Genome Sequencing for Cases Unsolved by Exome Sequencing: Identifying a Single-Exon Deletion in TBCK in a Case from 30 Years Ago.

In patients with neurodevelopmental disorders (NDDs), exome sequencing (ES), the diagnostic gold standard, reveals an underlying monogenic condition in only approximately 40% of cases. We report the case of a female patient with profound NDD who died 30 years ago at the age of 3 years and for whom genome sequencing (GS) now identified a single-exon deletion in TBCK previously missed by ExomeDepth, the copy number variation (CNV) detection algorithm in ES.Deoxyribonucleic acid (DNA) was extracted from frozen muscle tissue of the index patient and the parents' blood. Genome data were analyzed for structural variants and single nucleotide variants (SUVs)/indels as part of the Bavarian Genomes consortium project.Biallelic variants in TBCK, which are linked to the autosomal recessive disorder TBCK syndrome, were detected in the affected individual: a novel frameshift variant and a deletion of exon 23, previously established as common but underrecognized pathogenic variant in individuals with TBCK syndrome. While in the foregoing ES analysis, calling algorithms for (SNVs)/indels were able to identify the frameshift variant, ExomeDepth failed to call the intragenic deletion.Our case illustrates the added value of GS for the detection of single-exon deletions for which calling from ES data remains challenging and confirms that the deletion of exon 23 in TBCK may be underdiagnosed in patients with NDDs. Furthermore, it shows the importance of "molecular or genetic autopsy" allowing genetic risk counseling for family members as well as the end of a diagnostic odyssey of 30 years.

De novo missense variants in RRAGC lead to a fatal mTORopathy of early childhood.

PURPOSE: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell growth in response to nutritional status. Central to the mTORC1 function is the Rag-GTPase heterodimer. One component of the Rag heterodimer is RagC (Ras-related GTP-binding protein C), which is encoded by the RRAGC gene. METHODS: Genetic testing via trio exome sequencing was applied to identify the underlying disease cause in 3 infants with dilated cardiomyopathy, hepatopathy, and brain abnormalities, including pachygyria, polymicrogyria, and septo-optic dysplasia. Studies in patient-derived skin fibroblasts and in a HEK293 cell model were performed to investigate the cellular consequences. RESULTS: We identified 3 de novo missense variants in RRAGC (NM_022157.4: c.269C>A, p.(Thr90Asn), c.353C>T, p.(Pro118Leu), and c.343T>C, p.(Trp115Arg)), which were previously reported as occurring somatically in follicular lymphoma. Studies of patient-derived fibroblasts carrying the p.(Thr90Asn) variant revealed increased cell size, as well as dysregulation of mTOR-related p70S6K (ribosomal protein S6 kinase 1) and transcription factor EB signaling. Moreover, subcellular localization of mTOR was decoupled from metabolic state. We confirmed the key findings for all RRAGC variants described in this study in a HEK293 cell model. CONCLUSION: The above results are in line with a constitutive overactivation of the mTORC1 pathway. Our study establishes de novo missense variants in RRAGC as cause of an early-onset mTORopathy with unfavorable prognosis.

Defining the genotypic and phenotypic spectrum of X-linked MSL3-related disorder.

PURPOSE: We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). METHODS: Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. RESULTS: We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. CONCLUSION: Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals.

De novo variants in neurodevelopmental disorders-experiences from a tertiary care center.

Up to 40% of neurodevelopmental disorders (NDDs) such as intellectual disability, developmental delay, autism spectrum disorder, and developmental motor abnormalities have a documented underlying monogenic defect, primarily due to de novo variants. Still, the overall burden of de novo variants as well as novel disease genes in NDDs await discovery. We performed parent-offspring trio exome sequencing in 231 individuals with NDDs. Phenotypes were compiled using human phenotype ontology terms. The overall diagnostic yield was 49.8% (n = 115/231) with de novo variants contributing to more than 80% (n = 93/115) of all solved cases. De novo variants affected 72 different-mostly constrained-genes. In addition, we identified putative pathogenic variants in 16 genes not linked to NDDs to date. Reanalysis performed in 80 initially unsolved cases revealed a definitive diagnosis in two additional cases. Our study consolidates the contribution and genetic heterogeneity of de novo variants in NDDs highlighting trio exome sequencing as effective diagnostic tool for NDDs. Besides, we illustrate the potential of a trio-approach for candidate gene discovery and the power of systematic reanalysis of unsolved cases.

Next-generation sequencing and comprehensive data reassessment in 263 adult patients with neuromuscular disorders: insights into the gray zone of molecular diagnoses.

BACKGROUND: Neuromuscular disorders (NMDs) are heterogeneous conditions with a considerable fraction attributed to monogenic defects. Despite the advancements in genomic medicine, many patients remain without a diagnosis. Here, we investigate whether a comprehensive reassessment strategy improves the diagnostic outcomes. METHODS: We analyzed 263 patients with NMD phenotypes that underwent diagnostic exome or genome sequencing at our tertiary referral center between 2015 and 2023. We applied a comprehensive reassessment encompassing variant reclassification, re-phenotyping and NGS data reanalysis. Multivariable logistic regression was performed to identify predictive factors associated with a molecular diagnosis. RESULTS: Initially, a molecular diagnosis was identified in 53 cases (20%), while an additional 23 (9%) had findings of uncertain significance. Following comprehensive reassessment, the diagnostic yield increased to 23%, revealing 44 distinct monogenic etiologies. Reasons for newly obtained molecular diagnoses were variant reclassifications in 7 and NGS data reanalysis in 3 cases including one recently described disease-gene association (DNAJB4). Male sex reduced the odds of receiving a molecular diagnosis (OR 0.42; 95%CI 0.21-0.82), while a positive family history (OR 5.46; 95%CI 2.60-11.76) and a myopathy phenotype (OR 2.72; 95%CI 1.11-7.14) increased the likelihood. 7% were resolved through targeted genetic testing or classified as acquired etiologies. CONCLUSION: Our findings reinforce the use of NGS in NMDs of suspected monogenic origin. We show that a comprehensive reassessment enhances diagnostic accuracy. However, one needs to be aware that genetic diagnoses are often made with uncertainty and can even be downgraded based on new evidence.

Prospects and challenges for the genetic counsellor profession in the German-speaking countries: report of a workshop.

The genetic counsellor profession has not yet been established in the German-speaking countries. In 2019 the Medical University of Innsbruck inaugurated the first German-taught Master's degree programme in Genetic and Genomic Counselling. In order to discuss prospects and challenges of the genetic counsellor profession in Germany, Austria and Switzerland (DACH region), the MSc programme team organized a two-day workshop with international speakers and medical geneticists from the DACH region. Day 1 was dedicated to the history, training and international profile of the genetic counsellor profession. Day 2 focused on four specific topics: (i) professional role, (ii) acceptance and job title, (iii) formal requirements and (iv) remuneration concepts for genetic counsellors in the DACH region. The workshop showed that the key factor for the successful implementation of the genetic counsellor profession is acceptance and trust within the medical genetics team. Genetic counsellors complement patient care in aspects that might be underserved considering the increasing demand of counselling in genomic medicine. Successful establishment of the genetic counsellor profession will entail the development of interprofessional teams under medical supervision and in the team of medical geneticists.