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To evaluate the impact of implementing commercial whole exome sequencing (WES) and targeted gene panel testing in pregnancies with fetal anomalies.A retrospective chart review of 124 patients with sequencing performed by commercial laboratories.The diagnostic yield of WES and panel testing was 21.5% and 26%, respectively, based on likely pathogenic (LP) or pathogenic (P) variants. Forty-two percent of exomes and 32% of panels analysed had one or more variants of uncertain significance (VUS) reported. A multidisciplinary in-depth review of the fetal phenotype, disease phenotype, variant data, and, in some patients, additional prenatal or postnatal investigations increased the diagnostic yield by 5% for exome analysis and 6% for panel analysis.The diagnostic yield of WES and panel testing combined was 23% based on LP and P variants. Although the reporting of VUS contributed to a 5% increase in diagnostic yield for WES and 6% for panels, the large number of VUS reported by commercial laboratories has significant resource implications. Our results support the need for greater adherence to the recommendations on the prenatal reporting of VUS and the importance of a multidisciplinary approach that brings together clinical and laboratory expertise in prenatal genetics and genomics.

Background Acid Sphingomyelinase Deficiency (ASMD) is a rare autosomal recessive disorder caused by mutations in the SMPD1 gene. This rarity contributes to misdiagnosis, delayed diagnosis and barriers to good care. There are no published national or international consensus guidelines for the diagnosis and management of patients with ASMD. For these reasons, we have developed clinical guidelines that defines standard of care for ASMD patients. Methods The information contained in these guidelines was obtained through a systematic literature review and the experiences of the authors in their care of patients with ASMD. We adopted the Appraisal of Guidelines for Research and Evaluation (AGREE II) system as method of choice for the guideline development process. Results The clinical spectrum of ASMD, although a continuum, varies substantially with subtypes ranging from a fatal infantile neurovisceral disorder to an adult-onset chronic visceral disease. We produced 39 conclusive statements and scored them according to level of evidence, strengths of recommendations and expert opinions. In addition, these guidelines have identified knowledge gaps that must be filled by future research. Conclusion These guidelines can inform care providers, care funders, patients and their carers about best clinical practice and leads to a step change in the quality of care for patients with ASMD with or without enzyme replacement therapy (ERT).

Background: Diet, with an emphasis on phenylalanine restriction, is recognized as an effective treatment for phenylketonuria (PKU). A similar approach is used in pregnant PKU patients to reduce teratogenic effects of elevated circulating phenylalanine. Our objective was to assess the dietary management of pregnant PKU individuals at Vancouver General Hospital, Canada, retrospectively including dietary recommendations, phenylalanine intake, patient compliance and a comparison with protein and phenylalanine requirements determined in healthy pregnancies. Methods: We reviewed dietitian’s health records for pregnant patients monitored for PKU between 1999-2019 at the Adult Metabolic Diseases Clinic (AMDC). Data were extracted on demographics, gestational weight gain, gestational age at delivery, pregnancy outcomes, plasma analyses, reported health concerns, and dietary intake of phenylalanine, tyrosine, and protein. We present descriptive statistics of subject characteristics and clinical results. Paired sample t-test assessed for differences between prescribed phenylalanine and phenylalanine intake. Generalized additive mixed models approach was used to observe changes in phenylalanine concentration in blood spots and phenylalanine tolerance throughout the pregnancy. Results: Eleven patients aged 20-40 years, with a total of 16 pregnancies, were included for analysis. Prescribed phenylalanine intake increased from 7.2 ± 4.3 to 16.7 ± 4.8 mg·kg-1·d-1 between early (13-19wk) and late (33-39wk) gestation. Actual phenylalanine intakes increased from 7.5.2 ± 4.3 to 17.7 ± 4.9 mg·kg-1·d-1 between these stages and were not statistically different. When compared to our previously determined mean phenylalanine requirements in healthy pregnancies during ~16wk pregnancy (15 mg·kg-1·d-1) and late (~36wk) pregnancy (21 mg·kg-1·d-1), phenylalanine intake was 50% lower at 13-19wk gestation and 16 % lower at 33-39wk gestation. Mean phenylalanine concentration in blood spots decreased as pregnancy progressed, with the mean concentration being highest preconception, and a slight increase between 8 and 12 wk. Conclusions: Current management practices at AMDC are working well to achieve targeted metabolic control. Increase in phenylalanine requirements during early healthy pregnancy, combined with slight increase in blood spot phenylalanine in early PKU pregnancy is intriguing and suggests a need to directly determine phenylalanne needs. This natural history analysis provides reference values and management practices of pregnant PKU patients, contributing to the limited data available.

