Multiomic ALS signatures highlight subclusters and sex differences suggesting the MAPK pathway as therapeutic target.

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.

Clinical and Genetic Findings in a Cohort of Patients with PRPF31-associated Retinal Dystrophy.

PURPOSE: The purpose of our study was to assess the phenotypic and genotypic spectrum in a large cohort of patients with PRPF31-associated retinal dystrophy. DESIGN: Retrospective cohort study METHODS: In this retrospective chart review study, we collected cross-sectional data on the phenotype and genotype of patients with PRPF31-associated retinal dystrophy from the clinics for inherited retinal dystrophies at the University of Tuebingen and the local RetDis database and biobank. Patients underwent thorough ophthalmological examinations and genetic testing. RESULTS: Eighty-six patients from 61 families were available for clinical assessment, while genomic DNA was available for 111 individuals (index patients and family members). Fifty-three different disease-associated variants were observed in our cohort. Point mutations were the most common class. All but two patients exhibited features of a typical Retinitis pigmentosa (RP). One patient showed a cone-rod-dystrophy pattern. One mutation carrier revealed no signs of a retinal dystrophy. There was a statistically significant better visual acuity for patients with large deletions in the 20-39 age group. Cystoid macular edema was common in those with preserved central retina and showed an association with female sex. CONCLUSION: Our study confirms high phenotypic variability in disease onset and age at which legal blindness is reached in PRPF31-linked RP. Non-penetrance is commonly documented in family history, although poorly represented in our study, possibly indicating that true asymptomatic mutation carriers are rare if followed-up over lifetime with thorough ophthalmologic workup.

Clinical, Neuroimaging, and Metabolic Footprint of the Neurodevelopmental Disorder Caused by Monoallelic HK1 Variants.

Background and Objectives: Hexokinase 1 (encoded by HK1) catalyzes the first step of glycolysis, the adenosine triphosphate-dependent phosphorylation of glucose to glucose-6-phosphate. Monoallelic HK1 variants causing a neurodevelopmental disorder (NDD) have been reported in 12 individuals. Methods: We investigated clinical phenotypes, brain MRIs, and the CSF of 15 previously unpublished individuals with monoallelic HK1 variants and an NDD phenotype. Results: All individuals had recurrent variants likely causing gain-of-function, representing mutational hot spots. Eight individuals (c.1370C>T) had a developmental and epileptic encephalopathy with infantile onset and virtually no development. Of the other 7 individuals (n = 6: c.1334C>T; n = 1: c.1240G>A), 3 adults showed a biphasic course of disease with a mild static encephalopathy since early childhood and an unanticipated progressive deterioration with, e.g., movement disorder, psychiatric disease, and stroke-like episodes, epilepsy, starting in adulthood. Individuals who clinically presented in the first months of life had (near)-normal initial neuroimaging and severe cerebral atrophy during follow-up. In older children and adults, we noted progressive involvement of basal ganglia including Leigh-like MRI patterns and cerebellar atrophy, with remarkable intraindividual variability. The CSF glucose and the CSF/blood glucose ratio were below the 5th percentile of normal in almost all CSF samples, while blood glucose was unremarkable. This biomarker profile resembles glucose transporter type 1 deficiency syndrome; however, in HK1-related NDD, CSF lactate was significantly increased in all patients resulting in a substantially different biomarker profile. Discussion: Genotype-phenotype correlations appear to exist for HK1 variants and can aid in counseling. A CSF biomarker profile with low glucose, low CSF/blood glucose, and high CSF lactate may point toward monoallelic HK1 variants causing an NDD. This can help in variant interpretation and may aid in understanding the pathomechanism. We hypothesize that progressive intoxication and/or ongoing energy deficiency lead to the clinical phenotypes and progressive neuroimaging findings.

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations.

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations; however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESCs), including a knockout and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-sequencing analyses in Zscan10-/- mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10.

Heterozygous loss-of-function SMC3 variants are associated with variable growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 14 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism, reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated an overall milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, and some had alternative symptomatologies with rational biological links to SMC3. Analyses of tumor and model system transcriptomic data and epigenetic data in a subset of cases suggest that SMC3 pLoF variants reduce SMC3 expression but do not strongly support clustering with functional genomic signatures of typical CdLS. Our finding of substantial population-scale LoF intolerance in concert with variable growth and developmental features in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multilayered genomic data paired with careful phenotyping.

