A New Case Series Suggests That SCA48 (ATX/STUB1) Is Primarily a Monogenic Disorder.

BACKGROUND: Monoallelic, pathogenic STUB1 variants cause autosomal dominant cerebellar ataxia (ATX-STUB1/SCA48). Recently, a genetic interaction between STUB1 variants and intermediate or high-normal CAG/CAA repeats in TBP was suggested, indicating digenic inheritance or a disease-modifying role for TBP expansions. OBJECTIVE: To determine the presence and impact of intermediate or high-normal TBP expansions in ataxic patients with heterozygous STUB1 variants. METHODS: We describe 21 patients with ataxia carrying a heterozygous STUB1 variant and determined TBP repeat length. RESULTS: A total of 15 of 21 patients (71%) carried a normal TBP<40 allele, 4 (19%) carried an intermediate TBP41-42 allele, and two carried a high-normal TBP40 allele (9.5%). Five of six carriers (83%) of both STUB1 variants and TBP40-42 alleles showed marked cognitive impairment. CONCLUSIONS: SCA48 is predominantly a monogenic disorder, because most patients carried an isolated, heterozygous STUB1 variant and presented with the typical combined phenotype of ataxia and cognitive dysfunction. Still, co-occurrence of TBP41-42 or high-normal TBP40 alleles was relatively frequent and associated with marked cognitive defects (28.5%), suggesting a modifying effect on clinical expression in some cases. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Optical Genome Mapping for Applications in Repeat Expansion Disorders.

Short tandem repeat (STR) expansions are associated with more than 60 genetic disorders. The size and stability of these expansions correlate with the severity and age of onset of the disease. Therefore, being able to accurately detect the absolute length of STRs is important. Current diagnostic assays include laborious lab experiments, including repeat-primed PCR and Southern blotting, that still cannot precisely determine the exact length of very long repeat expansions. Optical genome mapping (OGM) is a cost-effective and easy-to-use alternative to traditional cytogenetic techniques and allows the comprehensive detection of chromosomal aberrations and structural variants >500 bp in length, including insertions, deletions, duplications, inversions, translocations, and copy number variants. Here, we provide methodological guidance for preparing samples and performing OGM as well as running the analysis pipelines and using the specific repeat expansion workflows to determine the exact repeat length of repeat expansions expanded beyond 500 bp. Together these protocols provide all details needed to analyze the length and stability of any repeat expansion with an expected repeat size difference from the expected wild-type allele of >500 bp. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genomic ultra-high-molecular-weight DNA isolation, labeling, and staining Basic Protocol 2: Data generation and genome mapping using the Bionano Saphyr® System Basic Protocol 3: Manual De Novo Assembly workflow Basic Protocol 4: Local guided assembly workflow Basic Protocol 5: EnFocus Fragile X workflow Basic Protocol 6: Molecule distance script workflow.

Monoallelic de novo AJAP1 loss-of-function variants disrupt trans-synaptic control of neurotransmitter release.

Adherens junction-associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1-/- and Ajap1W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants.

The expanding clinical and genetic spectrum of DYNC1H1-related disorders.

Intracellular trafficking involves an intricate machinery of motor complexes including the dynein complex to shuttle cargo for autophagolysosomal degradation. Deficiency in dynein axonemal chains as well as cytoplasmic light and intermediate chains have been linked with ciliary dyskinesia and skeletal dysplasia. The cytoplasmic dynein 1 heavy chain protein (DYNC1H1) serves as a core complex for retrograde trafficking in neuronal axons. Dominant pathogenic variants in DYNC1H1 have been previously implicated in peripheral neuromuscular disorders (NMD) and neurodevelopmental disorders (NDD). As heavy-chain dynein is ubiquitously expressed, the apparent selectivity of heavy-chain dyneinopathy for motor neuronal phenotypes remains currently unaccounted for. Here, we aimed to evaluate the full DYNC1H1-related clinical, molecular and imaging spectrum, including multisystem features and novel phenotypes presenting throughout life. We identified 47 cases from 43 families with pathogenic heterozygous variants in DYNC1H1 (aged 0-59 years) and collected phenotypic data via a comprehensive standardized survey and clinical follow-up appointments. Most patients presented with divergent and previously unrecognized neurological and multisystem features, leading to significant delays in genetic testing and establishing the correct diagnosis. Neurological phenotypes include novel autonomic features, previously rarely described behavioral disorders, movement disorders, and periventricular lesions. Sensory neuropathy was identified in nine patients (median age of onset 10.6 years), of which five were only diagnosed after the second decade of life, and three had a progressive age-dependent sensory neuropathy. Novel multisystem features included primary immunodeficiency, bilateral sensorineural hearing loss, organ anomalies, and skeletal manifestations, resembling the phenotypic spectrum of other dyneinopathies. We also identified an age-dependent biphasic disease course with developmental regression in the first decade and, following a period of stability, neurodegenerative progression after the second decade of life. Of note, we observed several cases in whom neurodegeneration appeared to be prompted by intercurrent systemic infections with double-stranded DNA viruses (Herpesviridae) or single-stranded RNA viruses (Ross-River fever, SARS-CoV-2). Moreover, the disease course appeared to be exacerbated by viral infections regardless of age and/or severity of NDD manifestations, indicating a role of dynein in anti-viral immunity and neuronal health. In summary, our findings expand the clinical, imaging, and molecular spectrum of pathogenic DYNC1H1 variants beyond motor neuropathy disorders and suggest a life-long continuum and age-related progression due to deficient intracellular trafficking. This study will facilitate early diagnosis and improve counselling and health surveillance of affected patients.

Maternal cell contamination in postnatal umbilical cord blood samples implies a low risk for genetic misdiagnoses.

