identity within their shared regions. A 120bp deletion in exon 35 of TNXB is commonly used to differentiate the two homologues.

Objectives. To determine the reason behind discrepant results in an EDS diagnosis case; NGS revealed a heterozygous pathogenic variant in TNXB while Sanger sequencing did not detect the variant.

Design/Methods. EDS gene panel sequencing was done on the proband and her mother.  Sanger sequencing was carried out to confirm results. Various primer sets were designed and tested for Sanger, to attempt to confirm the NGS results. EDS MLPA was also done.

Results. MLPA was negative for pathogenic del/dups.  NGS on the proband revealed a heterozygous pathogenic variant (c.11381-2A>G) in the TNXB gene that was not detected using Sanger in either the proband or the mother.  Subsequent NGS on the mother confirmed the presence of the variant.  Several primer redesigns failed to pick up the variant until a primer 47bp from the start of exon 34 was moved to 45bp from the start of exon 35 (43bp upstream of the variant), which then confirmed the presence of the variant in both individuals.

Conclusions. The high homology of TNXB and TNXA makes it difficult to troubleshoot sequencing discrepancies.  It is possible that some type of genomic rearrangement in the TNXB gene in this mother and daughter could have let to the discrepant result between NGS and Sanger sequencing.

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Vascular Ehlers-Danlos Syndrome in two families presenting with extensive bruising

Dr. Shuaa Basalom

Background. Vascular Ehlers-Danlos Syndrome (vEDS) also known as Ehlers-Danlos type IV is dominantly inherited syndrome associated with mutations in COL3A1. The clinical criteria for vEDS are defined by the Villefranche classification.

Objectives. To present four patients from two families with molecularly confirmed vEDS who presented predominantly with easy bruising.

Clinical cases. The proband is a 7 year old girl who presented with excessive bruising, petechia and purpura that started at 10 months of age. The bruising appeared in unusual areas such as the soles of the feet.  She had no other Villefranche criteria. Family history was significant for similar bruising in her sister and father.  Molecular testing revealed a mutation in 3’ region of COL3A1 in all three.

The proband is a 17 year old male who presented with recurrent pneumothorax with pulmonary blebs, joint hyperlaxity and history of easy bruising. He also has a learning disability and history of left club foot.  Family history was unremarkable.  Physical examination revealed bruises on the legs and translucent skin.  Molecular testing revealed a splice site mutation in COL3A1.

Conclusions. Patients with vEDS are likely to have excessive bruising that is often mistaken for a coagulation disorder. These cases illustrate the importance of considering vEDS when excessive bruising is present, particularly in the pediatric age group.

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Homozygous mutation in RSPRY1 in a patient of Canadian indigenous descent results in skeletal dysplasia and intellectual disability: Expanding the phenotype of SEMD Faden-Alkuraya type

Dr. Inara Chacon

Background. The spondyloepimetaphyseal dysplasia Faden-Alkuraya type (SEMDFA), is characterized by  progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals and intellectual disability. It is caused by mutation in the RSPRY1 gene. Thus far, the only known individuals  with pathogenic variants in this gene are those from  two families from Saudi and Peruvian descent respectively.

Objective. We describe a 14 years old girl from aboriginal Canadian descent with a novel RSPRY1 pathologenic variant found by whole exome sequencing (WES).

Case description. Our patient had significant developmental regression since age 2. Very short stature (-6SD), hydrocephalia, facial dysmorphisms including hypertelorism, depressed nasal root and upturned nose,  mid-face hypoplasia, and  small pointy chin. The most striking skeletal findings included  thickend cranium, difuse osteopenia, generalized small flat epiphyses, dislocated patellas bilaterally, shortening of the fourth metatarsals and syndactyly of the toes. Brain MRI showed diffuse cerebral atrophy and right optic atrophy.  In addition to these findings, she has central apnea that required tracheostomy.

Results. Normal SNP microarray with several regions of homozygosity  representing at least 4.64% of the individual autosome. WES demonstrated a homozygous c.1285G>T(pGly429*) variant that codes for premature stop codon and predicts a loss of function.

Conclusions. RSPRY1 is a gene thought to be associated with nucleic acid processing but with a strong pattern of expression in the developing murine skeleton. This girl, from non-consanguineous parents, represents the third  described case  of SEMDFA and opens avenues for expanding research due to possible founder effect in the Canadian indigenous population.