Large single mtDNA (mitochondrial DNA) deletion syndrome is a rare inborn error of metabolism with variable heteroplasmy levels and clinical phenotype among affected individuals. Chronic progressive external ophthalmoplegia (CPEO) is the most common phenotype in adults with this form of mitochondrial disease [1-2]. The common CPEO clinical manifestations are ptosis and ophthalmoplegia. More variable phenotypic manifestations of CPEO (CPEO plus) include involvement of the peripheral nervous system and myopathy. Here, we describe a 62-year-old female with CPEO and the major mitochondrial DNA deletion present at 40% heteroplasmy, who had a coexistent previously undescribed CPEO phenotypic feature of persistent unexplained macrocytosis without anemia. Building on this case, we reviewed other major mtDNA deletion cases seen in our Adult Metabolic Diseases Clinic (AMDC) at the University of British Columbia, Vancouver, Canada from 2016 to 2022. The major mtDNA deletion cases (n=26) were compared with mtDNA missense variants identified in the clinic over the same period who acted as the comparison group (n=16). Of these, the most frequent diagnosis was maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). Ten out of 26 (38%) of mtDNA deletion patients had macrocytosis with elevated MCV (mean corpuscular volume), median (IQR, interquartile range) of 108fl (102-114fl). Seven of the patients with macrocytosis had no pertinent etiology. None of the comparison group had macrocytosis. There was a significant difference (P=0.000) between the MCV & MCH in the mtDNA deletion group compared to the comparison group. This communication sheds light on the association of macrocytosis with the mtDNA deletion syndrome. It would be of great interest to determine if the association is found in other mitochondrial disease clinic populations.

Purpose Phosphatidylinositol Glycan Anchor Biosynthesis, class G (PIGG) is an ethanolamine phosphate transferase catalyzing the modification of glycosylphosphatidylinositol (GPI). GPI serves as an anchor on the cell membrane for surface proteins called GPI-anchored proteins (GPI-APs). Pathogenic variants in genes involved in the biosynthesis of GPI cause inherited GPI deficiency (IGD), which still needs to be further characterized. Methods We describe 22 individuals from 19 unrelated families with biallelic variants in PIGG. We analyzed GPI-AP surface levels on granulocytes and fibroblasts for three and two individuals, respectively. We demonstrated enzymatic activity defects for PIGG variants in vitro in a PIGG/PIGO double knockout system. Results Phenotypic analysis of reported individuals reveals shared PIGG deficiency–associated features. All tested GPI-APs were unchanged on granulocytes whereas CD73 level in fibroblasts was decreased. In addition to classic IGD symptoms such as hypotonia, intellectual disability/developmental delay (ID/DD), and seizures, individuals with PIGG variants of null or severely decreased activity showed cerebellar atrophy, various neurological manifestations, and mitochondrial dysfunction, a feature increasingly recognized in IGDs. Individuals with mildly decreased activity showed autism spectrum disorder. Conclusion This in vitro system is a useful method to validate the pathogenicity of variants in PIGG and to study PIGG physiological functions. Unlabelled Image

© 2021 American Society of Human Genetics, EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG., This work was supported by the EU FP7 large-scale integrating project Genetic and Epigenetic Networks in Cognitive Dysfunction (241995) (to H.v.B.); National Institutes of Health (NIH) grants 5R01GM115730-03 (to M.H.), U54 NS115198 (to A.C.E. and M.H.), and T32GM008638 (to A.C.E.); and the Transatlantic Network of Excellence grant (10CVD03) from the Fondation Leducq and NIH NHGRI U01HG006398 (to D.J.R.). Family 4 was enrolled in the CAUSES Study; investigators include Shelin Adam, Christele Du Souich, Alison Elliott, Anna Lehman, Jill Mwenifumbo, Tanya Nelson, Clara Van Karnebeek, and Jan Friedman; it is funded by Mining for Miracles, British Columbia Children’s Hospital Foundation (grant number F15-01355) and Genome British Columbia (grant number F16-02276). D.L.P. is recipient of a CAPES Fellowship (99999.013311/2013-01). X.B. is supported by an AHA career development award (19CDA34630032).

Mosaic KRAS variants and other RASopathy genes cause oculoectodermal, encephalo-cranio-cutaneous lipomatosis, and Schimmelpenning-Feuerstein-Mims syndromes, and a spectrum of vascular malformations, overgrowth and other associated anomalies, the latter of which are only recently being characterized. We describe eight individuals in total (six unreported cases and two previously reported cases) with somatic KRAS variants and variably associated features. Given the findings of somatic overgrowth (in seven individuals) and vascular or lymphatic malformations (in eight individuals), we suggest mosaic RASopathies (mosaic KRAS variants) be considered in the differential diagnosis for individuals presenting with asymmetric overgrowth and lymphatic or vascular anomalies. We expand the association with embryonal tumors, including the third report of embryonal rhabdomyosarcoma, as well as novel findings of Wilms tumor and nephroblastomatosis in two individuals. Rare or novel findings in our series include the presence of epilepsy, polycystic kidneys, and T-cell deficiency in one individual, and multifocal lytic bone lesions in two individuals. Finally, we describe the first use of targeted therapy with a MEK inhibitor for an individual with a mosaic KRAS variant. The purposes of this report are to expand the phenotypic spectrum of mosaic KRAS-related disorders, and to propose possible mechanisms of pathogenesis, and surveillance of its associated findings.

