Biallelic loss-of-function variants of ZFTRAF1 cause neurodevelopmental disorder with microcephaly and hypotonia.

PURPOSE: Neurodevelopmental disorders exhibit clinical and genetic heterogeneity, ergo manifest dysfunction in components of diverse cellular pathways; the precise pathomechanism for the majority remains elusive. METHODS: We studied 5 affected individuals from 3 unrelated families manifesting global developmental delay, postnatal microcephaly, and hypotonia. We used exome sequencing and prioritized variants that were subsequently characterized using immunofluorescence, immunoblotting, pulldown assays, and RNA sequencing. RESULTS: We identified biallelic variants in ZFTRAF1, encoding a protein of yet unknown function. Four affected individuals from 2 unrelated families segregated 2 homozygous frameshift variants in ZFTRAF1, whereas, in the third family, an intronic splice site variant was detected. We investigated ZFTRAF1 at the cellular level and signified it as a nucleocytoplasmic protein in different human cell lines. ZFTRAF1 was completely absent in the fibroblasts of 2 affected individuals. We also identified 110 interacting proteins enriched in mRNA processing and autophagy-related pathways. Based on profiling of autophagy markers, patient-derived fibroblasts show irregularities in the protein degradation process. CONCLUSION: Thus, our findings suggest that biallelic variants of ZFTRAF1 cause a severe neurodevelopmental disorder.

Biallelic missense variants in COG3 cause a congenital disorder of glycosylation with impairment of retrograde vesicular trafficking.

Biallelic variants in genes for seven out of eight subunits of the conserved oligomeric Golgi complex (COG) are known to cause recessive congenital disorders of glycosylation (CDG) with variable clinical manifestations. COG3 encodes a constituent subunit of the COG complex that has not been associated with disease traits in humans. Herein, we report two COG3 homozygous missense variants in four individuals from two unrelated consanguineous families that co-segregated with COG3-CDG presentations. Clinical phenotypes of affected individuals include global developmental delay, severe intellectual disability, microcephaly, epilepsy, facial dysmorphism, and variable neurological findings. Biochemical analysis of serum transferrin from one family showed the loss of a single sialic acid. Western blotting on patient-derived fibroblasts revealed reduced COG3 and COG4. Further experiments showed delayed retrograde vesicular recycling in patient cells. This report adds to the knowledge of the COG-CDG network by providing collective evidence for a COG3-CDG rare disease trait and implicating a likely pathology of the disorder as the perturbation of Golgi trafficking.

A homozygous founder variant in PDE2A causes paroxysmal dyskinesia with intellectual disability.

Intellectual developmental disorder with paroxysmal dyskinesia or seizures (IDDPADS, OMIM#619150) is an ultra-rare childhood-onset autosomal recessive movement disorder manifesting paroxysmal dyskinesia, global developmental delay, impaired cognition, progressive psychomotor deterioration and/or drug-refractory seizures. We investigated three consanguineous Pakistani families with six affected individuals presenting overlapping phenotypes partially consistent with the reported characteristics of IDDPADS. Whole exome sequencing identified a novel missense variant in Phosphodiesterase 2A (PDE2A): NM_002599.4: c.1514T > C p.(Phe505Ser) that segregated with the disease status of individuals in these families. Retrospectively, we performed haplotype analysis that revealed a 3.16 Mb shared haplotype at 11q13.4 among three families suggesting a founder effect in this region. Moreover, we also observed abnormal mitochondrial morphology in patient fibroblasts compared to controls. Belonging to diverse age groups (13 years-60 years), patients presented paroxysmal dyskinesia, developmental delay, cognitive abnormalities, speech impairment, and drug-refractory seizures with variable onset of disease (as early as 3 months of age to 7 years). Together with the previous reports, we observed that intellectual disability, progressive psychomotor deterioration, and drug-refractory seizures are consistent outcomes of the disease. However, permanent choreodystonia showed variability. We also noticed that the later onset of paroxysmal dyskinesia manifests severe attacks in terms of duration. Being the first report from Pakistan, we add to the clinical and mutation spectrum of PDE2A-related recessive disease raising the total number of patients from six to 12 and variants from five to six. Together, with our findings, the role of PDE2A is strengthened in critical physio-neurological processes.

