Search

Sphingolipids (SL) represent a structurally diverse class of lipids that are central to cellular physiology and neuronal development and function. Defects in the sphingolipid metabolism are typically associated with nervous system disorders. The C4-dihydroceramide desaturase (DEGS1) catalyzes the conversion of dihydroceramide to ceramide, the final step in the SL de-novo synthesis. Loss of function mutations in DEGS1 cause a hypomyelinating leukodystrophy, which is associated with increased plasma dihydrosphingolipids (dhSL) and with the formation of an atypical SPB 18:1(14Z);O2 metabolite. Here, we characterize two novel DEGS1 variants of unknown significance (VUS), provide a structural model with a predicted substrate binding site and propose a regulatory link between DEGS1 and fatty acid desaturase 3 (FADS3). Both VUS involve single amino acid substitutions near the C-terminus within conserved regions of the enzyme. Patient 1 (p.R311K variant) shows severe progressive tetraspasticity, intellectual disability, and epilepsy in combination with brain magnetic resonance imaging (MRI) findings, typical for DEGS1-related leukodystrophy. Patient 2 (p.G270E variant) presents with delayed psychomotor development, oculomotor apraxia, and a normal brain MRI. Plasma from the p.R311K carrier showed a significantly elevated dhSL species and the presence of SPB 18:1(14Z);O2, while the plasma SL profile for the p.G270E variant was not altered. This suggests the p.R331K variant is pathogenic, while the p.G270E appears benign. As an increase in dihydroSL species is also seen in other pathological disorders of the SL metabolism, the SPB 18:1(14Z);O2 seems to be a more specific biomarker to discriminate between pathogenic and benign DEGS1 variants.

Costello syndrome is a clinically recognizable, severe neurodevelopmental disorder caused by heterozygous activating variants in HRAS. The vast majority of affected patients share recurring variants affecting HRAS codons 12 and 13 and a relatively uniform phenotype. Here, we report the unique and attenuated phenotype of six individuals of an extended family affected by the HRAS variant c.176C>T p.(Ala59Gly), which, to our knowledge, has never been reported as a germline variant in patients so far. HRAS Alanine 59 has been previously functionally investigated as an oncogenic hotspot and the p.Ala59Gly substitution was shown to impair intrinsic GTP hydrolysis. All six individuals we report share a phenotype of ectodermal anomalies and mild features suggestive of a RASopathy, reminiscent of patients with Noonan syndrome-like disorder with loose anagen hair. All six are of normal intelligence, none have a history of failure to thrive or malignancy, and they have no known cardiac or neurologic pathologies. Our report adds to the previous reports of patients with rare variants affecting amino acids located in the SWITCH II/G3 region of HRAS and suggests a consistent, attenuated phenotype distinct from classical Costello syndrome. We propose the definition of a new distinct HRAS-related RASopathy for patients carrying HRAS variants affecting codons 58, 59, 60.

The magnitude of clinical utility of preconception expanded carrier screening (ECS) concerning its potential to reduce the risk of affected offspring is unknown. Since neurodevelopmental disorders (NDDs) in their offspring is a major concern of parents-to-be, we addressed the question of residual risk by assessing the risk-reduction potential for NDDs in a retrospective study investigating ECS with different criteria for gene selection and definition of pathogenicity. We used exome sequencing data from 700 parents of children with NDDs and blindly screened for carrier-alleles in up to 3046 recessive/X-linked genes. Depending on variant pathogenicity thresholds and gene content, NDD-risk-reduction potential was up to 43.5% in consanguineous, and 5.1% in nonconsanguineous couples. The risk-reduction-potential was compromised by underestimation of pathogenicity of missense variants (false-negative-rate 4.6%), inherited copy-number variants and compound heterozygosity of one inherited and one de novo variant (0.9% each). Adherence to the ACMG recommendations of restricting ECS to high-frequency genes in nonconsanguineous couples would more than halve the detectable inherited NDD-risk. Thus, for optimized clinical utility of ECS, screening in recessive/X-linked genes regardless of their frequency (ACMG Tier-4) and sensible pathogenicity thresholds should be considered for all couples seeking ECS.

