Diagnostic testing laboratories are valuable partners for disease gene discovery: 5-year experience with GeneMatcher.

Although the rates of disease gene discovery have steadily increased with the expanding use of genome and exome sequencing by clinical and research laboratories, only ~16% of genes in the genome have confirmed disease associations. Here we describe our clinical laboratory's experience utilizing GeneMatcher, an online portal designed to promote disease gene discovery and data sharing. Since 2016, we submitted 246 candidates from 243 unique genes to GeneMatcher, of which 111 (45%) are now clinically characterized. Submissions meeting our candidate gene-reporting criteria based on a scoring system using patient and molecular-weighted evidence were significantly more likely to be characterized as of October 2021 versus genes that did not meet our clinical-reporting criteria (p = 0.025). We reported relevant findings related to these newly characterized gene-disease associations in 477 probands. In 218 (46%) instances, we issued reclassifications after an initial negative or candidate gene (uncertain) report. We coauthored 104 publications delineating gene-disease relationships, including descriptions of new associations (60%), additional supportive evidence (13%), subsequent descriptive cohorts (23%), and phenotypic expansions (4%). Clinical laboratories are pivotal for disease gene discovery efforts and can screen phenotypes based on genotype matches, contact clinicians of relevant cases, and issue proactive reclassification reports.

TAOK1 is associated with neurodevelopmental disorder and essential for neuronal maturation and cortical development.

Thousand and one amino-acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen-activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant-negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems.

De novo variants in SLC12A6 cause sporadic early-onset progressive sensorimotor neuropathy.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous disorder of the peripheral nervous system. Biallelic variants in SLC12A6 have been associated with autosomal-recessive hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC). We identified heterozygous de novo variants in SLC12A6 in three unrelated patients with intermediate CMT. METHODS: We evaluated the clinical reports and electrophysiological data of three patients carrying de novo variants in SLC12A6 identified by diagnostic trio exome sequencing. For functional characterisation of the identified variants, potassium influx of mutated KCC3 cotransporters was measured in Xenopus oocytes. RESULTS: We identified two different de novo missense changes (p.Arg207His and p.Tyr679Cys) in SLC12A6 in three unrelated individuals with early-onset progressive CMT. All presented with axonal/demyelinating sensorimotor neuropathy accompanied by spasticity in one patient. Cognition and brain MRI were normal. Modelling of the mutant KCC3 cotransporter in Xenopus oocytes showed a significant reduction in potassium influx for both changes. CONCLUSION: Our findings expand the genotypic and phenotypic spectrum associated with SLC12A6 variants from autosomal-recessive HMSN/ACC to dominant-acting de novo variants causing a milder clinical presentation with early-onset neuropathy.

A retrospective review of multiple findings in diagnostic exome sequencing: half are distinct and half are overlapping diagnoses.

PURPOSE: We evaluated clinical and genetic features enriched in patients with multiple Mendelian conditions to determine which patients are more likely to have multiple potentially relevant genetic findings (MPRF). METHODS: Results of the first 7698 patients who underwent exome sequencing at Ambry Genetics were reviewed. Clinical and genetic features were examined and degree of phenotypic overlap between the genetic diagnoses was evaluated. RESULTS: Among patients referred for exome sequencing, 2% had MPRF. MPRF were more common in patients from consanguineous families and patients with greater clinical complexity. The difference in average number of organ systems affected is small: 4.3 (multiple findings) vs. 3.9 (single finding) and may not be distinguished in clinic. CONCLUSION: Patients with multiple genetic diagnoses had a slightly higher number of organ systems affected than patients with single genetic diagnoses, largely because the comorbid conditions affected overlapping organ systems. Exome testing may be beneficial for all cases with multiple organ systems affected. The identification of multiple relevant genetic findings in 2% of exome patients highlights the utility of a comprehensive molecular workup and updated interpretation of existing genomic data; a single definitive molecular diagnosis from analysis of a limited number of genes may not be the end of a diagnostic odyssey.

SYT1-associated neurodevelopmental disorder: a case series.

Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals' phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.

Correction: Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.

In the published version of this paper, some of the columns in the last three rows of Table 3 were mistakenly transposed. The corrected table appears below. In col. 6 of the row for DNMT3A, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S1". In col. 6 of the row for SON, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S2".

Outcomes of Diagnostic Exome Sequencing in Patients With Diagnosed or Suspected Autism Spectrum Disorders.

BACKGROUND: Exome sequencing has recently been proved to be a successful diagnostic method for complex neurodevelopmental disorders. However, the diagnostic yield of exome sequencing for autism spectrum disorders has not been extensively evaluated in large cohorts to date. MATERIALS AND METHODS: We performed diagnostic exome sequencing in a cohort of 163 individuals with autism spectrum disorder (66.3%) or autistic features (33.7%). RESULTS: The diagnostic yield observed in patients in our cohort was 25.8% (42 of 163) for positive or likely positive findings in characterized disease genes, while a candidate genetic etiology was reported for an additional 3.3% (4 of 120) of patients. Among the positive findings in the patients with autism spectrum disorder or autistic features, 61.9% were the result of de novo mutations. Patients presenting with psychiatric conditions or ataxia or paraplegia in addition to autism spectrum disorder or autistic features were significantly more likely to receive positive results compared with patients without these clinical features (95.6% vs 27.1%, P < 0.0001; 83.3% vs 21.2%, P < 0.0001, respectively). The majority of the positive findings were in recently identified autism spectrum disorder genes, supporting the importance of diagnostic exome sequencing for patients with autism spectrum disorder or autistic features as the causative genes might evade traditional sequential or panel testing. CONCLUSIONS: These results suggest that diagnostic exome sequencing would be an efficient primary diagnostic method for patients with autism spectrum disorders or autistic features. Moreover, our data may aid clinicians to better determine which subset of patients with autism spectrum disorder with additional clinical features would benefit the most from diagnostic exome sequencing.