A Novel Nonsense Variant in GRM1 Causes Autosomal Recessive Spinocerebellar Ataxia 13 in a Consanguineous Pakistani Family.

Background and objectives: Autosomal recessive spinocerebellar ataxia-13 (SCAR13) is an ultra-rare disorder characterized by slowly progressive cerebellar ataxia, cognitive deficiencies, and skeletal and oculomotor abnormalities. The objective of this case report is to expand the clinical and molecular spectrum of SCAR13. Methods: We investigated a consanguineous Pakistani family with four patients partially presenting with clinical features of SCAR13 using whole exome sequencing. Segregation analysis was performed by Sanger sequencing in all the available individuals of the family. Results: Patients presented with quadrupedal gait, delayed developmental milestones, non-progressive peripheral neuropathy, and cognitive impairment. Whole exome sequencing identified a novel pathogenic nonsense homozygous variant, Gly240*, in the gene GRM1 as a cause of SCAR13 that segregates with the recessive disease. Discussion: We report a novel homozygous nonsense variant in the GRM1 gene in four Pakistani patients presenting with clinical features that partially overlap with the already reported phenotype of SCAR13. In addition, the family presented quadrupedal gait and non-progressive symptoms, manifestations which have not been recognized previously. So far, only four variants in GRM1 have been reported, in families of Roma, Iranian, and Tunisian origins. The current study adds to the mutation spectrum of GRM1 and provides a rare presentation of SCAR13, the first from the Pakistani population.

Whole-Exome Sequencing of Pakistani Consanguineous Families Identified Pathogenic Variants in Genes of Intellectual Disability.

Intellectual disability (ID) is a condition of significant limitation of cognitive functioning and adaptive behavior, with 50% of etiology attributed to genetic predisposition. We recruited two consanguineous Pakistani families manifesting severe ID and developmental delay. The probands were subjected to whole exome sequencing (WES) and variants were further prioritized based on population frequency, predicted pathogenicity and functional relevance. The WES data analysis identified homozygous pathogenic variants in genes MBOAT7 and TRAPPC9. The pathogenicity of the variants was supported by co-segregation analysis and in silico tool. The findings of this study expand mutation spectrum and provide additional evidence to the role of MBOAT7 and TRAPPC9 in causation of ID.

A recurrent rare intronic variant in CAPN3 alters mRNA splicing and causes autosomal recessive limb-girdle muscular dystrophy-1 in three Pakistani pedigrees.

Autosomal recessive limb-girdle muscular dystrophy-1 (LGMDR1) is an autosomal recessive disorder characterized by progressive weakness of the proximal limb and girdle muscles. Biallelic mutations in CAPN3 are reported frequently to cause LGMDR1. Here, we describe 11 individuals from three unrelated consanguineous families that present with typical features of LGMDR1 that include proximal muscle wasting, weakness of the upper and lower limbs, and elevated serum creatine kinase. Whole-exome sequencing identified a rare homozygous CAPN3 variant near the exon 2 splice donor site that segregates with disease in all three families. mRNA splicing studies showed partial retention of intronic sequence and subsequent introduction of a premature stop codon (NM_000070.3: c.379 + 3A>G; p.Asp128Glyfs*15). Furthermore, we observe reduced CAPN3 expression in primary dermal fibroblasts derived from an affected individual, suggesting instability and/or nonsense-mediated decay of mutation-bearing mRNA. Genome-wide homozygosity mapping and single-nucleotide polymorphism analysis identified a shared haplotype and supports a possible founder effect for the CAPN3 variant. Together, our data extend the mutational spectrum of LGMDR1 and have implications for improved diagnostics for individuals of Pakistani origin.

Rare Pathogenic Variants in Genes Implicated in Glutamatergic Neurotransmission Pathway Segregate with Schizophrenia in Pakistani Families.

