De novo heterozygous missense variants in CELSR1 as cause of fetal pleural effusions and progressive fetal hydrops.

Fetal hydrops as detected by prenatal ultrasound usually carries a poor prognosis depending on the underlying aetiology. We describe the prenatal and postnatal clinical course of two unrelated female probands in whom de novo heterozygous missense variants in the planar cell polarity gene CELSR1 were detected using exome sequencing. Using several in vitro assays, we show that the CELSR1 p.(Cys1318Tyr) variant disrupted the subcellular localisation, affected cell-cell junction, impaired planar cell polarity signalling and lowered proliferation rate. These observations suggest that deleterious rare CELSR1 variants could be a possible cause of fetal hydrops.

Bi-allelic ACBD6 variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders.

The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Using exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with myristic acid alkyne (YnMyr) chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), aged 1-50 years, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%) and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%) and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%) and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each) as well as hypertrophy of the clava (24%) were common neuroimaging findings. Acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localization and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-myristoylation was similarly affected in acbd6-deficient zebrafish and X. tropicalis models, including Fus, Marcks and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders.

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.

Correction: Requirement of the acyl-CoA carrier ACBD6 in myristoylation of proteins: Activation by ligand binding and protein interaction.

[This corrects the article DOI: 10.1371/journal.pone.0229718.].

Impaired glucose-1,6-biphosphate production due to bi-allelic PGM2L1 mutations is associated with a neurodevelopmental disorder.

We describe a genetic syndrome due to PGM2L1 deficiency. PGM2 and PGM2L1 make hexose-bisphosphates, like glucose-1,6-bisphosphate, which are indispensable cofactors for sugar phosphomutases. These enzymes form the hexose-1-phosphates crucial for NDP-sugars synthesis and ensuing glycosylation reactions. While PGM2 has a wide tissue distribution, PGM2L1 is highly expressed in the brain, accounting for the elevated concentrations of glucose-1,6-bisphosphate found there. Four individuals (three females and one male aged between 2 and 7.5 years) with bi-allelic inactivating mutations of PGM2L1 were identified by exome sequencing. All four had severe developmental and speech delay, dysmorphic facial features, ear anomalies, high arched palate, strabismus, hypotonia, and keratosis pilaris. Early obesity and seizures were present in three individuals. Analysis of the children's fibroblasts showed that glucose-1,6-bisphosphate and other sugar bisphosphates were markedly reduced but still present at concentrations able to stimulate phosphomutases maximally. Hence, the concentrations of NDP-sugars and glycosylation of the heavily glycosylated protein LAMP2 were normal. Consistent with this, serum transferrin was normally glycosylated in affected individuals. PGM2L1 deficiency does not appear to be a glycosylation defect, but the clinical features observed in this neurodevelopmental disorder point toward an important but still unknown role of glucose-1,6-bisphosphate or other sugar bisphosphates in brain metabolism.

Bi-allelic loss-of-function variants in BCAS3 cause a syndromic neurodevelopmental disorder.

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands' primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands' fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development.

Monogenic variants in dystonia: an exome-wide sequencing study.

BACKGROUND: Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. METHODS: For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. FINDINGS: We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. INTERPRETATION: In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations. FUNDING: Else Kröner-Fresenius-Stiftung, Technische Universität München, Helmholtz Zentrum München, Medizinische Universität Innsbruck, Charles University in Prague, Czech Ministry of Education, the Slovak Grant and Development Agency, the Slovak Research and Grant Agency.

Mutations in MYLPF Cause a Novel Segmental Amyoplasia that Manifests as Distal Arthrogryposis.

We identified ten persons in six consanguineous families with distal arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T [p.Cys157Phe] or c.469T>C [p.Cys157Arg]) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A (p.Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly inherited DA, a c.98C>T (p.Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

Requirement of the acyl-CoA carrier ACBD6 in myristoylation of proteins: Activation by ligand binding and protein interaction.