Multicentric pilot study to standardize clinical whole exome sequencing (WES) for cancer patients.

A growing number of druggable targets and national initiatives for precision oncology necessitate broad genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a positive percentage agreement (PPA) between 91 and 95% and a positive predictive value (PPV) between 82 and 95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Explanations for deviations included low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79-1, TMB: 0.97-0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.

A PAK1 Mutational Hotspot Within the Regulatory CRIPaK Domain is Associated With Severe Neurodevelopmental Disorders in Children.

BACKGROUND: P-21-activated kinases (PAKs) are protein serine/threonine kinases, part of the RAS/mitogen-activated protein kinase pathway. PAK1 is highly expressed in the central nervous system and crucially involved in neuronal migration and brain developmental processes. Recently, de novo heterozygous missense variants in PAK1 have been identified as an ultrarare cause of pediatric neurodevelopmental disorders. METHODS: We report a series of children affected with postnatal macrocephaly, neurodevelopmental impairment, and drug-resistant epilepsy. Repeated electroencephalographic (EEG) and video-EEG evaluations were performed over a two- to 10-year period during follow-up to delineate electroclinical histories. Genetic sequencing studies and computational evaluation of the identified variants were performed in our patient cohort. RESULTS: We identified by whole-exome sequencing three novel de novo variants in PAK1 (NM_001128620: c.427A>G, p.Met143Val; c.428T>C, p.Met143Thr; c.428T>A, p.Met143Lys) as the underlying cause of the disease in our families. The three variants affected the same highly conserved Met143 residue within the cysteine-rich inhibitor of PAK1 (CRIPaK) domain, which was identified before as a PAK1 inhibitor target. Computational studies suggested a defective autoinhibition presumably due to impaired PAK1 autoregulation as a result of the recurrent substitution. CONCLUSIONS: We delineated the electroclinical phenotypes of PAK1-related neurological disorders and highlight a novel mutational hotspot that may involve defective autoinhibition of the PAK1 protein. The three novel variants affecting the same hotspot residue within the CRIPaK domain highlight potentially impaired PAK1-CRIPaK interaction as a novel disease mechanism. These findings shed light on possible future treatments targeted at the CRIPaK domain, to modulate PAK1 activity and function.

Heterozygous loss-of-function SMC3 variants are associated with variable and incompletely penetrant growth and developmental features.

Heterozygous missense variants and in-frame indels in SMC3 are a cause of Cornelia de Lange syndrome (CdLS), marked by intellectual disability, growth deficiency, and dysmorphism, via an apparent dominant-negative mechanism. However, the spectrum of manifestations associated with SMC3 loss-of-function variants has not been reported, leading to hypotheses of alternative phenotypes or even developmental lethality. We used matchmaking servers, patient registries, and other resources to identify individuals with heterozygous, predicted loss-of-function (pLoF) variants in SMC3, and analyzed population databases to characterize mutational intolerance in this gene. Here, we show that SMC3 behaves as an archetypal haploinsufficient gene: it is highly constrained against pLoF variants, strongly depleted for missense variants, and pLoF variants are associated with a range of developmental phenotypes. Among 13 individuals with SMC3 pLoF variants, phenotypes were variable but coalesced on low growth parameters, developmental delay/intellectual disability, and dysmorphism reminiscent of atypical CdLS. Comparisons to individuals with SMC3 missense/in-frame indel variants demonstrated a milder presentation in pLoF carriers. Furthermore, several individuals harboring pLoF variants in SMC3 were nonpenetrant for growth, developmental, and/or dysmorphic features, some instead having intriguing symptomatologies with rational biological links to SMC3 including bone marrow failure, acute myeloid leukemia, and Coats retinal vasculopathy. Analyses of transcriptomic and epigenetic data suggest that SMC3 pLoF variants reduce SMC3 expression but do not result in a blood DNA methylation signature clustering with that of CdLS, and that the global transcriptional signature of SMC3 loss is model-dependent. Our finding of substantial population-scale LoF intolerance in concert with variable penetrance in subjects with SMC3 pLoF variants expands the scope of cohesinopathies, informs on their allelic architecture, and suggests the existence of additional clearly LoF-constrained genes whose disease links will be confirmed only by multi-layered genomic data paired with careful phenotyping.

