Prenatal diagnosis of diaphanospondylodysostosis (DSD) : a case report

Peer reviewe

Variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy

Bott et al. here reports that de novo and biallelic variants in ATP6V0A1 gene affect the ability of the V-ATPase complex to translocate protons and acidify the endolysosomal compartment in neurons, causing a severe neurological phenotype ranging from developmental and epileptic encephalopathy to progressive myoclonus epilepsy. The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here, we identified 17 individuals from 14 unrelated families with both with new and previously characterized variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in Caenorhabditis elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.Peer reviewe

A patient with pontocerebellar hypoplasia type 6 : Novel RARS2 mutations, comparison to previously published patients and clinical distinction from PEHO syndrome

Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with edema, Hypsarrhythmia and Optic atrophy). The proband presented with severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly conserved amino acid and a frameshift variant with consequent degradation of the transcript resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO syndrome patients. The study highlights the challenges in clinical diagnosis of patients with neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI findings.Peer reviewe

Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production

Optic atrophy 1 (OPA1) is a mitochondrial inner membrane protein that has an important role in mitochondrial fusion and structural integrity. Dysfunctional OPA1 mutations cause atrophy of the optic nerve leading to blindness. Here, we show that OPA1 has an important role in the innate immune system. Using conditional knockout mice lacking Opa1 in neutrophils (Opa1(N Delta)), we report that lack of OPA1 reduces the activity of mitochondrial electron transport complex I in neutrophils. This then causes a decline in adenosine-triphosphate (ATP) production through glycolysis due to lowered NAD(+) availability. Additionally, we show that OPA1-dependent ATP production in these cells is required for microtubule network assembly and for the formation of neutrophil extracellular traps. Finally, we show that Opa1(N Delta) mice exhibit a reduced antibacterial defense capability against Pseudomonas aeruginosa.Peer reviewe

Progressive Myoclonus Epilepsy Caused by a Homozygous Splicing Variant of SLC7A6OS

Exome sequencing was performed in 2 unrelated families with progressive myoclonus epilepsy. Affected individuals from both families shared a rare, homozygous c.191A > G variant affecting a splice site in SLC7A6OS. Analysis of cDNA from lymphoblastoid cells demonstrated partial splice site abolition and the creation of an abnormal isoform. Quantitative reverse transcriptase polymerase chain reaction and Western blot showed a marked reduction of protein expression. Haplotype analysis identified a similar to 0.85cM shared genomic region on chromosome 16q encompassing the c.191A > G variant, consistent with a distant ancestor common to both families. Our results suggest that biallelic loss-of-function variants in SLC7A6OS are a novel genetic cause of progressive myoclonus epilepsy. ANN NEUROL 2020Peer reviewe

Novel synonymous and missense variants in FGFR1 causing Hartsfield syndrome

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1-related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson–Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly)

Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes

Progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous rare diseases. Over 70% of PME cases can now be molecularly solved. Known PME genes encode a variety of proteins, many involved in lysosomal and endosomal function. We performed whole-exome sequencing (WES) in 84 (78 unrelated) unsolved PME-affected individuals, with or without additional family members, to discover novel causes. We identified likely disease-causing variants in 24 out of 78 (31%) unrelated individuals, despite previous genetic analyses. The diagnostic yield was significantly higher for individuals studied as trios or families (14/28) versus singletons (10/50) (OR = 3.9, p value = 0.01, Fisher's exact test). The 24 likely solved cases of PME involved 18 genes. First, we found and functionally validated five heterozygous variants in NUS1 and DHDDS and a homozygous variant in ALG10, with no previous disease associations. All three genes are involved in dolichol-dependent protein glycosylation, a pathway not previously implicated in PME. Second, we independently validate SEMA6B as a dominant PME gene in two unrelated individuals. Third, in five families, we identified variants in established PME genes; three with intronic or copy-number changes (CLN6, GBA, NEU1) and two very rare causes (ASAH1, CERS1). Fourth, we found a group of genes usually associated with developmental and epileptic encephalopathies, but here, remarkably, presenting as PME, with or without prior developmental delay. Our systematic analysis of these cases suggests that the small residuum of unsolved cases will most likely be a collection of very rare, genetically heterogeneous etiologies.Peer reviewe

