ABSTRACT Background and Objectives Spinocerebellar ataxias (SCA) represent a clinically and genetically heterogeneous group of progressive neurodegenerative diseases with prominent cerebellar atrophy. Recently, a novel pathogenic repeat expansion in intron 1 of FGF14 was identified, causing adult‐onset SCA (SCA27B). We aimed to determine the proportion of our unsolved adult‐onset ataxia cohort harboring this expansion using several technologies, and to characterize the phenotypic presentation within our population. Methods Individuals presenting with adult‐onset ataxia (> 30 years old) and negative previous genetic testing were selected from the Care4Rare patient repository. Affected individuals were from all ethnicities, and 90% had a family history suggestive of dominant ataxia, representing 19 of the 23 families included. We used multiple tools (PCR, long‐read genome sequencing and optical genome mapping (OGM)) to identify the pathogenic GAA repeat in FGF14. Results Of the 23 families included in this study, 65.2% harbored a pathogenic GAA expansion in FGF14. Individuals of French‐Canadian descent (FC) represented most of our cohort and had a 64.7% diagnostic yield. Affected individuals presented with gaze‐evoked nystagmus, gait ataxia, cerebellar dysarthria, and early episodic features. The GAA expansion in FGF14 was visible by OGM in all individuals tested. Interpretation Our diagnostic yield demonstrates this expansion may be the most common cause of adult‐onset SCA in dominant families of FC ancestry. Our FC participants have a phenotype distinct from previously published FC patients, with gaze‐evoked nystagmus being the most common eye anomaly. From a diagnostic standpoint, the pathogenic GAA repeat can be identified by OGM, but additional tests are required to complement the interpretation.

Canada is a federation of 14 governments: one federal, ten provincial, and three territorial, that represent small and ethnically diverse populations. Although healthcare is publicly funded, its delivery is a provincial/territorial responsibility. As a result, while rare genetic diseases (RD) affect people across Canada, availability and eligibility for clinical genome-wide sequencing (GWS) varies considerably by region. Until recently, most GWS tests for RD were sent out of country, resulting in a large cost to the healthcare system, long turn-around times, and loss of opportunity to develop clinical knowledge.

Background Acute change in gait speed while performing a mental task [dual-task gait cost (DTC)], and hyperintensity magnetic resonance imaging signals in white matter are both important disability predictors in older individuals with history of stroke (poststroke). It is still unclear, however, whether DTC is associated with overall hyperintensity volume from specific major brain regions in poststroke. Methods This is a cohort study with a total of 123 older (69 ± 7 years of age) participants with history of stroke were included from the Ontario Neurodegenerative Disease Research Initiative. Participants were clinically assessed and had gait performance assessed under single- and dual-task conditions. Structural neuroimaging data were analyzed to measure both, white matter hyperintensity (WMH) and normal appearing volumes. Percentage of WMH volume in frontal, parietal, occipital, and temporal lobes as well as subcortical hyperintensities in basal ganglia + thalamus were the main outcomes. Multivariate models investigated associations between DTC and hyperintensity volumes, adjusted for age, sex, years of education, global cognition, vascular risk factors, APOE4 genotype, residual sensorimotor symptoms from previous stroke and brain volume. Results There was a significant positive global linear association between DTC and hyperintensity burden (adjusted Wilks’ λ = .87, P = .01). Amongst all WMH volumes, hyperintensity burden from basal ganglia + thalamus provided the most significant contribution to the global association (adjusted β = .008, η2 = .03; P = .04), independently of brain atrophy. Conclusions In poststroke, increased DTC may be an indicator of larger white matter damages, specifically in subcortical regions, which can potentially affect the overall cognitive processing and decrease gait automaticity by increasing the cortical control over patients’ locomotion.