Classification of Genes: Standardized Clinical Validity Assessment of Gene-Disease Associations Aids Diagnostic Exome Analysis and Reclassifications.

Ascertaining a diagnosis through exome sequencing can provide potential benefits to patients, insurance companies, and the healthcare system. Yet, as diagnostic sequencing is increasingly employed, vast amounts of human genetic data are produced that need careful curation. We discuss methods for accurately assessing the clinical validity of gene-disease relationships to interpret new research findings in a clinical context and increase the diagnostic rate. The specifics of a gene-disease scoring system adapted for use in a clinical laboratory are described. In turn, clinical validity scoring of gene-disease relationships can inform exome reporting for the identification of new or the upgrade of previous, clinically relevant gene findings. Our retrospective analysis of all reclassification reports from the first 4 years of diagnostic exome sequencing showed that 78% were due to new gene-disease discoveries published in the literature. Among all exome positive/likely positive findings in characterized genes, 32% were in genetic etiologies that were discovered after 2010. Our data underscore the importance and benefits of active and up-to-date curation of a gene-disease database combined with critical clinical validity scoring and proactive reanalysis in the clinical genomics era.

Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.

PURPOSE: Diagnostic exome sequencing (DES) is now a commonly ordered test for individuals with undiagnosed genetic disorders. In addition to providing a diagnosis for characterized diseases, exome sequencing has the capacity to uncover novel candidate genes for disease. METHODS: Family-based DES included analysis of both characterized and novel genetic etiologies. To evaluate candidate genes for disease in the clinical setting, we developed a systematic, rule-based classification schema. RESULTS: Testing identified a candidate gene among 7.7% (72/934) of patients referred for DES; 37 (4.0%) and 35 (3.7%) of the genes received evidence scores of "candidate" and "suspected candidate," respectively. A total of 71 independent candidate genes were reported among the 72 patients, and 38% (27/71) were subsequently corroborated in the peer-reviewed literature. This rate of corroboration increased to 51.9% (27/52) among patients whose gene was reported at least 12 months previously. CONCLUSIONS: Herein, we provide transparent, comprehensive, and standardized scoring criteria for the clinical reporting of candidate genes. These results demonstrate that DES is an integral tool for genetic diagnosis, especially for elucidating the molecular basis for both characterized and novel candidate genetic etiologies. Gene discoveries also advance the understanding of normal human biology and more common diseases.Genet Med 19 2, 224-235.

Allele-specific regulation of DISC1 expression by miR-135b-5p.

Disrupted-in-schizophrenia-1 (DISC1) gene has been established as a risk factor for various neuropsychiatric phenotypes. Both coding and regulatory variants in DISC1 have been identified and associated with these phenotypes in genetic studies. MicroRNAs (miRNAs) are important regulators of protein coding genes. Since the miRNA-mRNA target recognition mechanism is vulnerable to disruption by DNA polymorphisms, we investigated whether polymorphisms in the DISC1 3'UTR affect binding of miRNAs and lead to allele-specific regulation of DISC1. We identified four predicted polymorphic miRNA target sites in the DISC1 3'UTR, and demonstrated that miR-135b-5p regulates the level of DISC1 mRNA. Moreover, DISC1 regulation by miR-135b-5p is allele specific: miR-135b-5p only binds to the major allele (A) of rs11122396, not to the minor allele (G). Thus, the G allele may be functionally related to the DISC1-associated phenotypes by abolishing regulation by miR-135b-5p, leading to elevated DISC1 levels.

Analysis of X chromosome inactivation in autism spectrum disorders.

Autism spectrum disorders (ASD) are complex genetic disorders more frequently observed in males. Skewed X chromosome inactivation (XCI) is observed in heterozygous females carrying gene mutations involved in several X-linked syndromes. In this study, we aimed to estimate the role of X-linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 163 affected girls. The XCI pattern was also determined in two control groups (144 adult females and 40 young females) with a similar age distribution to the mothers sample and affected girls sample, respectively. We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X-linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X-linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (> or = 80:20) and a modest increased allele sharing was obtained in the Xq27-Xq28 region, with a peak Z-score of 1.75 close to rs719489. In summary, our results suggest that there is no major X-linked gene subject to XCI and expressed in blood cells conferring susceptibility to ASD. However, the possibility that rare mutations in X-linked genes could contribute to ASD cannot be excluded. We propose that the XCI profile could be a useful criteria to prioritize families for mutation screening of X-linked candidate genes.