Classical homocystinuria presenting with transient basal ganglia pathology and dystonia.

Classical homocystinuria is caused by pathogenic variants in the CBS gene leading to a deficiency of the vitamin B6-dependent enzyme cystathionine beta synthase. The disease is typically associated with high blood homocysteine concentrations. Clinical features include developmental delay/intellectual disability, psychiatric problems, thromboembolism, lens dislocation, and marfanoid habitus. We report on a child with classical homocystinuria presenting with acute episodes of dystonia and symmetrical basal ganglia abnormalities mimicking a mitochondrial disease. After starting treatment with vitamin B6, homocysteine levels rapidly normalized and dystonic episodes did not re-occur. Moreover, brain-imaging findings almost completely disappeared. The case illustrates that homocystinuria should be considered as a treatable differential diagnosis of dystonia.

Variants in EFCAB7 underlie nonsyndromic postaxial polydactyly.

Polydactyly is the most common limb malformation that occurs in 1.6-10.6 per one thousand live births, with incidence varying with ancestry. The underlying gene has been identified for many of the ~100 syndromes that include polydactyly. While for the more common form, nonsydromic polydactyly, eleven candidate genes have been reported. We investigated the underlying genetic cause of autosomal recessive nonsyndromic postaxial polydactyly in four consanguineous Pakistani families. Some family members with postaxial polydactyly also present with syndactyly, camptodactyly, or clinodactyly. Analysis of the exome sequence data revealed two novel homozygous frameshift deletions in EFCAB7: [c.830delG;p.(Gly277Valfs*5)]; in three families and [c.1350_1351delGA;p.(Asn451Phefs*2)] in one family. Sanger sequencing confirmed that these variants segregated with postaxial polydactyly, i.e., family members with postaxial polydactyly were found to be homozygous while unaffected members were heterozygous or wild type. EFCAB7 displays expressions in the skeletal muscle and on the cellular level in cilia. IQCE-EFCAB7 and EVC-EVC2 are part of the heterotetramer EvC complex, which is a positive regulator of the Hedgehog (Hh) pathway, that plays a key role in limb formation. Depletion of either EFCAB7 or IQCE inhibits induction of Gli1, a direct Hh target gene. Variants in IQCE and GLI1 have been shown to cause nonsyndromic postaxial polydactyly, while variants in EVC and EVC2 underlie Ellis van Creveld and Weyers syndromes, which include postaxial polydactyly as a phenotype. This is the first report of the involvement of EFCAB7 in human disease etiology.

Hemizygous variants in protein phosphatase 1 regulatory subunit 3F (PPP1R3F) are associated with a neurodevelopmental disorder characterized by developmental delay, intellectual disability and autistic features.

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.

Novel homozygous LAMB1 in-frame deletion in a pediatric patient with brain anomalies and cerebrovascular event.

Biallelic pathogenic variants in LAMB1 have been associated with autosomal recessive lissencephaly 5 (OMIM 615191), which is characterized by brain malformations (cobblestone lissencephaly, hydrocephalus), developmental delay, and epilepsy. Pathogenic variants in LAMB1 are rare, with only 11 pathogenic variants and 11 patients reported to date. Here, we report on a 6-year-old patient from a consanguineous family with profound developmental delay, microcephaly, and a history of a perinatal cerebrovascular event. Brain magnetic resonance imaging (MRI) showed cerebellar cystic defects, signal intensity abnormalities, and a hypoplastic corpus callosum. Trio-exome analysis revealed a homozygous in-frame deletion of Exons 23 and 24 of LAMB1 affecting 104 amino acids including the epidermal growth factor (EGF)-like units 11 and 12 in Domain III. To our knowledge, this is the first reported in-frame deletion in LAMB1. Our findings broaden the clinical and molecular spectrum of LAMB1-associated syndromes.

Variability in Cochlear Implantation Outcomes in a Large German Cohort With a Genetic Etiology of Hearing Loss.