Background: Although pathogenic 22q11.2 deletions are an important cause of developmental delays and lifelong disease burden, their variable and complex clinical expression contributes to under-recognition, delayed molecular diagnosis and uncertainty about prevalence. We sought to estimate the contemporary live-birth prevalence of typical 22q11.2 deletions using a population-based newborn screening sample and to examine data available for associated clinical features. Methods: Using DNA available from an unbiased sample of about 12% of all dried blood spots collected for newborn screening in Ontario between January 2017 and September 2018, we prospectively screened for 22q11.2 deletions using multiplex quantitative polymerase chain reaction assays and conducted independent confirmatory studies. We used cross-sectional analyses to compare available clinical and T-cell receptor excision circle (TREC, used in newborn screening for severe combined immunodeficiency) data between samples with and without 22q11.2 deletions. Results: The estimated minimum prevalence of 22q11.2 deletions was 1 in 2148 (4.7 per 10 000) live births (95% confidence interval [CI] 2.5 to 7.8 per 10 000), based on a total of 30 074 samples screened, with 14 having confirmed 22q11.2 deletions. Of term singletons, samples with 22q11.2 deletions had significantly younger median maternal age (25.5 v. 32.0 yr, difference −6.5 yr, 95% CI −7 to −2 yr), a greater proportion with small birth weight for gestational age (odds ratio 7.00, 95% CI 2.36 to 23.18) and lower median TREC levels (108.9 v. 602.5 copies/3 μL, p < 0.001). Interpretation: These results indicate that the 22q11.2 deletion syndrome is one of the most common of rare genetic conditions and may be associated with relatively younger maternal ages and with prenatal growth abnormalities. The findings support the public health importance of early — prenatal and neonatal — diagnosis that would enable prompt screening for and management of well-known actionable features associated with 22q11.2 deletions.

Introduction Myoclonus-dystonia is an inherited disorder characterized by a combination of myoclonic jerks and dystonia. Mutations in the epsilon-sarcoglycan gene ( SGCE ) represent the main known genetic cause. In the last few years, deep brain stimulation (DBS) has shown significant promise in treating these patients. There is only one report in the literature of a patient with positive SGCE mutation and isolated myoclonus phenotype who has been successfully treated with DBS. Case Presentation We present a case of a 16-year-old young man with a history of quick jerks since childhood. They progressed gradually over the years involving the entire body and interfering with most of his daily activities. He had no dystonia. Genetic testing identified a single base deletion in exon 3 of the SGCE gene, considered very likely pathogenic. After unsuccessfully trying several oral medications, he underwent DBS of the globus pallidus internus (GPi). His Unified Myoclonus Rating Scale score during rest and with action improved by 92.8% and 82.6%, respectively. Discussion The striking effect of DBS on myoclonic jerks confirms the superior benefit of DBS over oral medications. Further study is needed to determine the role of mutation status in predicting DBS response, especially considering that myoclonus-dystonia is genetically heterogeneous. Conclusion Our case confirms the poor response to oral medications and supports the use of GPi DBS for patients with genetically confirmed myoclonus-dystonia and isolated-myoclonus phenotype. In addition, our case represents familial myoclonus-dystonia due to a novel SGCE mutation.

Background: Artemis enzyme, encoded by the DCLRE1C gene, is essential to V(D)J recombination in both T and B lymphocytes. Artemis functions as an important component of the nonhomologous end-joining DNA double-strand break repair pathway. Artemis deficiency leads to a T-B-NK+ severe combined immune deficiency (SCID) associated with radiosensitivity. Clinical presentation: We present a case of a positive newborn screen for SCID in a patient who was subsequently shown to have a T-B-NK+ phenotype. Further immune evaluation showed profound T and B lymphopenia, near-absent response to mitogen stimulation, and absent immunoglobulins A and M. Genetic investigation demonstrated a novel and putative pathogenic variant in the DCLRE1C gene. Conclusion: This case identifies a novel variant in the DCLRE1C gene in a patient with SCID identified by newborn screening. Statement of novelty: This case report identifies a novel variant in the DCLRE1C gene in a patient with T-B-NK+ SCID.

Carnitine Uptake Defect (CUD) is an autosomal recessive disorder due to mutations in the SLC22A5 gene. Classically patients present in infancy with profound muscle weakness and cardiomyopathy with characteristic EKG findings. Later presentations include recurrent hypoketotic hypoglycemia, proximal limb girdle myopathy, and/or recurrent muscle pain. Newborn screening detects most of these clinical variants but in addition has identified maternal CUD often in asymptomatic women. We describe a family ascertained through 3 newborn screening (NBS) positive infants found to be unaffected themselves but in whom the mothers (sisters) were affected. There were also two affected children born to an affected male and his heterozygous wife who were false negatives on NBS but had increased fractional excretion of free carnitine in the urine. Analysis on a Next Generation Sequencing panel specifically designed to fully cover newborn screening disease targets showed a homozygous change in the five probands (SLC22A5; NM_003060:c.-149G > A; p.?). The mutation segregates with the CUD within the family. It is in the 5' UTR and has a frequency within the gnomAd database of 0.001198. Plasma carnitine was decreased and fractional excretion of free carnitine was increased in all affected individuals. Functional carnitine uptake studies in cultured skin fibroblasts of one proband showed carnitine uptake at the 5 mu M concentration to be 6% of controls. Relative expression of OCTN2 mRNA to beta-actin mRNA by qRT-PCR was increased in a proband relative to controls by a factor of 465-fold. Western blotting revealed a 120 kDa protein band, as well as a weaker 240 kDa band in the proband, the significance of which is unknown at this time.