Structural brain anomalies in patients with FOXG1 syndrome and in Foxg1+/- mice

Objective FOXG1 syndrome is a rare neurodevelopmental disorder associated with heterozygous FOXG1 variants or chromosomal microaberrations in 14q12. The study aimed at assessing the scope of structural cerebral anomalies revealed by neuroimaging to delineate the genotype and neuroimaging phenotype associations. Methods We compiled 34 patients with a heterozygous (likely) pathogenic FOXG1 variant. Qualitative assessment of cerebral anomalies was performed by standardized re-analysis of all 34 MRI data sets. Statistical analysis of genetic, clinical and neuroimaging data were performed. We quantified clinical and neuroimaging phenotypes using severity scores. Telencephalic phenotypes of adult Foxg1+/- mice were examined using immunohistological stainings followed by quantitative evaluation of structural anomalies. Results Characteristic neuroimaging features included corpus callosum anomalies (82%), thickening of the fornix (74%), simplified gyral pattern (56%), enlargement of inner CSF spaces (44%), hypoplasia of basal ganglia (38%), and hypoplasia of frontal lobes (29%). We observed a marked, filiform thinning of the rostrum as recurrent highly typical pattern of corpus callosum anomaly in combination with distinct thickening of the fornix as a characteristic feature. Thickening of the fornices was not reported previously in FOXG1 syndrome. Simplified gyral pattern occurred significantly more frequently in patients with early truncating variants. Higher clinical severity scores were significantly associated with higher neuroimaging severity scores. Modeling of Foxg1 heterozygosity in mouse brain recapitulated the associated abnormal cerebral morphology phenotypes, including the striking enlargement of the fornix. Interpretation Combination of specific corpus callosum anomalies with simplified gyral pattern and hyperplasia of the fornices is highly characteristic for FOXG1 syndrome.Peer reviewe

Delineating the GRIN1 phenotypic spectrum: a distinct genetic NMDA receptor encephalopathy

Objective:To determine the phenotypic spectrum caused by mutations in GRIN1 encoding the NMDA receptor subunit GluN1 and to investigate their underlying functional pathophysiology.Methods:We collected molecular and clinical data from several diagnostic and research cohorts. Functional consequences of GRIN1 mutations were investigated in Xenopus laevis oocytes.Results:We identified heterozygous de novo GRIN1 mutations in 14 individuals and reviewed the phenotypes of all 9 previously reported patients. These 23 individuals presented with a distinct phenotype of profound developmental delay, severe intellectual disability with absent speech, muscular hypotonia, hyperkinetic movement disorder, oculogyric crises, cortical blindness, generalized cerebral atrophy, and epilepsy. Mutations cluster within transmembrane segments and result in loss of channel function of varying severity with a dominant-negative effect. In addition, we describe 2 homozygous GRIN1 mutations (1 missense, 1 truncation), each segregating with severe neurodevelopmental phenotypes in consanguineous families.Conclusions:De novo GRIN1 mutations are associated with severe intellectual disability with cortical visual impairment as well as oculomotor and movement disorders being discriminating phenotypic features. Loss of NMDA receptor function appears to be the underlying disease mechanism. The identification of both heterozygous and homozygous mutations blurs the borders of dominant and recessive inheritance of GRIN1-associated disorders.Johannes R. Lemke (32EP30_136042/1) and Peter De Jonghe (G.A.136.11.N and FWO/ESF-ECRP) received financial support within the EuroEPINOMICS-RES network (www.euroepinomics.org) within the Eurocores framework of the European Science Foundation (ESF). Saskia Biskup and Henrike Heyne received financial support from the German Federal Ministry for Education and Research (BMBF IonNeurONet: 01 GM1105A and FKZ: 01EO1501). Katia Hardies is a PhD fellow of the Institute for Science and Technology (IWT) Flanders. Ingo Helbig was supported by intramural funds of the University of Kiel, by a grant from the German Research Foundation (HE5415/3-1) within the EuroEPINOMICS framework of the European Science Foundation, and additional grants of the German Research Foundation (DFG, HE5415/5-1, HE 5415/6-1), German Ministry for Education and Research (01DH12033, MAR 10/012), and grant by the German chapter of the International League against Epilepsy (DGfE). The project also received infrastructural support through the Institute of Clinical Molecular Biology in Kiel, supported in part by DFG Cluster of Excellence "Inflammation at Interfaces" and "Future Ocean." The project was also supported by the popgen 2.0 network (P2N) through a grant from the German Ministry for Education and Research (01EY1103) and by the International Coordination Action (ICA) grant G0E8614N. Christel Depienne, Caroline Nava, and Delphine Heron received financial support for exome analyses by the Centre National de Genotypage (CNG, Evry, France)

Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines : a case report

Abstract Background Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. Methods Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ+ T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and TH1 polarization. Results A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. Conclusions Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial