Biallelic NUDT2 variants defective in mRNA decapping cause a neurodevelopmental disease.

Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present. The disorder is associated with rare variants in NUDT2, a mRNA decapping and Ap4A hydrolysing enzyme, including novel missense and in-frame deletion variants. We show that these NUDT2 variants lead to a marked loss of enzymatic activity, strongly implicating loss of NUDT2 function as the cause of the disorder. NUDT2-deficient patient fibroblasts exhibit a markedly altered transcriptome, accompanied by changes in mRNA half-life and stability. Amongst the most up-regulated mRNAs in NUDT2-deficient cells, we identified host response and interferon-responsive genes. Importantly, add-back experiments using an Ap4A hydrolase defective in mRNA decapping highlighted loss of NUDT2 decapping as the activity implicated in altered mRNA homeostasis. Our results confirm that reduction or loss of NUDT2 hydrolase activity is associated with a neurological disease, highlighting the importance of a physiologically balanced mRNA processing machinery for neuronal development and homeostasis.

Bi-allelic variants of FILIP1 cause congenital myopathy, dysmorphism and neurological defects.

Filamin-A-interacting protein 1 (FILIP1) is a structural protein that is involved in neuronal and muscle function and integrity and interacts with FLNa and FLNc. Pathogenic variants in filamin-encoding genes have been linked to neurological disorders (FLNA) and muscle diseases characterized by myofibrillar perturbations (FLNC), but human diseases associated with FILIP1 variants have not yet been described. Here, we report on five patients from four unrelated consanguineous families with homozygous FILIP1 variants (two nonsense and two missense). Functional studies indicated altered stability of the FILIP1 protein carrying the p.[Pro1133Leu] variant. Patients exhibit a broad spectrum of neurological symptoms including brain malformations, neurodevelopmental delay, muscle weakness and pathology and dysmorphic features. Electron and immunofluorescence microscopy on the muscle biopsy derived from the patient harbouring the homozygous p.[Pro1133Leu] missense variant revealed core-like zones of myofibrillar disintegration, autophagic vacuoles and accumulation of FLNc. Proteomic studies on the fibroblasts derived from the same patient showed dysregulation of a variety of proteins including FLNc and alpha-B-crystallin, a finding (confirmed by immunofluorescence) which is in line with the manifestation of symptoms associated with the syndromic phenotype of FILIP1opathy. The combined findings of this study show that the loss of functional FILIP1 leads to a recessive disorder characterized by neurological and muscular manifestations as well as dysmorphic features accompanied by perturbed proteostasis and myopathology.

Bi-allelic loss-of-function variants in KIF21A cause severe fetal akinesia with arthrogryposis multiplex.

BACKGROUND: Fetal akinesia (FA) results in variable clinical presentations and has been associated with more than 166 different disease loci. However, the underlying molecular cause remains unclear in many individuals. We aimed to further define the set of genes involved. METHODS: We performed in-depth clinical characterisation and exome sequencing on a cohort of 23 FA index cases sharing arthrogryposis as a common feature. RESULTS: We identified likely pathogenic or pathogenic variants in 12 different established disease genes explaining the disease phenotype in 13 index cases and report 12 novel variants. In the unsolved families, a search for recessive-type variants affecting the same gene was performed; and in five affected fetuses of two unrelated families, a homozygous loss-of-function variant in the kinesin family member 21A gene (KIF21A) was found. CONCLUSION: Our study underlines the broad locus heterogeneity of FA with well-established and atypical genotype-phenotype associations. We describe KIF21A as a new factor implicated in the pathogenesis of severe neurogenic FA sequence with arthrogryposis of multiple joints, pulmonary hypoplasia and facial dysmorphisms. This hypothesis is further corroborated by a recent report on overlapping phenotypes observed in Kif21a null piglets.

Novel Variants of SOX4 in Patients with Intellectual Disability.