PURPOSE Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf-Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. METHODS We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. RESULTS The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2's folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. CONCLUSION NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch-Steindl syndrome after the delineators of this phenotype.

Optic nerve hypoplasia (ONH) is a congenital optic nerve abnormality caused by underdevelopment of retinal ganglion cells (RGCs). Despite being a rare disease, ONH is the most common optic disc anomaly in ophthalmological practice. So far, mutations in several genes have been identified as causative, however many cases of ONH remain without a molecular explanation. The early transcription factor atonal basic-helix-loop-helix (bHLH) transcription factor 7 (ATOH7) is expressed in retinal progenitor cells and has a crucial role in RGC development. Previous studies have identified several mutations in the ATOH7 locus in cases of eye developmental diseases such as nonsyndromic congenital retinal nonattachment and persistent hyperplasia of the primary vitreous. Here we present two siblings with a phenotype predominated by bilateral ONH, with additional features of foveal hypoplasia and distinct vascular abnormalities, where whole-exome sequencing identified two compound heterozygous missense mutations affecting a conserved amino acid residue within the bHLH domain of ATOH7 (NM_145178.3:c.175G>A; p.(Ala59Thr) and c.176C>T; p.(Ala59Val)). ATOH7 expression constructs with patient single nucleotide variants were cloned for functional characterization. Protein analyses revealed decreased protein amounts and significantly enhanced degradation in the presence of E47, a putative bHLH dimerization partner. Protein interaction assays revealed decreased heterodimerization and DNA-binding of ATOH7 variants, resulting in total loss of transcriptional activation of luciferase reporter gene expression. These findings strongly support pathogenicity of the two ATOH7 mutations, one of which is novel. Additionally, this report highlights the possible impact of altered ATOH7 dimerization on protein stability and function.

Purpose The aim of this study was to investigate the molecular basis of childhood glaucoma in Switzerland to recommend future targeted genetic analysis in the Swiss population. Methods Whole-exome sequencing and copy number variation (CNV) analysis was performed in a Swiss cohort of 18 patients from 14 unrelated families. Identified variants were validated by Sanger sequencing and multiplex ligation-dependent probe amplification. Breakpoints of structural variants were determined by a microarray. A minigene assay was conducted for functional analysis of a splice site variant. Results A diagnosis of primary congenital glaucoma was made in 14 patients, of which six (43%) harbored pathogenic variants in CYP1B1, one (7%) a frameshift variant in FOXC1, and seven (50%) remained without a genetic diagnosis. Three patients were diagnosed with glaucoma associated with nonacquired ocular anomalies, of which two patients with mild ocular features of Axenfeld-Rieger syndrome harbored a FOXC1 duplication plus an additional FOXC1 missense variant, and one patient with a Barkan membrane remained without genetic diagnosis. A diagnosis of juvenile open-angle glaucoma was made in one patient, and genetic analysis revealed a FOXC1 duplication. Conclusions Sequencing of CYP1B1 and FOXC1, as well as analysis of CNVs in FOXC1, should be performed before extended gene panel sequencing. Translational Relevance The identification of the molecular cause of childhood glaucoma is a prerequisite for genetic counseling and personalized care for patients and families.