Glycine N-myristoylation is an essential acylation modification modulating the functions, stability, and membrane association of diverse cytosolic proteins in human cells. Myristoyl-CoA is the 14-carbon acyl donor of the acyltransferase reaction. Acyl-CoAs of a chain length compatible with the binding site of the N-myristoyltransferase enzymes (NMT) are competitive inhibitors, and the mechanism protecting these enzymes from unwanted acyl-CoA species requires the acyl-CoA binding protein ACBD6. The acyl-CoA binding domain (ACB) and the ankyrin-repeat motifs (ANK) of ACBD6 can perform their functions independently. Interaction of ANK with human NMT2 was necessary and sufficient to provide protection. Fusion of the ANK module to the acyl-CoA binding protein ACBD1 was sufficient to confer the NMT-stimulatory property of ACBD6 to the chimera. The ACB domain is dispensable and sequestration of the competitor was not the basis for NMT2 protection. Acyl-CoAs bound to ACB modulate the function of the ANK module and act as positive effector of the allosteric activation of the enzyme. The functional relevance of homozygous mutations in ACBD6 gene, which have not been associated with a disease so far, is presented. Skin-derived fibroblasts of two unrelated individuals with neurodevelopmental disorder and carrying loss of function mutations in the ACBD6 gene were deficient in protein N-myristoylation. These cells were sensitive to substrate analog competing for myristoyl-CoA binding to NMT. These findings account for the requirement of an ANK-containing acyl-CoA binding protein in the cellular mechanism protecting the NMT enzymes and establish that in human cells, ACBD6 supports the N-myristoylation of proteins.

Spektrum der Ichthyosen in einer österreichischen Ichthyosekohorte von 2004-2007.

HINTERGRUND: Ichthyosen sind eine heterogene Gruppe von Krankheiten, deren klinische Klassifizierung schwierig ist. Hier wird die Ichthyosekohorte eines Expertisezentrums für Genodermatosen im Detail beschrieben. PATIENTEN UND METHODIK: Eingeschlossen wurden Patienten mit klinisch oder genetisch bestätigter Ichthyose, die zwischen 2004 und 2017 untersucht und in einer Datenbank aufgenommen wurden. Krankheitsbeginn, Phänotyp, Histologie, Komorbiditäten und Familienanamnese wurden detailliert beschrieben. Bei den genetisch getesteten Patienten wurden Jahr und Methode der genetischen Testung protokolliert und die Prävalenz der unterschiedlichen Autosomal-rezessive-kongenitale Ichthyose (ARCI)-Gene und -Phänotypen, die Prävalenz der syndromalen Ichthyosen und die Genotyp-Phänotyp-Korrelationen analysiert. ERGEBNISSE UND METHODIK: Von den insgesamt 198 eingeschlossenen Patienten wurden 151 genetisch getestet. 81 Patienten hatten eine Ichthyosis vulgaris (IV), 43 eine X-chromosomale Ichthyose (XLI), 38 eine ARCI, 9 eine keratinopathische Ichthyose (KPI) und ein Patient eine Exfoliative Ichthyose. 26 Patienten litten an einer syndromalen Ichthyose. Im Vergleich zu den syndromalen Ichthyosen wurde bei den häufigen Ichthyosen (IV, XLI) und KPI eine gute Phänotyp-Genotyp-Korrelation beobachtet. In 91 % der ARCI-Patienten konnte die exakte Diagnose durch genetische Testung gestellt werden. Lediglich bei 33 % der Patienten mit syndromaler Ichthyose bestand vor der genetischen Testung ein Verdacht auf die tatsächliche Diagnose. In 86 % der Fälle wurde eine kausale Mutation nachgewiesen. SCHLUSSFOLGERUNGEN: Die Arbeit beschreibt das Spektrum der Ichthyosen an einem Expertisezentrum und zeigt, dass für diese Gruppe die genetische Testung von Genodermatosen ein diagnostischer Standard werden sollte.

A Syrian patient with Steel syndrome due to compound heterozygous COL27A1 mutations with colobomata of the eye.