SOX4 is a transcription factor with pleiotropic functions required for different developmental processes, such as corticogenesis. As with all SOX proteins, it contains a conserved high mobility group (HMG) and exerts its function via interaction with other transcription factors, such as POU3F2. Recently, pathogenic SOX4 variants have been identified in several patients who had clinical features overlapping with Coffin-Siris syndrome. In this study, we identified three novel variants in unrelated patients with intellectual disability, two of which were de novo (c.79G>T, p.Glu27*; c.182G>A p.Arg61Gln) and one inherited (c.355C>T, p.His119Tyr). All three variants affected the HMG box and were suspected to influence SOX4 function. We investigated the effects of these variants on transcriptional activation by co-expressing either wildtype (wt) or mutant SOX4 with its co-activator POU3F2 and measuring their activity in reporter assays. All variants abolished SOX4 activity. While our experiments provide further support for the pathogenicity of SOX4 loss-of-function (LOF) variants as a cause of syndromic intellectual disability (ID), our results also indicate incomplete penetrance associated with one variant. These findings will improve classification of novel, putatively pathogenic SOX4 variants.

Patient-individual phenotypes of glioblastoma stem cells are conserved in culture and associate with radioresistance, brain infiltration and patient prognosis.

Identification of prognostic or predictive molecular markers in glioblastoma resection specimens may lead to strategies for therapy stratification and personalized treatment planning. Here, we analyzed in primary glioblastoma stem cell (pGSC) cultures the mRNA abundances of seven stem cell (MSI1, Notch1, nestin, Sox2, Oct4, FABP7 and ALDH1A3), and three radioresistance or invasion markers (CXCR4, IKCa and BKCa ). From these abundances, an mRNA signature was deduced which describes the mesenchymal-to-proneural expression profile of an individual GSC culture. To assess its functional significance, we associated the GSC mRNA signature with the clonogenic survival after irradiation with 4 Gy and the fibrin matrix invasion of the GSC cells. In addition, we compared the molecular pGSC mRNA signature with the tumor recurrence pattern and the overall survival of the glioblastoma patients from whom the pGSC cultures were derived. As a result, the molecular pGSC mRNA signature correlated positively with the pGSC radioresistance and matrix invasion capability in vitro. Moreover, patients with a mesenchymal (>median) mRNA signature in their pGSC cultures exhibited predominantly a multifocal tumor recurrence and a significantly (univariate log rank test) shorter overall survival than patients with proneural (≤median mRNA signature) pGSCs. The tumors of the latter recurred predominately unifocally. We conclude that our pGSC cultures induce/select those cell subpopulations of the heterogeneous brain tumor that determine disease progression and therapy outcome. In addition, we further postulate a clinically relevant prognostic/predictive value for the 10 mRNAs-based mesenchymal-to-proneural signature of the GSC subpopulations in glioblastoma.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome.

PURPOSE: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. METHODS: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. RESULTS: Computational facial and Human Phenotype Ontology-based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. CONCLUSION: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

Predicting clinical phenotypes of metachromatic leukodystrophy based on the arylsulfatase A activity and the ARSA genotype? - Chances and challenges.

OBJECTIVES: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficiency of arylsulfatase A (ARSA). Subsequent accumulation of sulfatides leads to demyelination and neurodegeneration in the central and peripheral nervous system. To date MLD is classified based on the age at onset, however, especially for late onset forms this classification provides only limited projection regarding the clinical disease course. Moreover, evolving newborn screening approaches raise the need to predict the disease onset and course in pre-symptomatic individuals. Here, we correlate the ARSA activity and the ARSA-genotype with clinical parameters in a large cohort of 96 affected individuals. MATERIALS AND METHODS: Clinical data of 96 affected individuals with genetically and/or biochemically confirmed MLD were collected from a national database. Leukocyte samples from 69 affected individuals were re-analyzed for the ARSA activity using p-nitrocatecholsulfate as substrate with a refined ARSA assay towards the lower limit of detection. For 84 individuals genetic sequencing was conducted by Sanger or next generation sequencing (NGS). RESULTS: The adapted ARSA assay revealed the discriminatory power to differentiate MLD subtypes as the residual enzyme activity was low in late infantile and early juvenile forms, and clearly higher in late juvenile and adult MLD (p < 0.001). A residual enzyme activity below 1% compared to controls predicted an early onset (late-infantile or early-juvenile) and rapid disease progression. A firm genotype-phenotype correlation was proven as reliable for bi-allelic protein-truncating variants in the ARSA gene resulting in minimal residual ARSA activity, an early onset of the disease and initial decline of motor functions. Although the impact of missense variants was equivocal, few variants with a recognizable clinical spectrum were identified. DISCUSSION: ARSA activity in leukocytes as well as the ARSA genotype can predict the age of disease onset and the dynamic of disease progression for most of the early onset forms. This knowledge is relevant for patient counseling and to guide treatment decisions, especially when identifying pre-symptomatic individuals, e.g., in newborn screening. However, due to the high cumulative frequency of rare disease-causing missense variants in the ARSA gene that lead to highly variable residual enzyme activity, reiterated biochemical and genetic studies are needed to improve disease course prediction.