Purpose To assess the clinical utility of next-generation sequencing (NGS) for the diagnosis of patients with optic atrophy (OA). Design Retrospective cohort study. Methods 97 patients were referred to the McMaster University Medical Center (Hamilton, Ontario) for evaluation of bilateral OA. All patients were sent for NGS including a 22 nuclear gene panel and/or complete mitochondrial DNA (mtDNA) sequencing. Positive genetic test results and abnormal vibration sensation were compared in patients +/- environmental exposures or a family history. Results 19/94 (20.2%) had a positive nuclear variant, of which 15/19 (78.9%) were in the OPA1 gene. No positive mtDNA variants were identified. The detection of a positive genetic variant was significantly different in patients who reported excessive ethanol use, but not in patients who smoke (0/19 (0%) vs. 19/78 (24.4%), P = 0.0164 and 4/22 (18.2%) vs. 15/74 (20.3%), P = 0.829, respectively). Patients with a positive family history were more likely to have a positive genetic variant compared to patients with a negative family history (P = 0.0112). There were significantly more excessive drinkers with an abnormal vibration sensation (P = 0.026), and with a similar trend in smokers (P = 0.074). Conclusions All positive genetic variants were identified in nuclear genes. We identified a potential independent pathophysiological link between a history of excessive ethanol consumption and bilateral OA. Further investigations should evaluate and identify potential environmental risk factors for OA.

Measurement of T-cell receptor (TCR) excision circles (TRECs) as a screening tool has revolutionized the diagnosis and management of many T-cell primary immunodeficiencies by enabling early diagnosis.1,2 The screening test is based on quantitative analysis of a DNA byproduct of T-cell maturation, the δRec-ΨJα TREC,3 which is produced late in T-cell maturation by the majority of αβ T cells. TREC levels have been shown to correlate well with thymic output of new T-cell emigrants.1,3

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.

Ataxia telangiectasia (AT) is a genetic condition caused by mutations involving ATM (Ataxia Telangiectasia Mutated). This gene is responsible for the expression of a DNA double stranded break repair kinase, the ATM protein kinase. The syndrome encompasses combined immunodeficiency and various degrees of neurological abnormalities and increased risk of malignancy. Typically, patients present early in life with delay in neurological milestones, but very infrequently, with life threatening infections typical of a profound T cell deficiency. It would therefore be unexpected to identify this condition immediately after birth using T cell receptor excision circle (TREC)-based newborn screening (NBS) for SCID. We sought to evaluate the frequency of AT detected by NBS, and to assess immunity as well as the genetic aberrations associated with this early presentation. Here, we describe the clinical, laboratory, and genetic features of patients diagnosed with AT through the Ontario NBS program for SCID, and followed in our center since its inception in 2013. Four patients were diagnosed with AT as a result of low TRECs on NBS. In each case, whole exome sequencing was diagnostic. All of our patients had compound heterozygous mutations involving the FRAP-ATM-TRRAP (FAT) domain of the ATM gene, which appears critical for kinase activity and is highly sensitive to mutagenesis. Our patients presented with profound lymphopenia involving both B and T cells. The ratio of naïve/memory CD45+RA/RO T cells population was variable. T cell repertoire showed decreased T cell diversity. Two out of four patients had decreased specific antibody response to vaccination and hypogammaglobulinemia requiring IVIG replacement. In two patients, profound decreased responses to phytohemagglutinin stimulation was observed. In the other two patients, the initial robust response declined with time. In summary, the rate of detection of AT through NBS had been surprisingly high at our center. One case was identified per year, while the total rate for SCID has been five new cases per year. This early detection may allow for better prospective evaluation of AT shortly after birth, and may assist in formulating early and more effective interventions both for the neurological as well as the immune abnormalities in this syndrome.