OBJECTIVE: Maternal cell contamination (MCC) poses a risk for misdiagnosis in prenatal genetic testing, and is examined in accredited diagnostic laboratories However, the awareness of possible MCC in perinatal/postnatal genetic testing, mainly of umbilical cord blood (CB), is lower. METHOD: We investigated the rate of MCC in DNA from both umbilical CB samples and umbilical cord samples that were sent to our diagnostic laboratory for diagnostic testing between 1995 and 2021 (n = 236). RESULTS: MCC was detected in 4% of umbilical CB samples, and in one umbilical cord sample. Particularly tests enriching for a specific variant are very sensitive for low amounts of MCC, as we emphasize here with a false positive diagnosis of myotonic dystrophy type 1 in a newborn. CONCLUSIONS: Overall, with appropriate collection and use, umbilical CB and umbilical cord samples are suitable for genetic testing based on the low rates of MCC and misdiagnosis. These findings do however underline the importance of routine MCC testing in umbilical CB samples and umbilical cord samples for both requesting clinicians and diagnostic genetic laboratories.

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy.

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.

Genome sequencing as a generic diagnostic strategy for rare disease.

BACKGROUND: To diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease. METHODS: We performed short-read GS (NovaSeq™6000; 150 bp paired-end reads, 37 × mean coverage) on 1000 cases with 1271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels < 50 bp), large variants (copy number variants and short tandem repeats) and other variants (structural variants and aneuploidies). Variant calling format files were queried per variant, from which workflow-specific true positive rates (TPRs) for detection were determined. A TPR of ≥ 98% was considered the threshold for transition to GS. A GS-first scenario was generated for our laboratory, using diagnostic efficacy and predicted false negative as primary outcome measures. As input, we modeled the diagnostic path for all 24,570 individuals referred in 2022, combining the clinical referral, the transition of the underlying workflow(s) to GS, and the variant type(s) to be detected. RESULTS: Overall, 95% (1206/1271) of variants were detected. Detection rates differed per variant category: small variants in 96% (826/860), large variants in 93% (341/366), and other variants in 87% (39/45). TPRs varied between workflows (79-100%), with 7/10 being replaceable by GS. Models for our laboratory indicate that a GS-first strategy would be feasible for 84.9% of clinical referrals (750/883), translating to 71% of all individuals (17,444/24,570) receiving GS as their primary test. An estimated false negative rate of 0.3% could be expected. CONCLUSIONS: GS can capture clinically relevant germline variants in a 'GS-first strategy' for the majority of clinical indications in a genetics diagnostic lab.

Brody Disease, an Early-Onset Myopathy With Delayed Relaxation and Abnormal Gait: A Case Series of 9 Children.

Brody disease is a rare autosomal recessive myopathy, caused by pathogenic variants in the ATP2A1 gene. It is characterized by an exercise-induced delay in muscle relaxation, often reported as muscle stiffness. Children may manifest with an abnormal gait and difficulty running. Delayed relaxation is commonly undetected, resulting in a long diagnostic delay. Almost all published cases so far were adults with childhood onset and adult diagnosis. With diagnostic next-generation sequencing, an increasing number of patients are diagnosed in childhood. We describe the clinical and genetic features of 9 children from 6 families with Brody disease. All presented with exercise-induced delayed relaxation, reported as difficulty running and performing sports. Muscle strength and mass was normal, and several children even had an athletic appearance. However, the walking and running patterns were abnormal. The diagnostic delay ranged between 2 and 7 years. Uniformly, a wide range of other disorders were considered before genetic testing was performed, revealing pathogenic genetic variants in ATP2A1. To conclude, this case series is expected to improve clinical recognition and timely diagnosis of Brody disease in children. We propose that ATP2A1 should be added to gene panels for congenital myopathies, developmental and movement disorders, and muscle channelopathies.

Lessons learned from rapid exome sequencing for 575 critically ill patients across the broad spectrum of rare disease.

Introduction: Rapid exome sequencing (rES) has become the first-choice genetic test for critically ill patients, mostly neonates, young infants, or fetuses in prenatal care, in time-sensitive situations and when it is expected that the genetic test result may guide clinical decision making. The implementation of rES has revolutionized medicine by enabling timely identification of genetic causes for various rare diseases. The utilization of rES has increasingly been recognized as an essential diagnostic tool for the identification of complex and undiagnosed genetic disorders. Methods: We conducted a retrospective evaluation of our experiences with rES performed on 575 critically ill patients from various age groups (prenatal to adulthood), over a four-year period (2016-2019). These patients presented with a wide spectrum of rare diseases, including but not limited to neurological disorders, severe combined immune deficiency, and cancer. Results: During the study period, there was a significant increase in rES referrals, with a rise from a total of two referrals in Q1-2016 to 10 referrals per week in Q4-2019. The median turnaround time (TAT) decreased from 17 to 11 days in the period 2016-2019, with an overall median TAT of 11 days (IQR 8-15 days). The overall diagnostic yield for this cohort was 30.4%, and did not significantly differ between the different age groups (e.g. adults 22.2% vs children 31.0%; p-value 0.35). However, variability in yield was observed between clinical entities: craniofacial anomalies yielded 58.3%, while for three clinical entities (severe combined immune deficiency, aneurysm, and hypogonadotropic hypogonadism) no diagnoses were obtained. Discussion: Importantly, whereas clinical significance is often only attributed to a conclusive diagnosis, we also observed impact on clinical decision-making for individuals in whom no genetic diagnosis was established. Hence, our experience shows that rES has an important role for patients of all ages and across the broad spectrum of rare diseases to impact clinical outcomes.