OBJECTIVES: The variability in outcomes of cochlear implantation is largely unexplained, and clinical factors are not sufficient for predicting performance. Genetic factors have been suggested to impact outcomes, but the clinical and genetic heterogeneity of hereditary hearing loss makes it difficult to determine and interpret postoperative performance. It is hypothesized that genetic mutations that affect the neuronal components of the cochlea and auditory pathway, targeted by the cochlear implant (CI), may lead to poor performance. A large cohort of CI recipients was studied to verify this hypothesis. DESIGN: This study included a large German cohort of CI recipients (n = 123 implanted ears; n = 76 probands) with a definitive genetic etiology of hearing loss according to the American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP) guidelines and documented postoperative audiological outcomes. All patients underwent preoperative clinical and audiological examinations. Postoperative CI outcome measures were based on at least 1 year of postoperative audiological follow-up for patients with postlingual hearing loss onset (>6 years) and 5 years for children with congenital or pre/perilingual hearing loss onset (≤6 years). Genetic analysis was performed based on three different methods that included single-gene screening, custom-designed hearing loss gene panel sequencing, targeting known syndromic and nonsyndromic hearing loss genes, and whole-genome sequencing. RESULTS: The genetic diagnosis of the 76 probands in the genetic cohort involved 35 genes and 61 different clinically relevant (pathogenic, likely pathogenic) variants. With regard to implanted ears (n = 123), the six most frequently affected genes affecting nearly one-half of implanted ears were GJB2 (21%; n = 26), TMPRSS3 (7%; n = 9), MYO15A (7%; n = 8), SLC26A4 (5%; n = 6), and LOXHD1 and USH2A (each 4%; n = 5). CI recipients with pathogenic variants that influence the sensory nonneural structures performed at or above the median level of speech performance of all ears at 70% [monosyllable word recognition score in quiet at 65 decibels sound pressure level (SPL)]. When gene expression categories were compared to demographic and clinical categories (total number of compared categories: n = 30), mutations in genes expressed in the spiral ganglion emerged as a significant factor more negatively affecting cochlear implantation outcomes than all clinical parameters. An ANOVA of a reduced set of genetic and clinical categories (n = 10) identified five detrimental factors leading to poorer performance with highly significant effects ( p < 0.001), accounting for a total of 11.8% of the observed variance. The single strongest category was neural gene expression accounting for 3.1% of the variance. CONCLUSIONS: The analysis of the relationship between the molecular genetic diagnoses of a hereditary etiology of hearing loss and cochlear implantation outcomes in a large German cohort of CI recipients revealed significant variabilities. Poor performance was observed with genetic mutations that affected the neural components of the cochlea, supporting the "spiral ganglion hypothesis."

Clinical trio genome sequencing facilitates the interpretation of variants in cancer predisposition genes in paediatric tumour patients.

The prevalence of pathogenic and likely pathogenic (P/LP) variants in genes associated with cancer predisposition syndromes (CPS) is estimated to be 8-18% for paediatric cancer patients. In more than half of the carriers, the family history is unsuspicious for CPS. Therefore, broad genetic testing could identify germline predisposition in additional children with cancer resulting in important implications for themselves and their families. We thus evaluated clinical trio genome sequencing (TGS) in a cohort of 72 paediatric patients with solid cancers other than retinoblastoma or CNS-tumours. The most prevalent cancer types were sarcoma (n = 26), neuroblastoma (n = 15), and nephroblastoma (n = 10). Overall, P/LP variants in CPS genes were identified in 18.1% of patients (13/72) and P/LP variants in autosomal-dominant CPS genes in 9.7% (7/72). Genetic evaluation would have been recommended for the majority of patients with P/LP variants according to the Jongmans criteria. Four patients (5.6%, 4/72) carried P/LP variants in autosomal-dominant genes known to be associated with their tumour type. With the immediate information on variant inheritance, TGS facilitated the identification of a de novo P/LP in NF1, a gonadosomatic mosaic in WT1 and two pathogenic variants in one patient (DICER1 and PALB2). TGS allows a more detailed characterization of structural variants with base-pair resolution of breakpoints which can be relevant for the interpretation of copy number variants. Altogether, TGS allows comprehensive identification of children with a CPS and supports the individualised clinical management of index patients and high-risk relatives.

First case of adult onset neuronal intranuclear inclusion disease with both typical radiological signs and NOTCH2NLC repeat expansions in a Caucasian individual.