Angiokeratoma corporis diffusum with severe acroparesthesia, an endothelial abnormality, and inconspicuous genetic findings.

Angiokeratoma corporis diffusum (ACD) was long thought to be a specific dermal sign of Fabry disease (FD, X-linked alpha-galactosidase A [GLA] deficiency). However, other lysosomal storage diseases (LSDs) have also been identified as triggers of ACD. Generalized vasculopathy is an important pathogenetic factor in FD and may also lead to the acroparesthesia (AP) often predominant in FD. We report on an 85-year-old woman with ACD present since her youth and associated with severe AP. Ultrastructure of the dermal lesion showed no lysosomal involvement, but the absence of the basement membrane of the endothelial cells of the capillary vessels was noteworthy. Repeated analyses of the GLA gene revealed no evidence of FD. Whole-exome sequencing was negative for FD and other LSDs, and allowed us to also study FD-related intronic regions of the GLA gene. This is the first report of a patient with FD-like ACD with an endothelial abnormality, otherwise unexplained vasculopathy and severe AP, which are not due to FD or another LSD. Based on family history, another genetic, yet unidentified, defect may cause the disease in this patient. In unexplained ACD, extended genetic analysis is required to exclude particular pathogenic variants of the GLA gene and other genes.

A single center experience of prenatal parent-fetus trio exome sequencing for pregnancies with congenital anomalies.

OBJECTIVES: To examine the diagnostic yield of trio exome sequencing in fetuses with multiple structural defects with no pathogenic findings in cytogenetic and microarray analyses. METHODS: We recruited 51 fetuses with two or more defects, non-immune fetal hydrops or fetal akinesia deformation syndrome|or fetal akinesia deformation sequence (FADS). Trio exome sequencing was performed on DNA from chorionic villi samples and parental blood. Detection of genomic variation and prioritization of clinically relevant variants was performed according to in-house standard operating procedures. RESULTS: Median maternal and gestational age was 32.0 years and 21.0 weeks, respectively. Forty-three (84.3%) fetuses had two or more affected organ systems. The remaining fetuses had isolated fetal hydrops or FADS. In total, the exome analysis established the genetic cause for the clinical abnormalities in 22 (43.1%, 95% CI 29.4%-57.8%) pregnancies. CONCLUSIONS: In fetuses with multiple defects, hydrops or FADS and normal standard genetic results, trio exome sequencing has the potential to identify genetic anomalies in more than 40% of cases.

Assay of ß-glucosidase 2 (GBA2) activity using lithocholic acid ß-3-O-glucoside substrate for cultured fibroblasts and glucosylceramide for brain tissue.

Beta (ß)-glucosidase 2 (GBA2) is deficient in a form of human spastic paraplegia due to defects in GBA2 (SPG46). GBA2 was proposed as a modifier of Gaucher disease, a lysosomal storage disease resulting from deficient ß-glucosidase 1; GBA1. Current GBA2 activity assays using artificial substrates incompletely model the activity encountered in vivo. We studied GBA2 activity, using lithocholic acid ß-glucoside or glucosylceramide as natural ß-glucosidase substrates in murine tissues or cultured patient fibroblasts with the pathologic genotypes: Gba1-/-; Gba2-/-; GBA1-/-; GBA2+/- and found expected and unexpected deviations from normal controls.