Specifications of the ACMG/AMP guidelines for ACADVL variant interpretation.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a relatively common inborn error of metabolism, but due to difficulty in accurately predicting affected status through newborn screening, molecular confirmation of the causative variants by sequencing of the ACADVL gene is necessary. Although the ACMG/AMP guidelines have helped standardize variant classification, ACADVL variant classification remains disparate due to a phenotype that can be nonspecific, the possibility of variants that produce late-onset disease, and relatively high carrier frequency, amongst other challenges. Therefore, an ACADVL-specific variant curation expert panel (VCEP) was created to facilitate the specification of the ACMG/AMP guidelines for VLCADD. We expect these guidelines to help streamline, increase concordance, and expedite the classification of ACADVL variants.

Interplay between probe design and test performance: overlap between genomic regions of interest, capture regions and high quality reference calls influence performance of WES-based assays.

Whole exome sequencing (WES)-based assays undergo rigorous validation before being implemented in diagnostic laboratories. This validation process generates experimental evidence that allows laboratories to predict the performance of the intended assay. The NA12878 Genome in a Bottle (GIAB) HapMap reference sample is commonly used for validation in diagnostic laboratories. We investigated what data points should be taken into consideration when validating WES-based assays using the GIAB reference in a diagnostic setting. We delineate specific factors that require special consideration and identify OMIM genes associated with diseases that may 'bypass' validation. Four replicates of the NA12878 sample were sequenced at the CHEO Genetics Diagnostic Laboratory on a NextSeq 500; the data were analyzed using the bcbio_nexgen v1.1.2 pipeline. The hap.py validation engine, Real Time Genomics vcfeval tool, and high confidence (HC) variant calls in HC regions available for the GIAB sample were used to validate the obtained variant calls. The same validation process was then used to evaluate variant calls obtained for the same sample by two other clinical diagnostic laboratories. We showed that variant calls in NA12878 can be confidently measured only in the regions that intersect between the GIAB HC regions and the target regions of exome capture. Of the 4139 (as of October 2019) OMIM genes associated with a phenotype and having a known molecular basis of disease, 84 were fully outside of the GIAB HC regions and many of the remaining OMIM genes were only partially covered by the HC regions. A significant proportion of variants identified in the NA12878 sample outside of the HC regions have unknown (UNK) status due to the absence of HC reference alleles. Verification of such calls is possible either by an alternative truth set or by orthogonal testing. Similarly, many variants outside of exome capture regions, if not accounted for, will be deemed false negatives due to insufficient probe coverage. Our results demonstrate the importance of the intersection between genomic regions of interest, capture regions, and the high confidence regions. If not considered, false and ambiguous variant calls could have a negative impact on diagnostic accuracy of the intended WES-based diagnostic assay and increase the need for confirmatory testing. To enable laboratories to identify 'problematic' regions and optimize validation efforts, we have made our VCF and BED files available in UCSC Genome Browser: NA12878 WES Benchmark. Relevant genes and genome annotations are evolving, we implemented a general purpose algorithm to cross-reference OMIM genes with the genomic regions of interest that can be applied to capture genes/regions outside HC regions (see repository of data material section).

Genetic Variation in the Ontario Neurodegenerative Disease Research Initiative.

BACKGROUND/OBJECTIVE: Apolipoprotein E (APOE) E4 is the main genetic risk factor for Alzheimer's disease (AD). Due to the consistent association, there is interest as to whether E4 influences the risk of other neurodegenerative diseases. Further, there is a constant search for other genetic biomarkers contributing to these phenotypes, such as microtubule-associated protein tau (MAPT) haplotypes. Here, participants from the Ontario Neurodegenerative Disease Research Initiative were genotyped to investigate whether the APOE E4 allele or MAPT H1 haplotype are associated with five neurodegenerative diseases: (1) AD and mild cognitive impairment (MCI), (2) amyotrophic lateral sclerosis, (3) frontotemporal dementia (FTD), (4) Parkinson's disease, and (5) vascular cognitive impairment. METHODS: Genotypes were defined for their respective APOE allele and MAPT haplotype calls for each participant, and logistic regression analyses were performed to identify the associations with the presentations of neurodegenerative diseases. RESULTS: Our work confirmed the association of the E4 allele with a dose-dependent increased presentation of AD, and an association between the E4 allele alone and MCI; however, the other four diseases were not associated with E4. Further, the APOE E2 allele was associated with decreased presentation of both AD and MCI. No associations were identified between MAPT haplotype and the neurodegenerative disease cohorts; but following subtyping of the FTD cohort, the H1 haplotype was significantly associated with progressive supranuclear palsy. CONCLUSION: This is the first study to concurrently analyze the association of APOE isoforms and MAPT haplotypes with five neurodegenerative diseases using consistent enrollment criteria and broad phenotypic analysis.