Schizophrenia is a disabling neuropsychiatric disorder of adulthood onset with high heritability. Worldwide collaborations have identified an association of ~270 common loci, with small individual effects and hence weak clinical implications. The recent technological feasibility of exome sequencing enables the identification of rare variants of high penetrance that refine previous findings and improve risk assessment and prognosis. We recruited two multiplex Pakistani families, having 11 patients and 19 unaffected individuals in three generations. We performed genome-wide SNP genotyping, next-generation mate pairing and whole-exome sequencing of selected members to unveil genetic components. Candidate variants were screened in unrelated cohorts of 508 cases, 300 controls and fifteen families (with 51 affected and 47 unaffected individuals) of Pakistani origin. The structural impact of substituted residues was assessed through in silico modeling using iTASSER. In one family, we identified a rare novel microduplication (5q14.1_q14.2) encompassing critical genes involved in glutamate signaling, such as CMYA5, HOMER and RasGRF2. The second family segregates two ultra-rare, predicted pathogenic variants in the GRIN2A (NM_001134407.3: c.3505C>T, (p.R1169W) and in the NRG3 NM_001010848.4: c.1951G>A, (p.E651K). These genes encode for parts of AMPA and NMDA receptors of glutamatergic neurotransmission, respectively, and the variants are predicted to compromise protein function by destabilizing their structures. The variants were absent in the aforementioned cohorts. Our findings suggest that rare, highly penetrant variants of genes involved in glutamatergic neurotransmission are contributing to the etiology of schizophrenia in these families. It also highlights that genetic investigations of multiplex, multigenerational families could be a powerful approach to identify rare genetic variants involved in complex disorders.

A recessive variant in TFAM causes mtDNA depletion associated with primary ovarian insufficiency, seizures, intellectual disability and hearing loss.

Mitochondrial disorders are collectively common, genetically heterogeneous disorders in both pediatric and adult populations. They are caused by molecular defects in oxidative phosphorylation, failure of essential bioenergetic supply to mitochondria, and apoptosis. Here, we present three affected individuals from a consanguineous family of Pakistani origin with variable seizures and intellectual disability. Both females display primary ovarian insufficiency (POI), while the male shows abnormal sex hormone levels. We performed whole exome sequencing and identified a recessive missense variant c.694C > T, p.Arg232Cys in TFAM that segregates with disease. TFAM (mitochondrial transcription factor A) is a component of the mitochondrial replisome machinery that maintains mtDNA transcription and replication. In primary dermal fibroblasts, we show depletion of mtDNA and significantly altered mitochondrial function and morphology. Moreover, we observed reduced nucleoid numbers with significant changes in nucleoid size or shape in fibroblasts from an affected individual compared to controls. We also investigated the effect of tfam impairment in zebrafish; homozygous tfam mutants carrying an in-frame c.141_149 deletion recapitulate the mtDNA depletion and ovarian dysgenesis phenotypes observed in affected humans. Together, our genetic and functional data confirm that TFAM plays a pivotal role in gonad development and expands the repertoire of mitochondrial disease phenotypes.

A GDF5 frameshift mutation segregating with Grebe type chondrodysplasia and brachydactyly type C+ in a 6 generations family: Clinical report and mini review.

Different mutations in the Growth/Differentiation Factor 5 gene (GDF5) have been associated with varying types of skeletal dysplasia, including Grebe type chondrodysplasia (GTC), Hunter-Thompson syndrome, Du Pan Syndrome and Brachydactyly type C (BDC). Heterozygous pathogenic mutations exert milder effects, whereas homozygous mutations are known to manifest more severe phenotypes. In this study, we report a GDF5 frameshift mutation (c.404delC) segregating over six generations in an extended consanguineous Pakistani family. The family confirmed that both GTC and BDC are part of the GDF5 mutational spectrum, with severe GTC associated with homozygosity, and with a wide phenotypic variability among heterozygous carriers, ranging from unaffected non-penetrant carriers, to classical BDC and to novel unclassified types of brachydactylies.

Monoallelic and bi-allelic variants in NCDN cause neurodevelopmental delay, intellectual disability, and epilepsy.

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development.

RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

Aberrant splicing due to a novel RPS7 variant causes Diamond-Blackfan Anemia associated with spontaneous remission and meningocele.

Diamond-Blackfan Anemia (DBA) is a congenital pure red cell aplasia caused by heterozygous variants in ribosomal protein genes. The hematological features associated with DBA are highly variable and non-hematological abnormalities are common. We report herein on an affected mother and her daughter presenting with transfusion-dependent anemia. The mother showed mild physical abnormalities and entered spontaneous remission at age 13 years. Her daughter was born with occipital meningocele. Exome sequencing of DNA from the mother revealed a heterozygous novel splice site variant (NM_001011.4:c.508-3T > G) in the Ribosomal Protein S7 gene (RPS7) inherited by the daughter. Functional analysis of the RPS7 variant expressed from a mini-gene construct revealed that the exon 7 acceptor splice site was replaced by a cryptic splice resulting in a transcript missing 64 bp of exon 7 (p.Val170Serfs*8). Our study confirms a pathogenic effect of a novel RPS7 variant in DBA associated with spontaneous remission in the mother and meningocele in her daughter, thus adding to the genotype-phenotype correlations in DBA.

Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy.

Intellectual disability (ID) is a heterogeneous clinical entity and includes an excess of males who harbor variants on the X-chromosome (XLID). We report rare FAM50A missense variants in the original Armfield XLID syndrome family localized in Xq28 and four additional unrelated males with overlapping features. Our fam50a knockout (KO) zebrafish model exhibits abnormal neurogenesis and craniofacial patterning, and in vivo complementation assays indicate that the patient-derived variants are hypomorphic. RNA sequencing analysis from fam50a KO zebrafish show dysregulation of the transcriptome, with augmented spliceosome mRNAs and depletion of transcripts involved in neurodevelopment. Zebrafish RNA-seq datasets show a preponderance of 3' alternative splicing events in fam50a KO, suggesting a role in the spliceosome C complex. These data are supported with transcriptomic signatures from cell lines derived from affected individuals and FAM50A protein-protein interaction data. In sum, Armfield XLID syndrome is a spliceosomopathy associated with aberrant mRNA processing during development.

Recessive variants in ZNF142 cause a complex neurodevelopmental disorder with intellectual disability, speech impairment, seizures, and dystonia.

PURPOSE: The purpose of this study was to expand the genetic architecture of neurodevelopmental disorders, and to characterize the clinical features of a novel cohort of affected individuals with variants in ZNF142, a C2H2 domain-containing transcription factor. METHODS: Four independent research centers used exome sequencing to elucidate the genetic basis of neurodevelopmental phenotypes in four unrelated families. Following bioinformatic filtering, query of control data sets, and secondary variant confirmation, we aggregated findings using an online data sharing platform. We performed in-depth clinical phenotyping in all affected individuals. RESULTS: We identified seven affected females in four pedigrees with likely pathogenic variants in ZNF142 that segregate with recessive disease. Affected cases in three families harbor either nonsense or frameshifting likely pathogenic variants predicted to undergo nonsense mediated decay. One additional trio bears ultrarare missense variants in conserved regions of ZNF142 that are predicted to be damaging to protein function. We performed clinical comparisons across our cohort and noted consistent presence of intellectual disability and speech impairment, with variable manifestation of seizures, tremor, and dystonia. CONCLUSION: Our aggregate data support a role for ZNF142 in nervous system development and add to the emergent list of zinc finger proteins that contribute to neurocognitive disorders.

Primary microcephaly, primordial dwarfism, and brachydactyly in adult cases with biallelic skipping of RTTN exon 42.

Biallelic and pathogenic variants in the RTTN gene, encoding the centrosomal protein Rotatin, are associated with variable degrees of neurodevelopmental abnormalities, microcephaly, and extracranial malformations. To date, no reported case has reached their third decade. Herein, we report on a consanguineous family with three adult members, age 43, 57, and 60 years respectively, with primary microcephaly, developmental delay, primordial dwarfism, and brachydactyly segregating a homozygous splice site variant NM_173630.3:c.5648-5T>A in RTTN. The variant RTTN allele results in a nonhypomorphic skipping of exon 42 and a frameshift [(NP_775901.3:p.Ala1883Glyfs*6)]. Brain MRI of one affected individual showed markedly reduced volume of cerebral lobes and enlarged sulci but without signs of neural migration defects. Our assessment of three adult cases with a biallelic RTTN variant shows that a predicted shortened Rotatin, lacking the C-terminal end, are associated with stationary clinical features into the seventh decade. Furthermore, our report adds brachydactyly to the phenotypic spectrum in this pleiotropic entity.

Mutations in NCAPG2 Cause a Severe Neurodevelopmental Syndrome that Expands the Phenotypic Spectrum of Condensinopathies.