Coffin–Siris and Nicolaides–Baraitser syndromes (CSS and NCBRS) are Mendelian disorders caused by mutations in subunits of the BAF chromatin remodeling complex. We report overlapping peripheral blood DNA methylation epi-signatures in individuals with various subtypes of CSS ( ARID1B , SMARCB1 , and SMARCA4 ) and NCBRS ( SMARCA2 ). We demonstrate that the degree of similarity in the epi-signatures of some CSS subtypes and NCBRS can be greater than that within CSS, indicating a link in the functional basis of the two syndromes. We show that chromosome 6q25 microdeletion syndrome, harboring ARID1B deletions, exhibits a similar CSS/NCBRS methylation profile. Specificity of this epi-signature was confirmed across a wide range of neurodevelopmental conditions including other chromatin remodeling and epigenetic machinery disorders. We demonstrate that a machine-learning model trained on this DNA methylation profile can resolve ambiguous clinical cases, reclassify those with variants of unknown significance, and identify previously undiagnosed subjects through targeted population screening.

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.

Primary CoQ10 deficiency is a clinically and genetically heterogeneous, autosomal recessive disorder resulting from mutations in genes involved in the synthesis of coenzyme Q10 (CoQ10). To date, mutations in nine proteins required for the biosynthesis of CoQ10 cause CoQ10 deficiency with varying clinical presentations. In 2009 the first patient with mutations in COQ9 was reported in an infant with a neonatal-onset, primary CoQ10 deficiency with multi-system disease. Here we describe four siblings with a previously undiagnosed lethal disorder characterized by oligohydramnios and intrauterine growth restriction, variable cardiomyopathy, anemia, and renal anomalies. The first and third pregnancy resulted in live born babies with abnormal tone who developed severe, treatment unresponsive lactic acidosis after birth and died hours later. Autopsy on one of the siblings demonstrated brain changes suggestive of the subacute necrotizing encephalopathy of Leigh disease. Whole-exome sequencing (WES) revealed the siblings shared compound heterozygous mutations in the COQ9 gene with both variants predicted to affect splicing. RT-PCR on RNA from patient fibroblasts revealed that the c.521 + 2 T > C variant resulted in splicing out of exons 4-5 and the c.711 + 3G > C variant spliced out exon 6, resulting in undetectable levels of COQ9 protein in patient fibroblasts. The biochemical profile of patient fibroblasts demonstrated a drastic reduction in CoQ10 levels. An additional peak on the chromatogram may represent accumulation of demethoxy coenzyme Q (DMQ), which was shown previously to accumulate as a result of a defect in COQ9. This family expands our understanding of this rare metabolic disease and highlights the prenatal onset, clinical variability, severity, and biochemical profile associated with COQ9-related CoQ10 deficiencies.

The inherited peripheral neuropathies ( IPNs ) are characterized by marked clinical and genetic heterogeneity and include relatively frequent presentations such as Charcot‐Marie‐Tooth disease and hereditary motor neuropathy, as well as more rare conditions where peripheral neuropathy is associated with additional features. There are over 250 genes known to cause IPN ‐related disorders but it is estimated that in approximately 50% of affected individuals a molecular diagnosis is not achieved. In this study, we examine the diagnostic utility of whole‐exome sequencing ( WES ) in a cohort of 50 families with 1 or more affected individuals with a molecularly undiagnosed IPN with or without additional features. Pathogenic or likely pathogenic variants in genes known to cause IPN were identified in 24% (12/50) of the families. A further 22% (11/50) of families carried sequence variants in IPN genes in which the significance remains unclear. An additional 12% (6/50) of families had variants in novel IPN candidate genes, 3 of which have been published thus far as novel discoveries ( KIF1A , TBCK , and MCM3AP ). This study highlights the use of WES in the molecular diagnostic approach of highly heterogeneous disorders, such as IPNs , places it in context of other published neuropathy cohorts, while further highlighting associated benefits for discovery.

