De novo missense variants in the E3 ubiquitin ligase adaptor KLHL20 cause a developmental disorder with intellectual disability,epilepsy, and autism spectrum disorder

PurposeKLHL20 is part of a CUL3-RING E3 ubiquitin ligase involved in protein ubiquitination. KLHL20 functions as the substrate adaptor that recognizes substrates and mediates the transfer of ubiquitin to the substrates. Although KLHL20 regulates neurite outgrowth and synaptic development in animal models, a role in human neurodevelopment has not yet been described. We report on a neurodevelopmental disorder caused by de novo missense variants in KLHL20.MethodsPatients were ascertained by the investigators through Matchmaker Exchange. Phenotyping of patients with de novo missense variants in KLHL20 was performed.ResultsWe studied 14 patients with de novo missense variants in KLHL20, delineating a genetic syndrome with patients having mild to severe intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, hyperactivity, and subtle dysmorphic facial features. We observed a recurrent de novo missense variant in 11 patients (NM_014458.4:c.1069G>A p.[Gly357Arg]). The recurrent missense and the 3 other missense variants all clustered in the Kelch-type β-propeller domain of the KLHL20 protein, which shapes the substrate binding surface.ConclusionOur findings implicate KLHL20 in a neurodevelopmental disorder characterized by intellectual disability, febrile seizures or epilepsy, autism spectrum disorder, and hyperactivity.     

Computational biology insights into genotype-clinical phenotype-protein phenotype relationships between novel SLC26A2 variants identified in inherited skeletal dysplasias

BackgroundPathogenic variants in the transmembrane sulfate transporter protein SLC26A2 are associated with different phenotypes of inherited chondrodysplasias. As limited data is published from India, in this study we sought to elucidate the molecular basis of inherited chondrodysplasias in an Indian cohort.MethodsMolecular screening of 32 fetuses with antenatally diagnosed lethal skeletal dysplasia was performed by next generation sequencing and Sanger sequencing. The genotype-protein phenotype characterization was done using computational biology techniques like homology modelling, stability and pathogenicity predictions.ResultsWe identified five rare autosomal recessive SLC26A2 [NM_000112.4] variants, including three homozygous c.796dupA(p.Thr266Asnfs*12), c.1724delA(p.Lys575Serfs*10), and c.1375_1377dup(p.Val459dup) and two heterozygous variants (c.532C > T(p.Arg178*)) and (c.1382C > T(p.Ala461Val)) in compound heterozygous form in a total of four foetuses. Genotype-protein phenotype annotations highlighted that the clinically severe achondrogenesis 1B causative c.796dupA(p.Thr266Asnfs*12) and c.1724delA(p.Lys575Serfs*10)variants impact SLC26A2 protein structure by deletion of the protein core and transmembrane STAS domains, respectively. In clinically moderate atelosteogenesis type 2 phenotype, the c.1382C > T(p.Ala461Val) variant is predicted to distort alpha helix conformation and alter the bonding properties and free energy dynamics of transmembrane domains and the c.532C > T(p.Arg178*) variant results in loss of both core transmembrane and STAS domains of the SLC26A2 protein. The c.1375_1377dup(p.Val459dup) variant identified in clinically milder atelosteogenesis type II-diastrophic dysplasia spectrum lethal phenotype is predicted to decrease the Qualitative Model Energy Analysis (QMean), which affects major geometrical aspects of the SLC26A2 protein structure.ConclusionWe expand the spectrum of SLC26A2 related lethal chondrodysplasia and report three novel variants correlating clinical severity and protein phenotype within the lethal spectrum of this rare dysplasia. We demonstrate the relevance of structural characterization to aid novel variant reclassification to provide better prenatal management and reproductive options to families with lethal antenatal skeletal disorder.     

Split hand-foot malformation and a novel WNT10B mutation

We report an Indian girl with split-hand/foot malformation (SHFM), sparse hair, and interrupted eyebrows, who carries a novel homozygous deletion c.695_697delACA in WNT10B. The variant is deduced to cause an in-frame deletion of Asn residue 232 (p.Asn232del). According to the protein model, this single amino acid deletion at the critical position in the protein structure is likely to severely affect the protein structure and function. This deletion is likely to lead decreased lifetime and make it unable to bind to its receptors and other ligands. The patient and all family members had normal bone density and they were not obese like some of the patients with WNT10B variants. Here we report a patient with SHFM6 who carried a novel WNT10B mutation. Sparse hair and interrupted eyebrows may be associated findings of SHFM6.     

Characterization of a novel variant in siblings with Asparagine Synthetase Deficiency

Asparagine Synthetase Deficiency (ASD) is a recently described inborn error of metabolism caused by bi-allelic pathogenic variants in the asparagine synthetase (ASNS) gene. ASD typically presents congenitally with microcephaly and severe, often medically refractory, epilepsy. Development is generally severely affected at birth. Tone is abnormal with axial hypotonia and progressive appendicular spasticity. Hyperekplexia has been reported. Neuroimaging typically demonstrates gyral simplification, abnormal myelination, and progressive cerebral atrophy. The present report describes two siblings from consanguineous parents with a homozygous Arg49Gln variant associated with a milder form of ASD that is characterized by later onset of symptoms. Both siblings had a period of normal development before onset of seizures, and development regression. Primary fibroblast studies of the siblings and their parents document that homozygosity for Arg49Gln blocks cell growth in the absence of extracellular asparagine. Functional studies with these cells suggest no impact of the Arg49Gln variant on basal ASNS mRNA or protein levels, nor on regulation of the gene itself. Molecular modelling of the ASNS protein structure indicates that the Arg49Gln variant lies near the substrate binding site for glutamine. Collectively, the results suggest that the Arg49Gln variant affects the enzymatic function of ASNS. The clinical, cellular, and molecular observations from these siblings expand the known phenotypic spectrum of ASD.     

c.1289G>A (p.Arg430His) variant in the epsilon isoform of the GFAP gene in a patient with adult onset Alexander disease

Alexander disease (AD) is a rare form of leukodystrophy caused by pathogenic variants in the GFAP gene. In young children the condition is fatal, while adults have variable neurological symptoms and prognosis. On magnetic resonance imaging, a pattern of atrophy of the medulla oblongata and cervical spinal cord with a ‘tadpole’ appearance is highly suggestive of adult-onset Alexander disease (AOAD). GFAP gene sequencing is used to confirm the diagnosis. Pre-mRNA of this gene undergoes alternative splicing resulting in formation of at least 8 different protein isoforms. Most patients with AD described to date have a pathogenic variant in the coding sequence of the main and the most abundant gene isoform, the GFAPα. Recently, two half-siblings with neurological symptoms and radiological signs of AOAD were reported and were not found to have any pathogenic variants in the GFAPα gene while further genetic testing by next generation sequencing revealed a c.1289G>A (p.Arg430His) variant in the alternative exon 7A of the GFAPε isoform.Here we present a case of another patient with symptoms and brain MRI pattern suggestive of AOAD. Similarly to the previously described patients, this patient did not have any pathogenic variants in the main gene isoform and had the same c.1289G>A (p.Arg430His) variant in the GFAPε. This report contributes to evidence of pathogenicity of the c.1289G>A (p.Arg430His) variant in the GFAPε.     

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay,movement disorder,and metabolic abnormalities

PurposeThis study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.MethodsWe performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells.ResultsIn the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells.ConclusionWe established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.     

Pontocerebellar hypoplasia due to bi-allelic variants in MINPP1

Pontocerebellar hypoplasia (PCH) describes a group of rare heterogeneous neurodegenerative diseases with prenatal onset. Here we describe eight children with PCH from four unrelated families harboring the homozygous MINPP1 (NM_004897.4) variants; c.75_94del, p.(Leu27Argfs*39), c.851 C > A, p.(Ala284Asp), c.1210 C > T, p.(Arg404*), and c.992 T > G, p.(Ile331Ser). The homozygous p.(Leu27Argfs*39) change is predicted to result in a complete absence of MINPP1. The p.(Arg404*) would likely lead to a nonsense mediated decay, or alternatively, a loss of several secondary structure elements impairing protein folding. The missense p.(Ala284Asp) affects a buried, hydrophobic residue within the globular domain. The introduction of aspartic acid is energetically highly unfavorable and therefore predicted to cause a significant reduction in protein stability. The missense p.(Ile331Ser) affects the tight hydrophobic interactions of the isoleucine by the disruption of the polar side chain of serine, destabilizing the structure of MINPP1. The overlap of the above-mentioned genotypes and phenotypes is highly improbable by chance. MINPP1 is the only enzyme that hydrolyses inositol phosphates in the endoplasmic reticulum lumen and several studies support its role in stress induced apoptosis. The pathomechanism explaining the disease mechanism remains unknown, however several others genes of the inositol phosphatase metabolism (e.g., INPP5K, FIG4, INPP5E, ITPR1) are correlated with phenotypes of neurodevelopmental disorders. Taken together, we present MINPP1 as a novel autosomal recessive pontocerebellar hypoplasia gene.Subject terms: Neurodevelopmental disorders, Mutation     