The joint occurrence of short stature, congenital dislocation of the hip, carpal coalition, dislocation of the radial head, cavus deformity, scoliosis, and vertebral anomalies was first described in 1993 by Steel et al. (OMIM #615155) in 23 children from Puerto Rico. The condition is caused by a deficient matrix protein, collagen type XXVII alpha 1 chain, due to bi-allelic loss of function mutations in the gene COL27A1. Outside of Puerto Rico, only four families have been described, in three of which the patients also had hearing loss. However, structural eye defects have not yet been reported in conjunction with this rare autosomal recessive syndrome. Here, we describe a 9-year-old girl born to nonconsanguineous Syrian parents with the characteristic features of Steel syndrome, including short stature, massive malalignment of large joints, kyphoscoliosis, hearing loss, and typical facial dysmorphism. However, she was also born with bilateral colobomata of the irides and choroido-retinae with unilateral affection of the macula. Whole exome sequencing identified two pathogenic compound heterozygous variants in COL27A1: c.93del, p.(Phe32Leufs*71) and c.3075del, p.(Lys1026Argfs*33). There was no discernible alternative cause for the colobomata. Our findings might indicate an association of this exceptionally rare disorder caused by COL27A1 mutations with developmental defects of the eye from the anophthalmia/microphthalmia/coloboma spectrum.

Spectrum of ichthyoses in an Austrian ichthyosis cohort from 2004 to 2017.

BACKGROUND: Ichthyoses are a heterogeneous disease group, which makes clinical classification challenging. An ichthyosis cohort at a center for genodermatoses is presented in detail. PATIENTS AND METHODS: Patients with clinically and/or genetically confirmed ichthyosis seen from 2004 to 2017 and listed in a database were included. Disease onset, phenotype, histology, comorbidities and family history were described in detail. In genetically tested patients, the prevalence of various ARCI genes, ARCI phenotypes and syndromic ichthyoses, as well as genotype-phenotype correlation and year/method of genetic testing was assessed. RESULTS: Of all 198 patients who were included in the cohort, 151 were genetically tested. 81 had ichthyosis vulgaris, 43 X-linked ichthyosis, 38 autosomal recessive congenital ichthyosis (ARCI), 9 keratinopathic ichthyosis (KPI) and one exfoliative ichthyosis. 26 individuals suffered from syndromic ichthyoses. A good genotype-phenotype correlation was observed for common ichthyoses and KPI; the correlation was less good in syndromic ichthyoses. In 91 % of ARCI patients an accurate diagnosis was obtained by genetic testing. In only 33 % of syndromic ichthyoses was the definitive diagnosis suspected before genetic testing, which revealed a causative mutation in 86 % of cases. CONCLUSION: This study describes the spectrum of ichthyoses in a center of expertise and shows that genetic testing should become a diagnostic standard for this disease group.

A further case of familial ring chromosome 20 mosaicism - molecular characterization of the ring and review of the literature.

Ring chromosome 20 syndrome is a rare chromosomal disorder characterized by childhood-onset drug-resistant epilepsy, behavioral problems and variable cognitive impairment. While most cases occur sporadically, parent-to-child transmission of ring 20 mosaicism has only been reported in a few exceptional families. We identified a further family with mother-to-child transmission of ring 20 mosaicism. Detailed characterization of the ring chromosome showed a complete ring with preserved telomere repetitive sequences. SNP genotyping excluded mosaic uniparental disomy and indicated that the chromosome was transmitted without recombination from mother to child. These results corroborate the findings of a previous study and support the hypothesis that inherited mosaicism is due to transmission of an unstable chromosome either prone to ring opening or to ring re-formation.

Isoform-specific domain organization determines conformation and function of the peroxisomal biogenesis factor PEX26.