Background: The protein encoded by interleukin-2 receptor common gamma chain (IL2RG) is an important signaling component of many interleukin receptors, including those of interleukin-2, -4, -7, and -21, known as the common gamma chain. Mutations in the gene encoding the common gamma chain of the interleukin-2 receptor cause X-linked severe combined immunodeficiency (SCID). In this report, we present an unknown genetic defect of a patient diagnosed with SCID whose genetic analysis was performed 2 decades later. Methods: Whole genome sequencing and Sanger confirmation were used to identify a novel frameshift mutation in IL2RG. Massively parallel sequencing of genes associated with SCID were performed on the patient's mother and sister. Results: Next generation sequencing techniques identified a heterozygous frame-shift deletion in the gene encoding the common gamma chain of IL2RG in our patient. The patient's mother had a low level mosaicism for the same deletion. The sister had no detectable deletion. Conclusion: We have identified a novel mutation in IL2RG resulting in an X-linked SCID phenotype. The genetic analysis of the patient's mother revealed a mosaicism which was not passed on to his sister. The importance of genetic analysis in family members and SCID patients with an unknown genetic defect should be emphasized for family planning and subsequent genetic counseling. Statement of novelty: Genetic testing is an extremely important component in evaluating severe combined immunodeficiency as it impacts treatment course and prognosis, and allows for genetic analysis and counselling of family members.

T-cell receptor excision circle levels are a surrogate marker of T-cell production and immune system function. We sought to determine whether non-pathological levels of infant T-cell receptor excision circles were associated with adverse events following immunization. A self-controlled case series design was applied on a sample of 231,693 children who completed newborn screening for severe combined immunodeficiency in Ontario, Canada between August 2013 and December 2015. Exposures included routinely administered pediatric vaccines up to 15 months of age. Main outcomes were combined health services utilization for recognized adverse events following immunization. 1,406,981 vaccination events were included in the final dataset. 103,007 children received the Pneu-C-13 or Men-C-C vaccine and 97,998 received the MMR vaccine at 12 months of age. 67,725 children received the varicella immunization at 15 months. Our analysis identified no association between newborn T-cell receptor excision circle levels and subsequent health services utilization events following DTa-IPV-Hib, Pneu-C-13, and Men-C-C vaccinations at 2-month (RI 0.94[95%CI 0.87-1.02]), 4-month (RI 0.82[95%CI 0.75-0.9]), 6-month (RI 0.63[95%CI 0.57-0.7]) and 12-month (RI 0.49[95%CI 0.44-0.55]). We also found no trends in health services utilization following MMR (RI 1.43[95%1.34-1.52]) or varicella (RI 1.14[95%CI 1.05-1.23]) vaccination. Our findings provide further support for the safety of pediatric vaccinations.

A novel autosomal recessive disorder characterized by pre‐ and postnatal growth restriction with microcephaly, distinctive craniofacial features, congenital alopecia, hypoplastic kidneys with renal insufficiency, global developmental delay, severe congenital sensorineural hearing loss, early mortality, hydrocephalus, and genital hypoplasia was observed in 4 children from 3 families of New Mexican Hispanic heritage. Three of the children died before 3 years of age from uremia and/or sepsis. Exome sequencing of the surviving individual identified a homozygous c.587T>C (p.Ile196Thr) mutation in ZPR1 Zinc Finger (ZPR1) that segregated appropriately in her family. In a second family, the identical variant was shown to be heterozygous in the affected individual's parents and not homozygous in any of her unaffected siblings. ZPR1 is a ubiquitously expressed, highly conserved protein postulated to transmit proliferative signals from the cell membrane to the nucleus. Structural modeling reveals that p.Ile196Thr disrupts the hydrophobic core of ZPR1. Patient fibroblast cells showed no detectable levels of ZPR1 and the cells showed a defect in cell cycle progression where a significant number of cells remained arrested in the G1 phase. We provide genetic and molecular evidence that a homozygous missense mutation in ZPR1 is associated with a rare and recognizable multisystem syndrome.

Of late, the attacks or breaching of software systems has increased compared with accounts in the past. Vulnerabilities present in software have become a serious threat to security, and many times, intruders exploit known (as well as unknown) vulnerabilities. There is a current need to protect computer systems from exploits and develop systems that are potentially robust from such security breechings. Thus it is important to analyze the optimal time when vulnerabilities should be made public and also deduce an optimal time to fix certain vulnerabilities before they can be exploited. Based on the scientific technique of calculating software safety integrity level (SIL) in the functional safety standards, we have proposed the quantitative method for ensuring SIL within software systems to determine an optimal patch release policy for fixing the vulnerabilities after the software has been launched into the field. Validation of the proposed policy has been done using real-life vulnerability data of Microsoft Windows, and obtained results are well-versed in explaining the proposition. Further sensitivity analysis has been performed to form a better understanding.