Item does not contain fulltext PURPOSE: JARID2, located on chromosome 6p22.3, is a regulator of histone methyltransferase complexes that is expressed in human neurons. So far, 13 individuals sharing clinical features including intellectual disability (ID) were reported with de novo heterozygous deletions in 6p22-p24 encompassing the full length JARID2 gene (OMIM 601594). However, all published individuals to date have a deletion of at least one other adjoining gene, making it difficult to determine if JARID2 is the critical gene responsible for the shared features. We aim to confirm JARID2 as a human disease gene and further elucidate the associated clinical phenotype. METHODS: Chromosome microarray analysis, exome sequencing, and an online matching platform (GeneMatcher) were used to identify individuals with single-nucleotide variants or deletions involving JARID2. RESULTS: We report 16 individuals in 15 families with a deletion or single-nucleotide variant in JARID2. Several of these variants are likely to result in haploinsufficiency due to nonsense-mediated messenger RNA (mRNA) decay. All individuals have developmental delay and/or ID and share some overlapping clinical characteristics such as facial features with those who have larger deletions involving JARID2. CONCLUSION: We report that JARID2 haploinsufficiency leads to a clinically distinct neurodevelopmental syndrome, thus establishing gene-disease validity for the purpose of diagnostic reporting.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell–omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

PURPOSE: Somatic variants in tumor necrosis factor receptor-associated factor 7 (TRAF7) cause meningioma, while germline variants have recently been identified in seven patients with developmental delay and cardiac, facial, and digital anomalies. We aimed to define the clinical and mutational spectrum associated with TRAF7 germline variants in a large series of patients, and to determine the molecular effects of the variants through transcriptomic analysis of patient fibroblasts.METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts.RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts.CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies.

Animal studies suggest that decreased vascular mitochondrial DNA copy number can promote hypertension. We conducted a chart review of blood pressure and hemodynamics in patients with either mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS, n = 36) or individuals with variants in the mitochondrial DNA polymerase gamma (POLG, n = 26). The latter included both pathogenic variants and variants of unknown significance (VUS). Hypertension rates (MELAS 50%, POLG 50%) were elevated relative to Canadian norms in 20-39 (MELAS) and 40-59 (MELAS and POLG) years of age groups. Peripheral resistance was high in the hypertensive versus normotensive patients, potentially indicative of microvascular disease. Despite antihypertensive treatment, systolic blood pressure remained elevated in the POLG versus MELAS group. The risk of hypertension was not associated with MELAS heteroplasmy. Hypertension rates were not different between individuals with known pathogenic POLG variants and those with VUS, including common variants. Hypertension (HT) also did not differ between patients with POLG variants with (n = 17) and without chronic progressive external opthalmoplegia (n = 9) (CPEO). HT was associated with variants in all three functional domains of POLG. These findings suggest that both pathogenic variants and several VUS in the POLG gene may promote human hypertension and extend our past reports that increased risk of HT is associated with MELAS.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

This study describes the cost trajectory of the standard diagnostic care pathway for children with suspected genetic disorders in British Columbia, Canada. Average annual per-patient costs were estimated using medical records review and a caregiver survey for a cohort of 498 children referred to BC Children’s and Women’s Hospitals (CW exome and genome sequencing) to 500 families of children with suspected genetic disorders. Direct costs peaked in the first year of patients’ diagnostic odyssey, with an average of C$2257 per patient (95% confidence interval [CI] C$2074, C$2441) for diagnostic testing and C$631 (95% CI C$543, C$727) for specialist consultations at C&W. In subsequent years, direct costs accrued at a constant rate, with an estimated annual per-patient cost of C$511 (95% CI C$473, C$551) for diagnostic testing and C$334 (95% CI C$295, C$369) for consultations at C&W. Travel costs and caregiver productivity loss associated with attending diagnosis-related physician appointments averaged C$1907/family/year. The continuing long-term accrual of costs by undiagnosed patients suggests that economic evaluations of diagnostic GWS services should use longer time horizons than have typically been used.

Heterozygous pathogenic variants in CIC, which encodes a transcriptional repressor, have been identified in individuals with neurodevelopmental phenotypes. To date, 11 CIC variants have been associated with the CIC-related neurodevelopmental syndrome. Here, we describe three novel and one previously reported CIC variants in four individuals with neurodevelopmental delay. Notably, we report for the first time a de novo frameshift variant specific to the long isoform of CIC (CIC-L, NM_001304815.1:c.1100dup, p.Pro368AlafsTer16) in an individual with speech delay, intellectual disability and autism spectrum disorder. Our investigation into the function of CIC-L reveals that partial loss of CIC-L leads transcriptional de-repression of CIC target genes. We also describe a missense variant (NM_015125.3:c.683G>A, p.Arg228Gln) in an individual with a history of speech delay and relapsed pre-B acute lymphoblastic leukemia (ALL). Functional studies of this variant suggest a partial loss of CIC transcriptional repressor activity. Our study expands the list of CIC pathogenic variants and contributes to the accumulating evidence that CIC haploinsufficiency or partial loss of function is a pathogenic mechanism causing neurodevelopmental phenotypes. This article is protected by copyright. All rights reserved.

Discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs*18) and presented with joint contractures, osteochondromas, and B cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in pro-survival and inflammatory genes. Systematic single-cell-omic analyses revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (with oncogenic signatures - MYC, JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further define detrimental effects a long-term use of calcineurin inhibitors.

The Chinese Communist Party and its supporters are increasingly using social media platforms to shape China’s public image. This online image is a means of strengthening domestic nationalism and of projecting “soft power” abroad. This paper examines various forms of anti-Westernism that are central to this image-making. It analyzes several recent topics—the Belt and Road Initiative, climate change, the COVID-19 vaccine, the Beijing Olympics, and the conflict in Ukraine—on the r/Sino subreddit page of Reddit and compares them with two online news outlets, the South China Morning Post and China Daily. The paper focuses on how these media frame the contest between a rising China and a failing West, so creating a discourse that competes with the negative portrayals of China outside the country. The paper contrasts the aggressive strengthening of China’s image against the West on social media with more sober accounts of the same topics in China’s official media and in commercial news outlets. The contribution of the paper is to document an emerging online anti-Westernism that is playing an increasing role in the changing geopolitical landscape.

This study aimed to compare downstream utilization of medical services among critically ill infants admitted to intensive care units who received rapid exome sequencing (ES) and those who followed alternative diagnostic testing pathways.Using propensity score-weighted regression models including sex, age at admission, and severity indicators, we compared a group of 47 infants who underwent rapid ES with a group of 211 infants who did not receive rapid ES. Utilization and cost indicators were compared between cohorts using negative binomial models for utilization and two-part models for costs.After controlling for patients' sociodemographic and clinical characteristics, we found no statistically significant difference in outpatient visits, hospitalizations, intensive care unit or total length of stay, or length of stay-associated costs between the cohorts at 12- or 26-month follow-up. Similarly, there was no evidence of higher utilization or costs by the ES group when infants who died were removed from the analysis.When examining utilization during and beyond the diagnostic trajectory, there is no evidence that ES changes frequency of outpatient visits or use of in-hospital resources in critically ill infants with suspected genetic disorders.

Purpose: Matchmaking has emerged as a useful strategy for building evidence toward causality of novel disease genes in patients with undiagnosed rare diseases. The Matchmaker Exchange (MME) is a collaborative initiative that facilitates international data sharing for matchmaking purposes; however, data on user experience is limited. Methods: Patients enrolled as part of the Finding of Rare Disease Genes in Canada (FORGE) and Care4Rare Canada research programs had their exome sequencing data reanalyzed by a multidisciplinary research team over a 2-year period. Compelling variants in genes not previously associated with a human phenotype were submitted through the MME node PhenomeCentral, and outcomes were collected. Results: In this study, 194 novel candidate genes were submitted to the MME, resulting in 1514 matches, and 15% of the genes submitted resulted in collaborations. Most submissions resulted in at least 1 match, and most matches were with GeneMatcher (82%), where additional email exchange was required to evaluate the match because of the lack of phenotypic or inheritance information. Conclusion: Matchmaking through the MME is an effective way to investigate novel candidate genes; however, it is a labor-intensive process. Engagement from the community to contribute phenotypic, genotypic, and inheritance data will ensure that matchmaking continues to be a useful approach in the future.

B-cell lymphoma/leukemia 11B (BCL11B) is a C2H2 zinc finger transcription factor that is critically important for regulating the development and function of a variety of systems including the central nervous system, the skin, and the immune system. Germline heterozygous variants are associated with a spectrum of clinical disorders, including severe combined immunodeficiency as well as neurological, craniofacial, and dermal defects. Of these individuals, ~50% present with severe allergic disease. Here, we report the detailed clinical and laboratory workup of one of the most severe BCL11B-dependent atopic cases to date. Leveraging a zebrafish model, we were able to confirm a strong T-cell defect in the patient. Based on these data, we classify germline BCL11B-dependent atopic disease as a novel primary atopic disorder.

Monoamine neurotransmitter disorders present predominantly with neurologic features, including dystonic or dyskinetic cerebral palsy and movement disorders. Genetic conditions that lead to secondary defects in the synthesis, catabolism, transport, and metabolism of biogenic amines can lead to neurotransmitter abnormalities, which can present with similar features. Eleven patients with secondary neurotransmitter abnormalities were enrolled between 2011 and 2015. All patients underwent research-based whole exome and/or whole genome sequencing (WES/WGS). A trial of treatment with levodopa/carbidopa and 5-hydroxytryptophan was initiated. In six families with abnormal neurotransmitter profiles and neurological phenotypes, variants in known disease-causing genes (KCNJ6, SCN2A, CSTB in 2 siblings, NRNX1, KIF1A and PAK3) were identified, while one patient had a variant of uncertain significance in a candidate gene (DLG4) that may explain her phenotype. In 3 patients, no compelling candidate genes were identified. A trial of neurotransmitter replacement therapy led to improvement in motor and behavioral symptoms in all but two patients. The patient with KCNJ6 variant did not respond to L-dopa therapy, but rather experienced increased dyskinetic movements even at low dose of medication. The patient’s symptoms harboring the NRNX1 deletion remained unaltered. This study demonstrates the utility of genome-wide sequencing in further understanding the etiology and pathophysiology of neurometabolic conditions, and the potential of secondary neurotransmitter deficiencies to serve as novel therapeutic targets. As there was a largely favorable response to therapy in our case series, a careful trial of neurotransmitter replacement therapy should be considered in patients with cerebrospinal fluid (CSF) monoamines below reference range.

Genome-wide sequencing (GWS) is a standard of care for diagnosis of suspected genetic disorders, but the proportion of patients found to have pathogenic or likely pathogenic variants ranges from less than 30% to more than 60% in reported studies. It has been suggested that the diagnostic rate can be improved by interpreting genomic variants in the context of each affected individual's full clinical picture and by regular follow-up and reinterpretation of GWS laboratory results. Trio exome sequencing was performed in 415 families and trio genome sequencing in 85 families in the CAUSES study. The variants observed were interpreted by a multidisciplinary team including laboratory geneticists, bioinformaticians, clinical geneticists, genetic counselors, pediatric subspecialists, and the referring physician, and independently by a clinical laboratory using standard American College of Medical Genetics and Genomics (ACMG) criteria. Individuals were followed for an average of 5.1 years after testing, with clinical reassessment and reinterpretation of the GWS results as necessary. The multidisciplinary team established a diagnosis of genetic disease in 43.0% of the families at the time of initial GWS interpretation, and longitudinal follow-up and reinterpretation of GWS results produced new diagnoses in 17.2% of families whose initial GWS interpretation was uninformative or uncertain. Reinterpretation also resulted in rescinding a diagnosis in four families (1.9%). Of the families studied, 33.6% had ACMG pathogenic or likely pathogenic variants related to the clinical indication. Close collaboration among clinical geneticists, genetic counselors, laboratory geneticists, bioinformaticians, and individuals’ primary physicians, with ongoing follow-up, reanalysis, and reinterpretation over time, can improve the clinical value of GWS.

Background The diagnostic journey for many rare disease patients remains challenging despite use of latest genetic technological advancements. We hypothesize that some patients remain undiagnosed due to more complex diagnostic scenarios that are currently not considered in genome analysis pipelines. To better understand this, we characterized the rare disorder (RD) spectrum using various bioinformatics resources (e.g., Orphanet/Orphadata, Human Phenotype Ontology, Reactome pathways) combined with custom-made R scripts. Results Our in silico characterization led to identification of 145 borderline-common, 412 rare and 2967 ultra-rare disorders. Based on these findings and point prevalence, we would expect that approximately 6.53%, 0.34%, and 0.30% of individuals in a randomly selected population have a borderline-common, rare, and ultra-rare disorder, respectively (equaling to 1 RD patient in 14 people). Importantly, our analyses revealed that (1) a higher proportion of borderline-common disorders were caused by multiple gene defects and/or other factors compared with the rare and ultra-rare disorders, (2) the phenotypic expressivity was more variable for the borderline-common disorders than for the rarer disorders, and (3) unique clinical characteristics were observed across the disorder categories forming the spectrum. Conclusions Recognizing that RD patients who remain unsolved even after genome sequencing might belong to the more common end of the RD spectrum support the usage of computational pipelines that account for more complex genetic and phenotypic scenarios.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl− channels and Cl−/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3−/− mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.

Purpose Intellectual disability (ID) results from a heterogeneous group of disorders and affects 1% to 2% of children. ID frequently occurs in association with other clinical features such as seizures or malformations. We suspected that strabismus might also be unusually frequent in this population and that it might be associated with ID groups affecting motor control. Methods We reviewed phenotypic descriptors, extracted from medical records, for a heterogeneous series of 222 probands with ID who had been enrolled in a study of clinical application of exome sequencing. We estimated the frequency of strabismus and other common clinical features and explored statistical associations between them. Data from Population Data British Columbia and Online Mendelian Inheritance in Man were also examined for confirmation of our observations. Results Strabismus had a higher prevalence among probands with ID than in the general population (odds ratio = 5.46). Moreover, probands with both ID and strabismus were more likely to have problems affecting motor control than those with ID and no strabismus (odds ratio = 2.84). Hypotonia was one of the most common motor control subgroups affecting the ID probands, and a frequent co-occurrence of strabismus and hypotonia was also observed (odds ratio = 2.51) and supported by related gene literature review. There was no evidence for associations between strabismus and other frequent clinical features. Conclusion Strabismus is a frequent feature in individuals with ID. The frequent co-occurrence of strabismus and motor control phenotypes, in particular hypotonia, suggests that a common cerebellar mechanism or pathway may underlie these phenotypes.

Genetic disorders are one of the leading causes of infant mortality and are frequent in neonatal intensive care units (NICUs). Rapid genome-wide sequencing (GWS; whole genome or exome sequencing (ES)), due to its diagnostic capabilities and immediate impacts on medical management, is becoming an appealing testing option in the NICU setting. RAPIDOMICS was a trio-based rapid ES pilot study of 25 babies with suspected genetic disorders in the BC Women's Hospital NICU. ES and bioinformatic analysis were performed after careful patient ascertainment. Trio analysis was performed using an in-house pipeline reporting variants in known disease-causing genes. Variants interpreted by the research team as definitely or possibly causal of the infant's phenotype were Sanger validated in a clinical laboratory. The average time to preliminary diagnosis was 7.2 days. Sanger validation was pursued in 15 patients for 13 autosomal dominant and 2 autosomal recessive disorders, with an overall diagnostic rate (partial or complete) of 60%.Conclusion: In total, 72% of patients enrolled had a genomic diagnosis achieved through ES, multi-gene panel testing or chromosomal microarray analysis. Among these, there was an 83% rate of significant and immediate impact on medical decision-making directly related to new knowledge of the diagnosis. Health service implementation challenges and successes are discussed. What is Known: • Rapid genome-wide sequencing in the neonatal intensive care setting has a greater diagnostic hit rate and impact on medical management than conventional genetic testing. However, the impact of consultation with genetics and patient ascertainment requires further investigation. What is New: • This study demonstrates the importance of genetic consultation and careful patient selection prior to pursuing exome sequencing (ES). • In total, 15/25 (60%) patients achieved a diagnosis through ES and 18/25 (72%) through ES, multi-gene panel testing or chromosomal microarray analysis with 83% of those having immediate effects on medical management.

Contains fulltext : 202928.pdf (Publisher’s version ) (Open Access) Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants.

Research teams developing biobanks and/or genomic databases must develop policies for the disclosure and reporting of potentially actionable genomic results to research participants. Currently, a broad range of approaches to the return of results exist, with some studies opting for non-disclosure of research results while others follow clinical guidelines for the return of potentially actionable findings from sequencing. In this review, we describe current practices and highlight decisions a research team must make when designing a return of results policy, from informed consent to disclosure practices and clinical validation options. The unique challenges of returning incidental findings in cardiac genes, including reduced penetrance and the lack of clinical screening standards for phenotype-negative individuals are discussed. Lastly, the National Hearts in Rhythm Organization (HiRO) Registry approach is described to provide a rationale for the selective return of field-specific variants to those participating in disease-specific research. Our goal is to provide researchers with a resource when developing a return of results policy tailored for their research program, based on unique factors related to study design, research team composition and availability of clinical resources.

Background: Following an unexplained cardiac arrest, clinical genetic testing is increasingly becoming standard of care. Periodic review of variant classification is required, as reinterpretation can change the diagnosis, prognosis, and management of patients and their relatives. Methods: This study aimed to develop and validate a standardized algorithm to facilitate clinical application of the 2015 American College of Medical Genetics and Association for Molecular Pathology guidelines for the interpretation of genetic variants. The algorithm was applied to genetic results in the Cardiac Arrest Survivors With Preserved Ejection Fraction Registry, to assess the rate of variant reclassification over time. Variant classifications were then compared with the classifications of 2 commercial laboratories to determine the rate and identify sources of variant interpretation discordance. Results: Thirty-one percent of participants (40 of 131) had at least 1 genetic variant with a clinically significant reclassification over time. Variants of uncertain significance were more likely to be downgraded (73%) to benign than upgraded to pathogenic (27%; P =0.03). For the second part of the study, 50% (70 of 139) of variants had discrepant interpretations (excluding benign variants), provided by at least 1 team. Conclusions: Periodic review of genetic variant classification is a key component of follow-up care given rapidly changing information in the field. There is potential for clinical care gaps with discrepant variant interpretations, based on the interpretation and application of current guidelines. The development of gene- and disease-specific guidelines and algorithms may provide an opportunity to further standardize variant interpretation reporting in the future. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT00292032.

Purpose: The presentation and underlying etiology of Cerebral Palsy (CP) in general are heterogenous. Clinical features present differently in pediatric versus adult patient populations. Many metabolic and genetic conditions present with clinical symptoms suggestive of CP. Precision medicine practices are currently a standard of care, and Next-Generation-Sequencing (NGS) tools are used for the purpose of diagnosis and management. We describe the diagnostic yield and impact on management of NGS comparing a cohort of 102 children and 37 adults with CP, referred to two tertiary care centres between 2015 and 2020 (adult cohort) and 2017–2020 (pediatric cohort) respectively. Principal results: In the adult cohort, 28 patients had a positive genetic diagnosis, giving a yield of 75.6%. Their age varied between 18 and 59 years, with a median of 28 years. Out of the positive diagnoses, 12 were consistent with an inborn error of metabolism and in 9 patients (32.1%) some form of treatment or management guideline was recommended. In the pediatric cohort 21 patients had a positive genetic diagnosis and 22 results are still pending, giving a yield of 32.8%. Age at diagnosis ranged between 18 months and 12 years. In 15 patients (71.4%) there was some form of management recommendation. All families benefited from genetic counseling. Major conclusions: Given the combined high yield of positive genetic diagnosis in pediatric and adult cases presenting with symptoms of Cerebral Palsy, and the more readily available Next Generation Sequencing testing in major academic centres, we recommend that either a referral to a pediatric or adult neurometabolic centre to be made, or genetic testing to be initiated where this is available.