Exome reports A de novo GNB2 variant associated with global developmental delay,intellectual disability,and dysmorphic features

Heterotrimeric G proteins are composed of α, β, and γ subunits and are involved in integrating signals between receptors and effector proteins. The 5 human Gβ proteins (encoded by GNB1, GNB2, GNB3, GNB4, and GNB5) are highly similar. Variants in GNB1 were identified as a genetic cause of developmental delay. De novo variant in GNB2 has recently been reported as a cause of sinus node dysfunction and atrioventricular block but not as a cause of developmental delay. Trio-based whole-exome sequencing was performed on an individual with global developmental delay, muscle hypotonia, multiple congenital joint contractures and dysmorphism such as brachycephalus, thick eyebrows, thin upper lip, micrognathia, prominent chin, and bilateral tapered fingers. We identified a de novo GNB2 variant c.229G>A, p.(Gly77Arg). Notably, pathogenic substitutions of the homologous Gly77 residue including an identical variant (p.Gly77Arg, p.Gly77Val, p.Gly77Ser, p.Gly77Ala) of GNB1, a paralog of GNB2, was reported in individuals with global developmental delay and hypotonia. Clinical features of our case overlap with those of GNB1 variants. Our study suggests that a GNB2 variant may be associated with syndromic global developmental delay.     

Heterozygous germline mutations in A2ML1 are associated with a disorder clinically related to Noonan syndrome

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphisms and congenital heart defects. To date, all mutations known to cause NS are dominant, activating mutations in signal transducers of the RAS/mitogen-activated protein kinase (MAPK) pathway. In 25% of cases, however, the genetic cause of NS remains elusive, suggesting that factors other than those involved in the canonical RAS/MAPK pathway may also have a role. Here, we used family-based whole exome sequencing of a case–parent trio and identified a de novo mutation, p.(Arg802His), in A2ML1, which encodes the secreted protease inhibitor α-2-macroglobulin (A2M)-like-1. Subsequent resequencing of A2ML1 in 155 cases with a clinical diagnosis of NS led to the identification of additional mutations in two families, p.(Arg802Leu) and p.(Arg592Leu). Functional characterization of these human A2ML1 mutations in zebrafish showed NS-like developmental defects, including a broad head, blunted face and cardiac malformations. Using the crystal structure of A2M, which is highly homologous to A2ML1, we identified the intramolecular interaction partner of p.Arg802. Mutation of this residue, p.Glu906, induced similar developmental defects in zebrafish, strengthening our conclusion that mutations in A2ML1 cause a disorder clinically related to NS. This is the first report of the involvement of an extracellular factor in a disorder clinically related to RASopathies, providing potential new leads for better understanding of the molecular basis of this family of developmental diseases.     

A pathogenic UFSP2 variant in an autosomal recessive form of pediatric neurodevelopmental anomalies and epilepsy

PurposeNeurodevelopmental disabilities are common and genetically heterogeneous. We identified a homozygous variant in the gene encoding UFM1-specific peptidase 2 (UFSP2), which participates in the UFMylation pathway of protein modification. UFSP2 variants are implicated in autosomal dominant skeletal dysplasias, but not neurodevelopmental disorders. Homozygosity for the variant occurred in eight children from four South Asian families with neurodevelopmental delay and epilepsy. We describe the clinical consequences of this variant and its effect on UFMylation.MethodsExome sequencing was used to detect potentially pathogenic variants and identify shared regions of homozygosity. Immunoblotting assessed protein expression and post-translational modifications in patient-derived fibroblasts.ResultsThe variant (c.344T>A; p.V115E) is rare and alters a conserved residue in UFSP2. Immunoblotting in patient-derived fibroblasts revealed reduced UFSP2 abundance and increased abundance of UFMylated targets, indicating the variant may impair de-UFMylation rather than UFMylation. Reconstituting patient-derived fibroblasts with wild-type UFSP2 reduced UFMylation marks. Analysis of UFSP2’s structure indicated that variants observed in skeletal disorders localize to the catalytic domain, whereas V115 resides in an N-terminal domain possibly involved in substrate binding.ConclusionDifferent UFSP2 variants cause markedly different diseases, with homozygosity for V115E causing a severe syndrome of neurodevelopmental disability and epilepsy.     