Purpose To compare phenotype variability in retinitis pigmentosa patients with recessive and dominant mutations in the SNRNP200 gene. Methods In a retrospective study, patients of two unrelated families were identified: family A, five patients aged 36 to 77 years; family B, one patient aged 9 years and his asymptomatic parents and sister. All patients received a comprehensive eye examination with a detailed retinal functional and morphologic assessment. Genetic testing was performed by whole exome sequencing (WES) in the index patient from each family. Genes described to be involved in eye diseases (n > 450) were screened for rare variants and segregation analysis was performed. Results A known heterozygous missense variant (c.3260C>T, p.(Ser1087Leu)) in the SNRNP200 gene was identified in the index patient of family A while a novel homozygous missense mutation (c.1634G>A, p.(Arg545His)) was found in the index patient of family B. Nyctalopia and photophobia were reported by 6/6 and 2/6 patients, respectively. The phenotype associated with the dominant mutation was characterized by variable disease onset (early childhood to the sixth decade of life), disease severity (visual acuity of 20/20-20/200 in the seventh to eighth decade), and advanced rod-cone dysfunction. Characteristics of recessive disease included distinct fundus changes of dot-like hypopigmentation together with retinal atrophy and severe rod-cone dysfunction. Conclusions The phenotype characteristics in autosomal dominant and recessive SNRNP200 mutations show distinct features, with earlier severe disease in the recessive case and a variable disease expression in the dominant inheritance pattern.

Inherited monogenic diseases of the retina and vitreous affect approximately 1 in 2000 individuals. They are characterized by tremendous genetic heterogeneity and clinical variability involving mutations in approximately 250 genes and more than 20 different clinical phenotypes. Clinical manifestations of retinal dystrophies (RDs) range from mild retinal dysfunctions to severe congenital forms of blindness. A detailed clinical diagnosis and the identification of causative mutations are crucial for genetic counseling of affected patients and their families, for understanding genotype-phenotype correlations and developing therapeutic approaches. Using whole exome sequencing (WES) we have established a reliable and efficient high-throughput analysis pipeline to identify disease-causing mutations. Our data indicate that this approach enables us to genetically diagnose approximately 64% of the patients (n = 58) with variant(s) in known disease-associated genes. We report 20 novel and 26 recurrent variants in genes associated with RDs. We also identified a novel phenotype for mutations in C2orf71 and provide functional evidence for exon skipping due to a splice-site variant identified in FLVCR1. In conclusion, WES can rapidly identify variants in various families affected with different forms of RDs. Our study also expands the clinical and allelic spectrum of genes associated with RDs in the Swiss population.

Purpose: To identify the genetic cause of autosomal recessive familial foveal retinoschisis (FFR). Methods: A female sibship with FFR was identified (Family-A; 17 and 16 years, respectively); panel based genetic sequencing (132 genes) and comparative genome hybridization (142 genes) were performed. Whole-exome sequencing (WES) was performed on both siblings using the Illumina-HiSeq-2500 platform. A sporadic male (Family-B; 35 years) with FFR underwent WES using Illumina NextSeq500. All three affected subjects underwent detailed ophthalmologic evaluation including fundus photography, autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinogram (ERG). Results: Panel-based genetic testing identified two presumed disease causing variants in CRB1 (p.Gly123Cys and p.Cys948Tyr) in Family-A sibship; no deletion or duplication was detected. WES analysis in the sibship identified nine genes with two or more shared nonsynonymous rare coding sequence variants; CRB1 remained a strong candidate gene, and CRB1 variants segregated with the disease. WES in Family-B identified two presumed disease causing variants in CRB1 (p.Ile167_Gly169del and p.Arg764Cys) that segregated with the disease phenotype. Distance visual acuity was 20/40 or better in all three affected except for the left eye of the older subject (Family-B), which showed macular atrophy. Fundus evaluation showed spoke-wheel appearance at the macula in five eyes. The SD-OCT showed macular schitic changes in inner and outer nuclear layers in all cases. The ERG responses were normal in all subjects. Conclusions: This is the first report to implicate CRB1 as the underlying cause of FFR. This phenotype forms the mildest end of the spectrum of CRB1-related diseases.