Introduction Genome-wide sequencing (exome or whole genome) is transforming the care and management of paediatric patients with a rare disease because of its diagnostic capabilities. Genome-wide sequencing is most effective when both parents and the child are sequenced as a trio. Genetic counselling is recommended for all families considering genome-wide sequencing. Although telehealth is well established in genetic counselling for hereditary cancer and prenatal genetics, its use with genome-wide sequencing has not been well studied. The CAUSES Clinic at BC Children’s and Women’s Hospitals was a translational paediatric trio-based genome-wide sequencing initiative. Pre-test genetic counselling via telehealth (at a clinical site near the family’s residence) was offered to families who had been previously evaluated by a clinical geneticist. We report on the first 300 families seen in the CAUSES clinic and compare health services implementation issues of families seen via telehealth versus on-site. Methods Demographics, cost to families (travel and time), time to first appointment, complete trio sample accrual and diagnostic rates were studied. Results Of the 300 patients, 58 (19%) were seen via telehealth and 242 (81%) were seen on-site for pre-test counselling. The mean time to completion of accrual of trio samples in the telehealth group was 56.3 (standard deviation ±87.3) days versus 18.9 (standard deviation ±62.4) days in the onsite group ( p −16). The mean per-family estimated actual or potential travel/time cost savings were greater in the telehealth group (Can$987; standard deviation = Can$1151) than for those seen on-site (Can$305; standard deviation = Can$589) ( p = 0.0004). Conclusions Telehealth allowed for access to genome-wide sequencing for families in remote communities and for them to avoid significant travel and time costs; however, there was a significant delay to accrual of the complete trio samples in the telehealth group, impacting on time of result reporting and delaying diagnoses for families for whom genome-wide sequencing was diagnostic.

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model (Kdm5a-/-) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a-/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function.

Monoamine neurotransmitter disorders present predominantly with neurologic features, including dystonic or dyskinetic cerebral palsy and movement disorders. Genetic conditions that lead to secondary defects in the synthesis, catabolism, transport, and metabolism of biogenic amines can lead to neurotransmitter abnormalities, which can present with similar features. Eleven patients with secondary neurotransmitter abnormalities were enrolled between 2011 and 2015. All patients underwent research-based whole exome and/or whole genome sequencing (WES/WGS). A trial of treatment with levodopa/carbidopa and 5-hydroxytryptophan was initiated. In six families with abnormal neurotransmitter profiles and neurological phenotypes, variants in known disease-causing genes (KCNJ6, SCN2A, CSTB in 2 siblings, NRNX1, KIF1A and PAK3) were identified, while one patient had a variant of uncertain significance in a candidate gene (DLG4) that may explain her phenotype. In 3 patients, no compelling candidate genes were identified. A trial of neurotransmitter replacement therapy led to improvement in motor and behavioral symptoms in all but two patients. The patient with KCNJ6 variant did not respond to L-dopa therapy, but rather experienced increased dyskinetic movements even at low dose of medication. The patient's symptoms harboring the NRNX1 deletion remained unaltered. This study demonstrates the utility of genome-wide sequencing in further understanding the etiology and pathophysiology of neurometabolic conditions, and the potential of secondary neurotransmitter deficiencies to serve as novel therapeutic targets. As there was a largely favorable response to therapy in our case series, a careful trial of neurotransmitter replacement therapy should be considered in patients with cerebrospinal fluid (CSF) monoamines below reference range.

Purpose Clinical diagnostic genome-wide (exome or genome) sequencing (GWS) in British Columbia requires funding approval by a provincial agency on a case-by-case basis. The CAUSES Clinic was a pediatric translational trio-based GWS study at BC Children's and Women's Hospitals. Referrals to the CAUSES Clinic were made through a Genomic Consultation Service (GCS), a multidisciplinary team led by genetic counsellors that provided advice regarding genomic testing for physicians considering GWS for their patients. Here we review the outcomes of the GCS, focusing on patients not recommended for the CAUSES Study. Methods Demographic, clinical, and testing data were abstracted from patient charts. Logistic regression analysis was used to explore associations between demographic and clinical variables and two outcomes: the type of recommendation and referring physicians’ decisions to follow the recommendation. Results Of 972 GCS referrals, 248 patients were not referred to the CAUSES Study. GWS (vs. a targeted test; e.g. multi-gene panel) was more likely to be recommended to physicians of patients with ID than physicians of patients without ID (OR = 2.98; 95% CI = 1.46 to 6.27; n = 149). In total, 40% of physicians who were recommended to pursue clinical genomic testing submitted an application for funding approval; 71% of applications were approved for funding. Among approved tests, 50% resulted in a diagnosis, including 33% of targeted tests and 82% of GWS tests (χ2 (1) = 5.0, p = 0.026). Conclusion The GCS provided an effective model in which physicians can interface with genetic specialists, including genetic counsellors, to facilitate appropriate genomic test selection.