An 18 bps in-frame deletion mutation in RUNX2 gene is a population polymorphism rather than a pathogenic variant

We recruited a family with an affected child exhibiting features of cleidocranial dysplasia with some phenotypic variations from reported cases. Whole exome sequencing data analysis identified an 18-bps heterozygous in-frame deletion variant (c.243-260delGGCGGCTGCGGCGGCGGC) in the RUNX2 gene. Sanger sequencing validated the presence of deletion in affected individual. Initially, we considered this variant as a causal mutation for the patient's phenotype based on previous report(s). However, further analysis of variant revealed that it is present in high frequency in variety of genome variation databases. Moreover, segregation analysis discovered the presence of variant in mother as well. Furthermore, screening of population matched control individuals revealed that the variant is present in apparently healthy individuals as well. Three-dimensional structures of the wild-type and mutant RUNX2 protein (p.Ala82_Ala87del) were analysed and it was found that both wild type and mutant protein show similar secondary structure pattern. Presence of RUNX2 deletion variant (c.243-260delGGCGGCTGCGGCGGCGGC) in control individuals, its high population frequency, benign effect on the overall protein structure lead to the argument that this variant is a population polymorphism and not a pathogenic mutation.     

PLA2G6 mutations associated with a continuous clinical spectrum from neuroaxonal dystrophy to hereditary spastic paraplegia

PLA2G6‐associated neurodegeneration (PLAN) and hereditary spastic paraplegia (HSP) are 2 groups of heterogeneous neurodegenerative diseases. In this study, we report PLA2G6 gene mutations in 3 families from Turkey, Morocco, and Romania. Two affected Turkish siblings presenting HSP adds the disease to PLAN phenotypes. They were homozygous for the PLA2G6 missense c.2239C>T, p.Arg747Trp variant and the ages of onset were 9 and 21. Parkinsonism, dystonia or cognitive decline were not the clinical elements in these patients contrary to the cases that has been previously reported with the same variant, however, iron accumulation was evident in their cranial magnetic resonance imaging. The Moroccan patient was homozygous for a novel missense c.1786C>T, p.Leu596Phe variant and the Romanian patient had 2 novel mutations; c.1898C>T, p.Ala633Val and c.1765_1768del, p.Ser589ThrfsTer76. Both of these patients conformed better to childhood onset PLAN with the age of onset at 4 and 7 years, respectively. Interestingly, all identified mutations were affecting the highly conserved patatin‐like phospholipase domain of the PLA2G6 protein.     

Heterozygous variants in MYBPC1 are associated with an expanded neuromuscular phenotype beyond arthrogryposis

Encoding the slow skeletal muscle isoform of myosin binding protein‐C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M‐motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M‐motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M‐motif to myosin, which likely impairs the formation of actomyosin cross‐bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early‐onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis.     

Genotyping of Italian patients with Behçet syndrome identified two novel ERAP1 polymorphisms using sequencing-based approach

The endoplasmic reticulum aminopeptidase protein 1 gene (ERAP1) is related to several human diseases, including Behçet syndrome (BS), a multisystemic disorder with unknown etiology. ERAP1 is involved in immune response and its role can be influenced by gene single nucleotide variations (SNVs).We genotyped the ERAP1 whole structure in 50 consecutive BS patients and 50 ethnically-matched healthy controls using both bioinformatics and molecular methodologies.We identified two novel heterozygous missense SNVs of ERAP1 exon3 responsible for the p.Glu183Val and p.Phe199Ser changes. The first variation was recognized in 7/50 (14%) BS patients and involved the substrate binding site (p.Glu183) required for the anchorage of the peptide N-terminal group. The SNV was predicted to be a damaging variation, as well as the p.Phe199Ser substitution (PolyPhen-2 and SIFT on line software). 3D protein structure prediction showed a change in energy score when the wild-type and the variant states were compared, probably influencing the substrate binding and the protein folding. The first variation was associated to a more stable protein chain, while the second polymorphism was related to a less stable protein chain.Our data need to be tested in larger genetic studies.     

NR5A1 gene variants repress the ovarian‐specific WNT signaling pathway in 46,XX disorders of sex development patients

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA‐binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti‐testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.