The use of whole-exome and whole-genome sequencing has been a catalyst for a genotype-first approach to diagnostics. Under this paradigm, we have implemented systematic sequencing of neonates and young children with a suspected genetic disorder. Here, we report on two families with recessive mutations in NCAPG2 and overlapping clinical phenotypes that include severe neurodevelopmental defects, failure to thrive, ocular abnormalities, and defects in urogenital and limb morphogenesis. NCAPG2 encodes a member of the condensin II complex, necessary for the condensation of chromosomes prior to cell division. Consistent with a causal role for NCAPG2, we found abnormal chromosome condensation, augmented anaphase chromatin-bridge formation, and micronuclei in daughter cells of proband skin fibroblasts. To test the functional relevance of the discovered variants, we generated an ncapg2 zebrafish model. Morphants displayed clinically relevant phenotypes, such as renal anomalies, microcephaly, and concomitant increases in apoptosis and altered mitotic progression. These could be rescued by wild-type but not mutant human NCAPG2 mRNA and were recapitulated in CRISPR-Cas9 F0 mutants. Finally, we noted that the individual with a complex urogenital defect also harbored a heterozygous NPHP1 deletion, a common contributor to nephronophthisis. To test whether sensitization at the NPHP1 locus might contribute to a more severe renal phenotype, we co-suppressed nphp1 and ncapg2, which resulted in significantly more dysplastic renal tubules in zebrafish larvae. Together, our data suggest that impaired function of NCAPG2 results in a severe condensinopathy, and they highlight the potential utility of examining candidate pathogenic lesions beyond the primary disease locus.

A hypomorphic inherited pathogenic variant in DDX3X causes male intellectual disability with additional neurodevelopmental and neurodegenerative features.

BACKGROUND: Intellectual disability (ID) is a common condition with a population prevalence frequency of 1-3% and an enrichment for males, driven in part by the contribution of mutant alleles on the X-chromosome. Among the more than 500 genes associated with ID, DDX3X represents an outlier in sex specificity. Nearly all reported pathogenic variants of DDX3X are de novo, affect mostly females, and appear to be loss of function variants, consistent with the hypothesis that haploinsufficiency at this locus on the X-chromosome is likely to be lethal in males. RESULTS: We evaluated two male siblings with syndromic features characterized by mild-to-moderate ID and progressive spasticity. Quad-based whole-exome sequencing revealed a maternally inherited missense variant encoding p.R79K in DDX3X in both siblings and no other apparent pathogenic variants. We assessed its possible relevance to their phenotype using an established functional assay for DDX3X activity in zebrafish embryos and found that this allele causes a partial loss of DDX3X function and thus represents a hypomorphic variant. CONCLUSIONS: Our genetic and functional data suggest that partial loss of function of DDX3X can cause syndromic ID. The p.R79K allele affects a region of the protein outside the critical RNA helicase domain, offering a credible explanation for the observed retention of partial function, viability in hemizygous males, and lack of pathology in females. These findings expand the gender spectrum of pathology of this locus and suggest that analysis for DDX3X variants should be considered relevant for both males and females.

Epigenetic regulation of HIV-1 latency: focus on polycomb group (PcG) proteins.

HIV-1 latency allows the virus to persist until reactivation, in a transcriptionally silent form in its cellular reservoirs despite the presence of effective cART. Such viral persistence represents a major barrier to HIV eradication since treatment interruption leads to rebound plasma viremia. Polycomb group (PcG) proteins have recently got a considerable attention in regulating HIV-1 post-integration latency as they are involved in the repression of proviral gene expression through the methylation of histones. This epigenetic regulation plays an important role in the establishment and maintenance of HIV-1 latency. In fact, PcG proteins act in complexes and modulate the epigenetic signatures of integrated HIV-1 promoter. Key role played by PcG proteins in the molecular control of HIV-1 latency has led to hypothesize that PcG proteins may represent a valuable target for future HIV-1 therapy in purging HIV-1 reservoirs. In this regard, various small molecules have been synthesized or explored to specifically block the epigenetic activity of PcG. In this review, we will highlight the possible therapeutic approaches to achieve either a functional or sterilizing cure of HIV-1 infection with special focus on histone methylation by PcG proteins together with current and novel pharmacological approaches to reactivate HIV-1 from latency that could ultimately lead towards a better clearance of viral latent reservoirs.

Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome.

BACKGROUND: Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). METHODS: Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. RESULTS: A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3, genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. CONCLUSION: Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.