Germ line mutations in H3F3A and H3F3B cause a previously unidentified neurodevelopmental syndrome., Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.

International audience; Congenital uterine anomalies (CUA) may have major impacts on the health and social well-being of affected individuals. Their expressivity is variable, with the most severe end of the spectrum being the absence of any fully or unilaterally developed uterus (aplastic uterus), which is a major feature in Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH). So far, etiologies of CUA remain largely unknown. As reports of familial occurrences argue for strong genetic contributors in some cases, we performed whole exome sequencing in nine multiplex families with recurrence of uterine and kidney malformations, a condition called hereditary urogenital adysplasia. Heterozygous likely causative variants in the gene GREB1L were identified in four of these families, confirming GREB1L as an important gene for proper uterine and kidney development. The apparent mode of inheritance was autosomal dominant with incomplete penetrance. The four families included fetuses with uterovaginal aplasia and bilateral renal agenesis, highlighting the importance to investigate GREB1L in such phenotypes. Subsequent sequencing of the gene in a cohort of 68 individuals with MRKH syndrome or uterine malformation (mostly sporadic cases) identified six additional variants of unknown significance. We therefore conclude that heterozygous GREB1L variants contribute to MRKH syndrome and this probably requires additional genetic or environmental factors for full penetrance.

Mutations in presenilin 1 (PSEN1) are the most common cause of autosomal dominant Alzheimer’s disease. Here, we report a Canadian-Vietnamese family carrying a PSEN1 p.Met233Val mutation with an exceptionally early and severe presentation that includes a wide range of atypical symptoms, including prominent ataxia, Parkinsonism, spasticity, dystonia, action tremor, myoclonus, bulbar symptoms, seizures, hallucinations and behavioral changes. Whole-exome sequencing (WES) was performed on the affected proband after many assessments over several years proved diagnostically inconclusive. The results were analyzed using the AnnEx “Annotated Exomes” browser (http://annex.can.ubc.ca), a web-based platform that facilitates WES variant annotation and interpretation. High-throughput sequencing can be especially informative for complex neurological disorders, and WES warrants consideration as a first-line clinical test. Data analyses facilitated by web-based bioinformatics tools have great potential for novel insight, although confirmatory, diagnostically accredited Sanger sequencing is recommended prior to reporting.

Mutations in presenilin 1 (PSEN1) are the most common cause of autosomal dominant Alzheimer’s disease. Here, we report a Canadian-Vietnamese family carrying a PSEN1 p.Met233Val mutation with an exceptionally early and severe presentation that includes a wide range of atypical symptoms, including prominent ataxia, Parkinsonism, spasticity, dystonia, action tremor, myoclonus, bulbar symptoms, seizures, hallucinations and behavioral changes. Whole-exome sequencing (WES) was performed on the affected proband after many assessments over several years proved diagnostically inconclusive. The results were analyzed using the AnnEx “Annotated Exomes” browser (http://annex.can.ubc.ca), a web-based platform that facilitates WES variant annotation and interpretation. High-throughput sequencing can be especially informative for complex neurological disorders, and WES warrants consideration as a first-line clinical test. Data analyses facilitated by web-based bioinformatics tools have great potential for novel insight, although confirmatory, diagnostically accredited Sanger sequencing is recommended prior to reporting.

Objective To describe the clinical presentation and genotype of subjects with aniridia with a particular focus on foveal hypoplasia. Design Prospective cohort study. Participants Thirty-three Canadian participants with aniridia and of various ethnic backgrounds residing in British Columbia. Methods Full ophthalmic examinations and posterior segment spectral domain-optical coherence tomography (SD-OCT) imaging were performed. Foveal hypoplasia was graded independently by 2 staff ophthalmologists. PAX6 sequencing was performed and chromosomal 11p anomalies investigated. Candidate gene and single-nucleotide polymorphism sequencing in genes functionally related to PAX6 were also studied. Results Best corrected visual acuities in the cohort ranged from 0.0 logMAR to no light perception. Total absence of iris tissue was seen in the majority (42 of 66 eyes). In those in whom SD-OCT was possible, foveal hypoplasia was seen in the majority (45 of 56 eyes, 80%). Molecular genetic defects involving PAX6 were identified in 30 participants (91%), including 4 novel PAX6 mutations (Gly18Val; Ser65ProfsX14; Met337ArgfsX18; Ser321CysfsX34) and 4 novel chromosome 11p deletions inclusive of PAX6 or a known PAX6 regulatory region. Conclusions The number of PAX6 mutations associated with aniridia continues to increase. Variable foveal architecture despite nearly identical anterior segment disease in 4 participants with an Ex9 ELP4-Ex4 DCDC1 deletion suggested that molecular cues causing variation in disease in the posterior segment differ from those at play in the anterior segment. Results in 3 patients without identifiable PAX6 mutations and a review of the literature suggest that such cases be described as phenocopies rather than actual cases of the syndrome of aniridia.