Peroxisomal biogenesis factor PEX26 is a membrane anchor for the multi-subunit PEX1-PEX6 protein complex that controls ubiquitination and dislocation of PEX5 cargo receptors for peroxisomal matrix protein import. PEX26 associates with the peroxisomal translocation pore via PEX14 and a splice variant (PEX26Δex5) of unknown function has been reported. Here, we demonstrate PEX26 homooligomerization mediated by two heptad repeat domains adjacent to the transmembrane domain. We show that isoform-specific domain organization determines PEX26 oligomerization and impacts peroxisomal ß-oxidation and proliferation. PEX26 and PEX26Δex5 displayed different patterns of interaction with PEX2-PEX10 or PEX13-PEX14 complexes, which relate to distinct pre-peroxisomes in the de novo synthesis pathway. Our data support an alternative PEX14-dependent mechanism of peroxisomal membrane association for the splice variant, which lacks a transmembrane domain. Structure-function relationships of PEX26 isoforms explain an extended function in peroxisomal homeostasis and these findings may improve our understanding of the broad phenotype of PEX26-associated human disorders.

Bi-allelic ADPRHL2 Mutations Cause Neurodegeneration with Developmental Delay, Ataxia, and Axonal Neuropathy.

ADP-ribosylation is a reversible posttranslational modification used to regulate protein function. ADP-ribosyltransferases transfer ADP-ribose from NAD+ to the target protein, and ADP-ribosylhydrolases, such as ADPRHL2, reverse the reaction. We used exome sequencing to identify five different bi-allelic pathogenic ADPRHL2 variants in 12 individuals from 8 families affected by a neurodegenerative disorder manifesting in childhood or adolescence with key clinical features including developmental delay or regression, seizures, ataxia, and axonal (sensori-)motor neuropathy. ADPRHL2 was virtually absent in available affected individuals' fibroblasts, and cell viability was reduced upon hydrogen peroxide exposure, although it was rescued by expression of wild-type ADPRHL2 mRNA as well as treatment with a PARP1 inhibitor. Our findings suggest impaired protein ribosylation as another pathway that, if disturbed, causes neurodegenerative diseases.

Evidence of mild founder LMOD3 mutations causing nemaline myopathy 10 in Germany and Austria.

OBJECTIVE: To expand the clinical and genetic spectrum of nemaline myopathy 10 by a series of Austrian and German patients with a milder disease course and missense mutations in LMOD3. METHODS: We characterized the clinical features and the genetic status of 4 unrelated adolescent or adult patients with nemaline myopathy. RESULTS: The 4 patients showed a relatively mild disease course. They all have survived into adulthood, 3 of 4 have remained ambulatory, and all showed marked facial weakness. Muscle biopsy specimens gave evidence of nemaline bodies. All patients were unrelated but originated from Austria (Tyrol and Upper Austria) and Southern Germany (Bavaria). All patients carried the missense variant c.1648C>T, p.(Leu550Phe) in the LMOD3 gene, either on both alleles or in trans with another missense variant (c.1004A>G, p.Gln335Arg). Both variants were not reported previously. CONCLUSIONS: In 2014, a severe form of congenital nemaline myopathy caused by disrupting mutations in LMOD3 was identified and denoted as NEM10. Unlike the previously reported patients, who had a severe clinical picture with a substantial risk of early death, our patients showed a relatively mild disease course. As the missense variant c.1648C>T is located further downstream compared to all previously published LMOD3 mutations, it might be associated with higher protein expression compared to the reported loss-of-function mutations. The apparent clusters of 2 mild mutations in Germany and Austria in 4 unrelated families may be explained by a founder effect.

The clinical manifestation of MCAD deficiency: challenges towards adulthood in the screened population.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common fatty acid oxidation disorder. Typically, undiagnosed individuals are asymptomatic until an episode of increased energy demand and fasting occurs, resulting in metabolic derangement. Phenotypic heterogeneity has been increasingly realized, with reports of both neonates and adults manifesting with life-threatening symptoms including encephalopathy, rhabdomyolysis, and cardiac failure. If diagnosed presymptomatically, outcome is favorable basically by avoidance of fasting. Early detection by newborn screening (NBS) has significantly reduced the incidence of severe adverse events including deaths. In this manuscript we focus on the natural course of the disease in both children and adults. Although NBS for MCADD has been successfully established, continuing efforts need to be made to avoid acute crises and deterioration of outcome in screened patients entering adolescence and adulthood.