Étude de variance génétique dans le cadre de l'initiative de recherche sur les maladies neurodégénératives en Ontario. Contexte/Objectif : L'apolipoprotéine E4 (ApoE4) constitue le principal facteur de risque génétique de la maladie d'Alzheimer. En raison de cette association systématique, il existe un intérêt certain à savoir dans quelle mesure cette classe d'apolipoprotéines peut influencer le risque d'autres maladies neurodégénératives. En outre, le milieu de la recherche n'a de cesse d'identifier d'autres biomarqueurs génétiques, par exemple les haplotypes H1 de la protéine tau associée aux microtubules, qui contribuent à certains phénotypes, Dans le cadre de cette étude, des participants à l'initiative de recherche sur les maladies neurodégénératives en Ontario ont été « génotypés ¼ afin de déterminer si l'ApoE4 ou l'haplotype H1 mentionné ci-dessus peuvent être associés à cinq maladies neurodégénératives : 1) la maladie d'Alzheimer et d'autres troubles cognitifs légers ; 2) la sclérose latérale amyotrophique ; 3) la démence fronto-temporale ; 4) la maladie de Parkinson ; 5) et finalement les déficits cognitifs d'origine vasculaire. Méthodes : Pour chaque participant, la cartographie des génotypes a été établie en fonction de leur ApoE4 respectif et de la présence d'haplotypes H1 de la protéine tau associée aux microtubules. Des analyses de régression logistique ont été ensuite effectuées dans le but d'identifier de possibles liens avec ces maladies neurodégénératives. Résultats : Nos travaux ont confirmé l'association entre l'ApoE4 et une plus grande occurrence de cas d'Alzheimer, et ce, en tenant compte de l'effet d'une dose de médicament. Ils ont aussi montré une association entre la seule ApoE4 et des troubles cognitifs légers. Cela dit, il convient de préciser que les quatre autres maladies n'ont pas été associées à cet allèle. Plus encore, nous avons trouvé que l'allèle E2 de l'apolipoprotéine était associé à une occurrence plus faible de cas d'Alzheimer et de troubles cognitifs légers. Fait à souligner, aucune association n'a été détectée entre l'haplotype H1 de la protéine tau associée aux microtubules et nos cohortes atteintes de maladies neurodégénératives. Toutefois, à la suite du sous-typage de la cohorte de participants atteints de démence fronto-temporale, il s'est avéré que l'haplotype H1 était associé de façon notable à la paralysie supra-nucléaire progressive. Conclusion : Il s'agit de la première étude à analyser simultanément, au moyen de critères de participation cohérents et d'une analyse phénotypique élargie, les associations entre les isoformes de l'ApoE, l'haplotype H1 de la protéine tau associée aux microtubules et cinq maladies neurodégénératives.

Mutations in the glucocerebrosidase gene are common in patients with Parkinson's disease from Eastern Canada.

BACKGROUND: Mutations in the ß-glucocerebrosidase gene (GBA) have been implicated as a risk factor for Parkinson's disease (PD). However, GBA mutations in PD patients of different ethnic origins were reported to be inconsistent. METHODS: We sequenced all exons of the GBA gene in 225 PD patients and 110 control individuals from Eastern Canada. RESULT: Two novel GBA variants of c.-119 A/G and S(-35)N, five known GBA mutations of R120W, N370S, L444P, RecNciI and RecTL mutation (del55/D409H/RecNciI) as well as two non-pathological variants of E326K and T369M were identified from PD patients while only one mutation of S13L and two non-pathological variants of E326K and T369M were found in the control individuals. The frequency of GBA mutations within PD patients (4.4%) is 4.8 times higher than the 0.91% observed in control individuals (X(2) = 2.91, p = 0.088; odds ratio = 4.835; 95% confidence interval = 2.524-9.123). The most common mutations of N370S and L444P accounted for 36.0% (9/25) of all the GBA mutations in this Eastern Canadian PD cohort. The frequency (6.67%) of E326K and T369M in PD patients is comparable to 7.27% in control individuals (X(2) = 0.042, p = 0.8376), further supporting that these two variants have no pathological effects on PD. Phenotype analysis showed that no significant difference in family history, age at onset and cognitive impairment was identified between the GBA mutation carriers and non-GBA mutation carriers. CONCLUSION: GBA mutations were found to be a common genetic risk factor for PD in Eastern Canadian patients.

Functional alteration of PARL contributes to mitochondrial dysregulation in Parkinson's disease.

Molecular genetics has linked mitochondrial dysfunction to the pathogenesis of Parkinson's disease by the discovery of rare, inherited mutations in gene products that associate with the mitochondria. Mutations in PTEN-induced kinase-1 (PINK1), which encodes a mitochondrial kinase, and PARKIN, encoding an E3 ubiquitin ligase, are the most frequent causes of recessive Parkinson's disease. Recent functional studies have revealed that PINK1 recruits PARKIN to mitochondria to initiate mitophagy, an important autophagic quality control mechanism that rids the cell of damaged mitochondria. PINK1 is post-translationally processed into a cleaved form whose levels are tightly regulated, although the significance of this processing is unknown. Here we demonstrate that the mitochondrial protease presenilin-associated rhomboid-like (PARL) can affect the proteolytic processing of PINK1 and that normal PINK1 localization and stability requires PARL's catalytic activity. PARL deficiency impairs PARKIN recruitment to mitochondria, suggesting PINK1's processing and localization are important in determining its interaction with PARKIN. We sequenced the PARL gene in Parkinson's disease patients and discovered a novel missense mutation in a functional domain of PARL's N-terminus. This PARL mutant is not sufficient to rescue PARKIN recruitment, suggesting that impaired mitophagy may be an underlying mechanism of disease pathogenesis in patients with PARL mutations.

Mutations in GDF5 presenting as semidominant brachydactyly A1.

Brachydactyly A1 (BDA1) is an autosomal dominant disorder characterized by shortness of all middle phalanges of the hands and toes, shortness of the proximal phalanges of the first digit, and short stature. Missense mutations in the Indian Hedgehog gene (IHH) are known to cause BDA1, and a second locus has been mapped to chromosome 5p. In a consanguineous French Canadian kindred with BDA1, both IHH and the 5p locus were excluded. Microsatellites flanking GDF5 on chromosome 20q were found to cosegregate with the disease. Sequencing of the GDF5 coding region revealed that a mildly affected individual in the family was heterozygous, and that all of the severely affected individuals were homozygous for a novel missense c.1195C>T mutation that predicts a p.Arg399Cys substitution at a highly conserved amino acid. Functional analysis demonstrated that although the p.Arg399Cys mutant is able to stimulate chondrogenesis, it is much less effective than wild-type GDF5. This data confirms genetic heterogeneity in BDA1, demonstrates that mutations upstream of IHH can result in BDA1, and shows that BDA1 can result from semidominant mutations in GDF5.

Large deletions account for an increasing number of mutations in SGCE.

Myoclonus-dystonia (M-D) (MIM 159900) is a rare "dystonia plus" syndrome, characterized by rapid myoclonic jerks, predominantly in the neck and upper limbs, in combination with dystonia. Mutations in the gene epsilon-sarcoglycan (SGCE) are known to be responsible for approximately one-third of cases. We screened 21 probands diagnosed with M-D for large deletions who were mutation negative as determined by PCR-direct sequencing. Multiplex PCR and quantification of PCR products was performed using a modified application of denaturing high performance liquid chromatography (dHPLC). We have identified two novel large multiexonic deletions of SGCE, which were confirmed by amplifying and sequencing the deletion breakpoints. Five other families were found to harbor small mutations identified by direct sequencing. Analysis of the region surrounding the deletions demonstrates that both deletions are the result of nonhomologous recombination with homologous end joining. This is only the second report of intragenic deletions with SGCE and it highlights the need to include exonic copy number variation when performing mutational analysis of SGCE.

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Voluntary Running Triggers VGF-Mediated Oligodendrogenesis to Prolong the Lifespan of Snf2h-Null Ataxic Mice.

Exercise has been argued to enhance cognitive function and slow progressive neurodegenerative disease. Although exercise promotes neurogenesis, oligodendrogenesis and adaptive myelination are also significant contributors to brain repair and brain health. Nonetheless, the molecular details underlying these effects remain poorly understood. Conditional ablation of the Snf2h gene impairs cerebellar development producing mice with poor motor function, progressive ataxia, and death between postnatal days 25-45. Here, we show that voluntary running induced an endogenous brain repair mechanism that resulted in a striking increase in hindbrain myelination and the long-term survival of Snf2h cKO mice. Further experiments identified the VGF growth factor as a major driver underlying this effect. VGF neuropeptides promote oligodendrogenesis in vitro, whereas Snf2h cKO mice treated with full-length VGF-encoding adenoviruses removed the requirement of exercise for survival. Together, these results suggest that VGF delivery could represent a therapeutic strategy for cerebellar ataxia and other pathologies of the CNS.

The ONDRISeq panel: custom-designed next-generation sequencing of genes related to neurodegeneration.

The Ontario Neurodegenerative Disease Research Initiative (ONDRI) is a multimodal, multi-year, prospective observational cohort study to characterise five diseases: (1) Alzheimer's disease (AD) or amnestic single or multidomain mild cognitive impairment (aMCI) (AD/MCI); (2) amyotrophic lateral sclerosis (ALS); (3) frontotemporal dementia (FTD); (4) Parkinson's disease (PD); and (5) vascular cognitive impairment (VCI). The ONDRI Genomics subgroup is investigating the genetic basis of neurodegeneration. We have developed a custom next-generation-sequencing-based panel, ONDRISeq that targets 80 genes known to be associated with neurodegeneration. We processed DNA collected from 216 individuals diagnosed with one of the five diseases, on ONDRISeq. All runs were executed on a MiSeq instrument and subjected to rigorous quality control assessments. We also independently validated a subset of the variant calls using NeuroX (a genome-wide array for neurodegenerative disorders), TaqMan allelic discrimination assay, or Sanger sequencing. ONDRISeq consistently generated high-quality genotyping calls and on average, 92% of targeted bases are covered by at least 30 reads. We also observed 100% concordance for the variants identified via ONDRISeq and validated by other genomic technologies. We were successful in detecting known as well as novel rare variants in 72.2% of cases although not all variants are disease-causing. Using ONDRISeq, we also found that the APOE E4 allele had a frequency of 0.167 in these samples. Our optimised workflow highlights next-generation sequencing as a robust tool in elucidating the genetic basis of neurodegenerative diseases by screening multiple candidate genes simultaneously.

Two novel disease-causing variants in BMPR1B are associated with brachydactyly type A1.

Brachydactyly type A1 is an autosomal dominant disorder primarily characterized by hypoplasia/aplasia of the middle phalanges of digits 2-5. Human and mouse genetic perturbations in the BMP-SMAD signaling pathway have been associated with many brachymesophalangies, including BDA1, as causative mutations in IHH and GDF5 have been previously identified. GDF5 interacts directly as the preferred ligand for the BMP type-1 receptor BMPR1B and is important for both chondrogenesis and digit formation. We report pathogenic variants in BMPR1B that are associated with complex BDA1. A c.975A>C (p.(Lys325Asn)) was identified in the first patient displaying absent middle phalanges and shortened distal phalanges of the toes in addition to the significant shortening of middle phalanges in digits 2, 3 and 5 of the hands. The second patient displayed a combination of brachydactyly and arachnodactyly. The sequencing of BMPR1B in this individual revealed a novel c.447-1G>A at a canonical acceptor splice site of exon 8, which is predicted to create a novel acceptor site, thus leading to a translational reading frameshift. Both mutations are most likely to act in a dominant-negative manner, similar to the effects observed in BMPR1B mutations that cause BDA2. These findings demonstrate that BMPR1B is another gene involved with the pathogenesis of BDA1 and illustrates the continuum of phenotypes between BDA1 and BDA2.

A mutation in the serine protease TMPRSS4 in a novel pediatric neurodegenerative disorder.

BACKGROUND: To elucidate the genetic basis of a novel neurodegenerative disorder in an Old Order Amish pedigree by combining homozygosity mapping with exome sequencing. METHODS AND RESULTS: We identified four individuals with an autosomal recessive condition affecting the central nervous system (CNS). Neuroimaging studies identified progressive global CNS tissue loss presenting early in life, associated with microcephaly, seizures, and psychomotor retardation; based on this, we named the condition Autosomal Recessive Cerebral Atrophy (ARCA). Using two unbiased genetic approaches, homozygosity mapping and exome sequencing, we narrowed the candidate region to chromosome 11q and identified the c.995C > T (p.Thr332Met) mutation in the TMPRSS4 gene. Sanger sequencing of additional relatives confirmed that the c.995C > T genotype segregates with the ARCA phenotype. Residue Thr332 is conserved across species and among various ethnic groups. The mutation is predicted to be deleterious, most likely due to a protein structure alteration as demonstrated with protein modelling. CONCLUSIONS: This novel disease is the first to demonstrate a neurological role for a transmembrane serine proteases family member. This study demonstrates a proof-of-concept whereby combining exome sequencing with homozygosity mapping can find the genetic cause of a rare disease and acquire better understanding of a poorly described protein in human development.