BACKGROUND AND PURPOSE: Adult onset neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder with a heterogeneous clinical presentation that can mimic stroke and various forms of dementia. To date, it has been described almost exclusively in Asian individuals. METHODS: This case presentation includes magnetic resonance imaging (MRI) of the neurocranium, histology by skin biopsy, and long-read genome sequencing. RESULTS: A 75-year-old Caucasian female presented with paroxysmal encephalopathy twice within a 14-month period. Brain MRI revealed high-intensity signals at the cerebral corticomedullary junction (diffusion-weighted imaging) and the paravermal area (fluid-attenuated inversion recovery), a typical distribution observed in adult onset NIID. The diagnosis was corroborated by skin biopsy, which demonstrated eosinophilic intranuclear inclusion bodies, and confirmed by long-read genome sequencing, showing an expansion of the GGC repeat in exon 1 of NOTCH2NLC. CONCLUSIONS: Our case proves adult onset NOTCH2NLC-GGC-positive NIID with typical findings on MRI and histology in a Caucasian patient and underscores the need to consider this diagnosis in non-Asian individuals.

Genome sequencing identifies complex structural MLH1 variant in unsolved Lynch syndrome.

BACKGROUND: Lynch syndrome is one of the most common cancer predisposition syndromes. It is caused by inherited changes in the mismatch repair pathway. With current diagnostic approaches, a causative genetic variant can be found in less than 50% of cases. A correct diagnosis is important for ensuring that an appropriate surveillance program is used and that additional high-risk family members are identified. METHODS: We used clinical genome sequencing on DNA from blood and subsequent transcriptome sequencing for confirmation. Data were analyzed using the megSAP pipeline and classified according to basic criteria in diagnostic laboratories. Segregation analyses in family members were conducted via breakpoint PCR. RESULTS: We present a family with the clinical diagnosis of Lynch syndrome in which standard diagnostic tests, such as panel or exome sequencing, were unable to detect the underlying genetic variant. Genome sequencing in the index patient confirmed the previous diagnostic results and identified an additional complex rearrangement with intronic breakpoints involving MLH1 and its neighboring gene LRRFIP2. The previously undetected structural variant was classified as medically relevant. Segregation analysis in the family identified additional at-risk individuals which were offered intensified cancer screening. DISCUSSION AND CONCLUSIONS: This case illustrates the advantages of clinical genome sequencing in detecting structural variants compared with current diagnostic approaches. Although structural variants are rare in Lynch syndrome families, they seem to be underreported, in part because of technical challenges. Clinical genome sequencing offers a comprehensive genetic characterization detecting a wide range of genetic variants.

Heterozygous pathogenic variants involving CBFB cause a new skeletal disorder resembling cleidocranial dysplasia.

BACKGROUND: Cleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (RUNX2) is currently the only known disease-causing gene for CCD, but several studies have suggested locus heterogeneity. METHODS: The cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level. RESULTS: In each subject a heterozygous pathogenic variant in CBFB was detected, whereas no genomic alteration involving RUNX2 was found. Three CBFB variants (one splice site alteration, one nonsense variant, one 2 bp duplication) were shown to result in a premature stop codon. A large intragenic deletion was found to delete exon 4, without affecting CBFB expression. The effect of a second splice site variant could not be determined but most likely results in a shortened or absent protein. Affected individuals showed similarities with RUNX2-related CCD, including dental and clavicular abnormalities. Normal stature and neurocognitive problems were however distinguishing features. CBFB encodes the core-binding factor ß subunit, which can interact with all RUNX proteins (RUNX1, RUNX2, RUNX3) to form heterodimeric transcription factors. This may explain the phenotypic differences between CBFB-related and RUNX2-related CCD. CONCLUSION: We confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants in CBFB in a cohort of eight individuals with clinical and radiographic features reminiscent of CCD.

Characterization of a novel non-canonical splice site variant (c.886-5T>A) in NBAS and description of the associated phenotype.

BACKGROUND: Biallelic pathogenic variants in the neuroblastoma-amplified sequence (NBAS) gene manifest in a broad spectrum of disorders, including, but not limited to recurrent acute liver failure, skeletal dysmorphism, susceptibility to infections, and SOPH syndrome with its cardinal symptoms of short stature, optic atrophy, and Pelger-Huët anomaly. We aimed to present clinical and genetic characteristics of two sisters (20 and 15 years old) who were diagnosed with optic atrophy and cone dystrophy in childhood. Genome sequencing revealed two novel variants in NBAS in compound heterozygous state in both sisters, namely a 1-bp deletion predicted to result in a premature termination codon (c.5104del; p.(Met1702*)), and a non-canonical splice site variant of unclear significance (c.886-5T>A; p.?). RESULTS: Clinical examination and history revealed cone dystrophy, optic atrophy, and Pelger-Huët anomaly, but no short stature, recurrent acute liver failure, or susceptibility to infections. RNA analysis revealed that the c.886-5T>A variant results in two aberrant transcripts that are predicted to lead to in frame amino acid changes in the ß-propeller region of the protein. CONCLUSION: We hypothesize that the phenotype of our subjects, which appears to be at the end of the spectrum of NBAS-related disorders, could be explained by residual protein function mediated by the non-canonical splice site variant c.886-5T>A. Our study contributes to the existing knowledge on the genotypic and phenotypic spectrum of NBAS-related disorders.

Germline findings in patients with advanced malignancies screened with paired blood-tumour testing for personalised treatment approaches.

BACKGROUND: Sequencing of tumour tissue with comprehensive gene panels is increasingly used to guide treatment in precision oncology. Analysis of tumour-normal pairs allows in contrast to tumour-only assessment direct discrimination between somatic and germline alterations, which might have important implications not only for the patients but also their families. METHODS: We performed tumour normal sequencing with a large gene panel in 1048 patients with advanced cancer to support treatment decision. Sequencing results were correlated with clinical and family data. RESULTS: We identified 156 likely pathogenic or pathogenic (LP/P) germline variants in cancer predisposition genes (CPGs) in 144 cases (13.7%). Of all patients, 8.8% had a LP/P variant in autosomal-dominant cancer predisposition genes (AD-CPGs), most of them being genes with high or moderate penetrance (ATM, BRCA2, CHEK2 and BRCA1). In 48 cases, the P/LP variant matched the expected tumour spectrum. A second variant in tumour tissue was found in 31 patients with AD-CPG variants. Low frequency mutations in either TP53, ATM or DNMT3A in the normal sample indicated clonal haematopoiesis in five cases. CONCLUSIONS: Tumour-normal testing for personalised treatment identifies germline LP/P variants in a relevant proportion of patients with cancer. The majority of them would not have been referred to genetic counselling based on family history. Indirect functional readouts of tumour-normal sequencing can provide novel links between CPGs and unexpected cancers. The interpretation of increasingly complex datasets in precision oncology is challenging and concepts of interdisciplinary personalised cancer prevention are needed to support patients and their families.

Exome-based gene panel analysis in a cohort of acute juvenile ischemic stroke patients:relevance of NOTCH3 and GLA variants.

BACKGROUND: Genetic variants are considered to have a crucial impact on the occurrence of ischemic stroke. In clinical routine, the diagnostic value of next-generation sequencing (NGS) in the medical clarification of acute juvenile stroke has not been investigated so far. MATERIAL AND METHODS: We analyzed an exome-based gene panel of 349 genes in 172 clinically well-characterized patients with magnetic resonance imaging (MRI)-proven, juvenile (age ≤ 55 years), ischemic stroke admitted to a single comprehensive stroke center. RESULTS: Monogenetic diseases causing ischemic stroke were observed in five patients (2.9%): In three patients with lacunar stroke (1.7%), we identified pathogenic variants in NOTCH3 causing cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Hence, CADASIL was identified at a frequency of 12.5% in the lacunar stroke subgroup. Further, in two male patients (1.2%) suffering from lacunar and cardioembolic stroke, pathogenic variants in GLA causing Fabry's disease were present. Additionally, genetic variants in monogenetic diseases lacking impact on stroke occurrence, variants of unclear significance (VUS) in monogenetic diseases, and (cardiovascular-) risk genes in ischemic stroke were observed in a total of 15 patients (15.7%). CONCLUSION: Genetic screening for Fabry's disease in cardioembolic and lacunar stroke as well as CADASIL in lacunar stroke might be beneficial in routine medical work-up of acute juvenile ischemic stroke.