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Specific Antibody Deficiency In A Patient With Ring Chromosome 18

Dr. Mehdi Yeganeh

Mehdi Yeganeh, Department of Medical Genetics, McGill University Health Centre; Duncan Lejtenyi, Division of Allergy and Immunology, Montreal Children’s Hospital; Bruce D. Mazer, Division of Allergy and Immunology, Montreal Children’s Hospital

Background: Primary antibody deficiency and autoimmunity have been previously reported in patients with chromosome 18 abnormalities. This phenotype resembles those of patients with non-syndromic antibody deficiency disorders. Therefore, studying immune defects associated with ring chromosome 18 could potentially help unveil the genetic basis of other primary antibody deficiencies associated with autoimmunity.

Objectives: We examined the immune system of a female patient with ring chromosome 18 affected by recurrent infections and autoimmunity, by assessing her specific antibody response and immunophenotyping.

Methods: Serum antibody titers to 23-multivalent pneumococcal polysaccharide and peptide vaccines were measured using ELISA. Lymphocyte sub-populations were determined by flow cytometry.

Case: The patient was diagnosed with ring chromosome 18 for delayed growth and dysmorphic features at the age of one month. Array comparative genomic hybridization showed deletions of 18p11.21-pter and 18q21.31-qter. At the age of 10 months, she started presenting with recurrent episodes of otitis media and pneumonia. Meanwhile, she developed hypothyroidism and polyarthritis with positive ANA and ANCA-C serum markers. The serum immunoglobulins and specific antibody levels were low. The CD19+CD27+ memory B cell and CD45RO+ T cell populations were decreased. Recurrent infections were controlled with parenteral immunoglobulin and autoimmune arthritis was treated with systemic and intra-articular corticosteroids.

Conclusions: Although selective IgA deficiency is the most common form of immunodeficiencies associated with chromosome 18 abnormalities, patients with ring chromosome 18 might be affected by specific antibody deficiency and autoimmunity. Our findings suggest that the pathogenesis likely involves late B cell maturation and lifelong immunoglobulin therapy is beneficial.

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Ambulatory status and quality of life in children with Friedreich ataxia

Dr. Resham Ejaz

Resham Ejaz, Division of Clinical and Metabolic Genetics, Department of Paediatrics, The Hospital for Sick Children; Shiyi Chen, The Hospital for Sick Children Research Institute, Child Health Evaluative Sciences/Biostatistics Design & Analysis Unit; Charles J. Isaacs, Department of Neurology, Children’s Hospital of Philadelphia, Department of Pediatrics; Amanda Carnevale, Division of Clinical and Metabolic Genetics, Department of Paediatrics, The Hospital for Sick Children; Judith Wilson, Cardiomyopathy and Heart Function Program, Labatt Family Heart Centre, The Hospital For Sick Children; Kristen George, Cardiomyopathy and Heart Function Program, Labatt Family Heart Centre, The Hospital For Sick Children; Martin B. Delatycki, Murdoch Childrens Research Institute, Victorian Clinical Genetics Services, Victoria, Australia;  Susan L. Perlman, Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA;  Katherine D. Mathews, Departments of Neurology and Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA;  George R. Wilmot, Department of Neurology, Emory University, Atlanta, GA; Chad Hoyle, Department of Neurology, Ohio State University College of Medicine, Columbus, OH;  Sub H. Subramony, Department of Neurology, University of Florida, College of Medicine, Gainesville, FL;  Theresa Zesiewicz, Department of Neurology, University of South Florida, Tampa FL;  Jennifer Farmer, Department of Neurology, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia PA;  David R. Lynch, Department of Neurology, Department of Pediatrics, Children’s Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA; Grace Yoon, Division of Clinical and Metabolic Genetics, Department of Paediatrics, Division of Neurology, Department of Paediatrics, The Hospital for Sick Children

Background: Friedreich ataxia (FRDA) is a recessively inherited, childhood-onset neurodegenerative disorder. Most patients present with ataxia and mobility devices are the mainstay of managing the resulting ambulation loss.

Objective: To determine the impact of loss of independent ambulation and use of a mobility device on quality of life (QOL) in children with FRDA.

Methods: Participants were 111 pediatric individuals from 9 institutions with genetically confirmed FRDA. Data was collected from a prospective natural history study with standardized clinical evaluations, including health-related QOL using the PedsQL 4.0 Generic Core Module as the main outcome measure. The association between mobility device use and QOL scores was evaluated using univariate and multiple regression. Longitudinal QOL data of 16 individuals who transitioned to or between mobility devices was also analyzed.

Results: Mobility device use was associated with worse mean PedsQL total scores, and physical, emotional, social, and academic subscores, after adjusting for gender, age of disease onset, and Friedreich Ataxia Rating Scale score. The magnitude of the difference was greatest for the physical subscore (19.5, p=0.0004) and least for the emotional subscore (10.61, p=0.031). Transition to or between mobility devices was associated with a worse physical subscore (18.13, p=0.012); there were no statistically significant changes in emotional (10.4, p=0.19), social (2.66, p=0.71) or academic subscores (4.0, p=0.6), and the emotional subscore trended toward improvement over time (9.55 per year, p=0.06).

Conclusions: Loss of independent ambulation has a significant impact on QOL in children with FRDA, with a larger effect in the physical domain than psychosocial health. 

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Understanding the Molecular Mechanisms in Pyruvate Dehydrogenase Deficiency Outcomes and Treatment

Dr. James O'Byrne

James J. O'Byrne, Division of Biochemical Genetics, BC Children’s Hospital, Vancouver; Klara Van Karnebeek, Centre for Molecular Medicine and Therapeutics, BCCH Research Institute, UBC Vancouver; Philipp Lange, Child and Family Research Institute, BC Children’s Hospital, Vancouver; Ramona Salvarinova, Division of Biochemical Genetics, BC Children’s Hospital, Vancouver;  Gabriella Horvath, Division of Biochemical Genetics, BC Children’s Hospital, Vancouver


Pyruvate Dehydrogenase Complex Deficiency (PDCD) is a clinically heterogeneous disorder, usually presenting with lactic acidemia and neurological manifestations that may be progressive or intermittent. Outcomes in PDCD vary from infantile death to severe intellectual disability, the basis of which is largely undetermined. Treatment with ketogenic diet or thiamine also has variable effectiveness. The objective of this study was to report five case of PDH deficiency and to examine for a genotype-phenotype correlation to outcome and responsiveness to treatment.


A retrospective chart analysis was performed on five cases of PDCD (3 female and 2 male).


The average age of symptom onset was 6 weeks (range1-3 months) in females and 18 months (range 9-24 months) in males. The average age of diagnosis is 5 months and 5 years in females and males respectively. 20% (1/5) have died and 80% (4/5) have cognitive delay. 20% (1/5) was responsive to thiamine treatment. 80% (4/5) have undergone molecular testing and mutations on PDHA1 have been identified in 75% (3/4).


The case reports highlight the phenotypic heterogeneity of PDCD. Interestingly, one case was very responsive to thiamine. This case had a mild mutation (in-frame insertion) and presented with a much milder phenotype than usually observed in males, suggesting there may be a genotype-phenotype correlation. We intend to perform E1 alpha subunit phosphorylation/dephosphorylation studies and structural modelling to investigate if this explains the wide variation in phenotypes and responsiveness to treatment.

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Tatton-Brown-Rahman Syndrome is a diagnostic consideration in individuals with Marfanoid features

Dr. James O'Byrne

James J. O’Byrne, Department of Medical Genetics, University of British Columbia, Vancouver, Canada;  Karen Y. Niederhoffer, Department of Medical Genetics, University of Alberta, Edmonton, Canada; CAUSES, Department of Medical Genetics, University of British Columbia, Vancouver, Canada; Linlea Armstrong, Department of Medical Genetics, University of British Columbia, Vancouver, Canada

Background.  Tatton- Brown- Rahman (TBR) syndrome is a recently described overgrowth syndrome with intellectual disability, associated with pathogenic variants in DNMT3A. 13/152 individuals with overgrowth of unknown etiology after analysis for Sotos, Weaver, PTEN Hamartoma and Beckwith Wiedemann syndromes were identified to have pathogenic mutations in DNMT3A, suggesting it is relatively common (1).  The series identified a distinctive facial appearance and suggested association with features such as scoliosis (1,2). 

Objectives. The purpose of this report is to expand the phenotype of TBR syndrome and highlight it as a diagnostic consideration in the differential diagnosis of Loeys Dietz and Marfan syndromes.

Design/Method. We report a 12 year old female referred for query connective tissue disorder.  She had tall stature 169cm  (> 99th percentile), mitral valve prolapse, arachnodactyly, thoracic lordosis and scoliosis, straight heavy eyebrows, short palpebral fissures ( < 3 rd centile), high arched palate, bifid uvula, droplet cortical  lens opacities, failure and retention of some primary dentition and hirsutism. There was mild ID and a history of three nocturnal seizures, A de novo pathogenic missense mutation in DNMT3A was identified by whole exome sequencing.

Results/Conclusion. The presented case has some hallmarks of TBR syndrome such as the mild ID, distinctive facies, tall stature and scoliosis. Other features such as bifid uvula, arachnodactyly and droplet  lens opacities have not been previously reported and mitral valve prolapse has only been reported once. This case expands the TBR syndrome phenotype and suggests it should be considered a differential diagnosis of patients being assessed for Marfan (like) conditions.