Brachydactyly A-1 mutations restricted to the central region of the N-terminal active fragment of Indian Hedgehog.

Mutations in the gene Indian Hedgehog (IHH) that cause Brachydactyly A-1 (BDA1) have been restricted to a specific region of the N-terminal active fragment of Indian Hedgehog involving codons 95, 100, 131, and 154. We describe two novel mutations in codons 128 and 130, not previously implicated in BDA1. Furthermore, we identified an independent mutation at codon 131 and we also describe a New Zealand family, which carries the 'Farabee' founder mutation and haplotype. All of the BDA1 mutations occur in a restricted area of the N-terminal active fragment of the IHH and are in contrast to those mutations causing an autosomal recessive acrocapitofemoral dysplasia, whose mutations are located at the distal N- and C-terminal regions of IHH-N and are physically separated from the BDA1-causing mutations. The identification of multiple independent mutations in codons 95, 100, and now in 131, implicate a discrete function for this region of the protein. Finally, we present a clinical review of all reported and confirmed cases of BDA1, highlighting features of the disorder, which add to the spectrum of the IHH mutations.

Refinement of the DYT15 locus in myoclonus dystonia.

Inherited myoclonus dystonia (MD) is an autosomal dominant disorder in which we previously mapped a novel locus to chromosome18p11 (OMIM number: 607488). Since no further informative STS markers were found within the flanking shared regions, we utilized single nucleotide polymorphisms (SNP) for fine-mapping. All known or predicted genes within this region were directly sequenced. We identified three recombinant SNPs in the distal region but none from the proximal region. Our previous linked region has now been reduced to 3.18 Mb but direct sequencing of all seven known and four predicted genes with EST support did not identify any mutations..

Translated mutation in the Nurr1 gene as a cause for Parkinson's disease.

Multiple genes have been now identified as causing Parkinson's disease (PD). In 2003, two mutations were identified in exon 1 of the Nurr1 gene in 10 of 107 individuals with familial PD. To date, investigators have only focused on screening for these known mutations of the Nurr1 gene. All individuals were recruited from two Parkinson's disease clinics in Canada. Following PCR amplification of each exon of the Nurr1 gene, samples underwent denaturing high-performance liquid chromatography (DHPLC) analysis. Ten individuals also underwent direct sequencing as well as any samples where variants were identified. The Nurr1 gene was evaluated for 202 PD individuals, 37% of whom had at least one relative with PD and 100 control non-PD individuals. Using DHPLC and direct sequencing, we did not detect any sequence variants in exon 1. Variants in amplicon 6 were seen and direct sequencing confirmed a known NI6P polymorphism in intron 6. Novel polymorphisms were also identified in exon 3 and intron 5. A novel mutation was identified in exon 3 in one nonfamilial PD individual. This heterozygous C-to-G transversion resulted in a serine-to-cysteine substitution and was not identified in any of the other 602 chromosomes screened. Mutations in the Nurr1 gene in our large cohort of familial and sporadic PD individuals are rare. The novel mutation in exon 3 is predicted to affect phosphorylation and functional studies to assess this are underway. This is the first coding mutation identified in the Nurr1 gene for Parkinson's disease.

A novel mutation in the IHH gene causes brachydactyly type A1: a 95-year-old mystery resolved.

Brachydactyly type A1 (BDA1) was the first disorder described in terms of autosomal dominant Mendelian inheritance. Early in the 1900s Farabee and Drinkwater described a number of families with BDA1. Examination of two of Drinkwater's families has revealed that, although they are not known to be related, both share a common mutation within the Indian hedgehog gene ( IHH). This novel mutation is a guanine to adenine transition at nucleotide 298, resulting in an Asn100Asp amino acid substitution. Both families demonstrate significant intrafamilial phenotypic heterogeneity among the affected individuals. Examination of single nucleotide polymorphisms (SNP) has shown that the affected individuals in both families share SNPs within IHH consistent with that of a common founder. The identification of the same mutation in these families has answered a question that is nearly a century old about the genetic cause of their disease and supports the hypothesis that IHH plays a pivotal role in normal human skeletogenesis.