Models of sexual selection often assume dichotomous sex roles, with one sex competing for access to mates, while the other sex is choosy. However, it is well known that mating decisions are realized by integrating information across multiple traits, the relative importance of which may be sex specific. While a large body of work has investigated the influence of sexual signals on mating behavior, such traits have typically been studied in isolation, oversimplifying the multimodal communication associated with natural mating behavior. We investigated the impact of 2 key traits (Major Histocompatibility Class II beta-chain [MHIIb] olfactory cues and body size) on mate choice decisions in the potbellied seahorse (Hippocampus abdominalis), a species considered to have female competition and male choice. We used a hierarchical experimental design (1. olfactory cues only, 2. olfactory and visual cues, and 3. free interaction) to investigate behavioral preferences and mating success of female and male seahorses under increasing levels of multimodal stimulation. Our data show that female seahorses prefer and mate with MHIIb-dissimilar males, while male seahorses mate randomly with respect to this trait. Conversely, males prefer and mate with large females, while females show no size-based mating preference. The multimodal integration of sex-specific mate preferences in mating behavior of the potbellied seahorse suggests the existence of mutual mate choice in this species. The results presented here suggest that more comprehensive studies of mating behavior, considering both female and male preferences for multiple traits, may lead to a more nuanced understanding of how sexual selection operates in natural populations.

Gene conversion, the unidirectional exchange of genetic material between homologous sequences, is thought to strongly influence patterns of genetic diversity. The high diversity of major histocompatibility complex (MHC) genes in many species is thought to reflect a long history of gene conversion events both within and among loci. Theoretical work suggests that intra- and interlocus gene conversion leave characteristic signatures of nucleotide diversity, but empirical studies of MHC variation have rarely been able to analyze the effects of conversion events in isolation, due to the presence of multiple gene copies in most species. The potbellied seahorse (Hippocampus abdominalis), a species with a single copy of the MH class II beta-chain gene (MHIIb), provides an ideal system in which to explore predictions on the effects of intralocus gene conversion on patterns of genetic diversity. The genetic diversity of the MHIIb peptide binding region (PBR) is high in the seahorse, similar to other vertebrate species. In contrast, the remainder of the gene shows a total absence of synonymous variation and low levels of intronic sequence diversity, concentrated in 3 short repetitive regions and 1-12 SNPs per intron. The distribution of substitutions across the gene results in a patchwork pattern of shared polymorphism between otherwise divergent sequences. The pattern of nucleotide diversity observed in the seahorse MHIIb gene is congruent with theoretical expectations for intralocus gene conversion, indicating that this evolutionary mechanism has played an important role in MHC gene evolution, contributing to both the high diversity in the PBR and the low diversity outside this region. Neutral variation at this locus may be further reduced due to biases in nucleotide composition and functional constraints.

Background: Both natural and sexual selection are thought to influence genetic diversity, but the study of the relative importance of these two factors on ecologically-relevant traits has traditionally focused on species with conventional sex-roles, with male-male competition and female-based mate choice. With its high variability and significance in both immune function and olfactory-mediated mate choice, the major histocompatibility complex(MHC/MH) is an ideal system in which to evaluate the relative contributions of these two selective forces to genetic diversity. Intrasexual competition and mate choice are both reversed in sex-role reversed species, and sexrelated differences in the detection and use of MH-odor cues are expected to influence the intensity of sexual selection in such species. The seahorse, Hippocampus abdominalis, has an exceptionally highly developed form of male parental care, with female-female competition and male mate choice. Results: Here, we demonstrate that the sex-role reversed seahorse has a single MH class II beta-chain gene and that the diversity of the seahorse MHIIb locus and its pattern of variation are comparable to those detected in species with conventional sex roles. Despite the presence of only a single gene copy, intralocus MHIIb allelic diversity in this species exceeds that observed in species with multiple copies of this locus. The MHIIb locus of the seahorse exhibits a novel expression domain in the male brood pouch. Conclusions: The high variation found at the seahorse MHIIb gene indicates that sex-role reversed species are capable of maintaining the high MHC diversity typical in most vertebrates. Whether such species have evolved the capacity to use MH-odor cues during mate choice is presently being investigated using mate choice experiments. If this possibility can be rejected, such systems would offer an exceptional opportunity to study the effects of natural selection in isolation, providing powerful comparative mode