Nature Genetics 2014;46:385-388
Clin Genet. 2016 Oct 4. doi: 10.1111/cge.12878. [Epub ahead of print]

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Investigation of simplex patients born to consanguineous parents: Whole exome sequencing has a high yield, with significant contribution of de novo mutations

Dr. Alison Eaton

Whole exome sequencing (WES) is an effective diagnostic and research tool. It is recommended as a frontline investigation in undiagnosed consanguineous rare disease patients. Here we review the results from two research initiatives, the FORGE Canada and Care4Rare consortia, which leveraged consanguinity for gene discovery. We analyzed the yield in isolated (simplex) subjects with confirmed or suspected (including founder populations) consanguinity and contrasted to projects with multiple affected individuals.

In simplex cases (n = 48), 73% were solved (known or novel genes). Of these, 71% were autosomal recessive (57% homozygous, 14% compound heterozygous) while 29% were de novo. When we defined two groups within this cohort based on degree of relatedness, in individuals born to second cousins or closer (n = 28), 75% were solved (90% homozygous and 10 % de novo). However, with more distant relatedness or presumed consanguinity (n = 20), 70% were solved, but strikingly, the variant was de novo in 57%. In consanguineous families with multiple affected individuals (n = 54) 78% were solved, with 97.5% of solved cases being inherited (90% homozygous) and apparent de novo variants accounting for only 2.5%.

Our data underscores the high yield of WES in the context of consanguinity. These results are influenced by the selected and highly studied nature of these research patients, and hence are higher than other unselected or non-consanguineous cohorts. Nevertheless, in simplex cases, particularly in the absence of close consanguinity, trio WES remains essential due to the higher proportion of de novo mutations.

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Nutritional Deficiency Complicating the Diagnosis of an Inborn Error of Metabolism: a Case Study

Dr. Estrella Lizabeth Mellin-Sanchez

Estrella-Lizbeth Mellin-Sancheza, The Hospital for Sick Children, Division of Clinical and Metabolic Genetics, University of Toronto, Toronto, ON, Canada; Dawn Cordeiro, The Hospital for Sick Children, Division of Clinical and Metabolic Genetics; Sarah Herd, The Hospital for Sick Children, Division of Clinical and Metabolic Genetics; Neal Sondheimer,The Hospital for Sick Children, Division of Clinical and Metabolic Genetics, University of Toronto, Toronto, ON, Canada

Objective. To describe a case presenting with malnutrition features overlapping and masking a metabolic disorder

Introduction. Nutritional deficiencies can alter metabolism in a pattern resembling inborn errors of metabolism (IEMs). In particular, the level of total homocysteine (Hcy) can be elevated by physiological, lifestyle, pharmacological, dietary and genetic factors. Hcy is a non-dietary non-structural sulfur-containing amino acid (AA) that is formed in the methionine catabolic pathway. Multiple enzymes and cofactors are involved. Most of them manageable by modifying diet, providing vitamins.

Case. An 11-month-old consanguineous Syrian refugee presented with a four-month history of intermittent diarrhea, fever and poor feeding. He was admitted upon arrival to Canada for weight loss with decreased urine output, cough, irritability, fever, loss of skills and oral aversion. At admission had hemoglobin (Hb) 45g/L (100-140), prompting a search for reversible causes of anemia. During this evaluation, he was found to have elevated total Hcy (tHcy) of 239µmol/L (2.9-10.0) and an abnormal acylcarnitine profile with elevations in propionyl-carnitine (C3). Urine organic acid analysis identified a large methylmalonic acid (MMA) peak. The pattern of elevated tHcy, C3 and MMA is consistent with defects in B12 metabolism or B12 deficiency. After the administration of intramuscular B12, his laboratory studies were repeated. The repeat studies found normalized C3 and MMA with a reduction of tHcy and profound elevation in methionine at 781µmol/L (nl 3-29). A diagnosis of cystathionine beta-synthetase deficiency (classical homocystinuria) was confirmed by sequencing of the CBS gene, which showed a known homozygous pathogenic variant c.526G>A.

Conclusion. The coincidental appearance of malnourishment, homocystinuria and B12 deficiency, raised initial suspicion for abnormalities in cobalamin transport and function. However, upon correction of the patient’s B12 deficiency, the true genetic diagnosis emerged. Classical homocystinuria is a diagnosis that is frequently missed, and the consequences in untreated patients include stroke, global developmental delay, ocular and skeletal manifestations. This case demonstrates the important principle that IEMs are difficult to diagnose in states of nutritional deprivation when normal metabolism is impaired.

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46th Annual Scientific Meeting - June 6-9, 2022 - Presented Virtually