Valproic Acid-Induced Thrombocytopenia in Treatment-Resistant GABRB3 Genetic Epilepsy: A Case Report.

Valproic acid (VPA) is utilized in the management of a variety of seizure and mood disorders. A rare side effect of this medication is dose-dependent thrombocytopenia. In this case, we report a patient with a treatment-resistant epilepsy GABRB3 genetic variant phenotype who was admitted for sepsis and found to have significant thrombocytopenia with clinical manifestations of epistaxis and easy bruising, which was found to be due to VPA use rather than secondary to other clinical pathologies. The patient's clinical condition improved with supportive treatment including fluid rehydration. Platelet counts normalized after a transfusion and holding of her valproate. She experienced breakthrough seizures despite the initiation of diazepam. The decision was made to restart VPA per Neurology consult recommendations for better seizure control. She had no breakthrough seizures reported after restarting VPA in the hospital. This case highlights the importance of monitoring antiseizure medication side effects, especially in populations at higher risk due to treatment resistance.

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.

Investigating the effects of a single ASPM variant (c.8508_8509) on brain architecture among siblings in a consanguineous Pakistani family.

BACKGROUND: Autosomal Recessive Primary Microcephaly (MCPH) is a rare, neurodevelopmental disorder associated with mild to severe mental retardation. It is characterized by reduced cerebral cortex that ultimately leads to reduction in skull size less than - 3 S.D below the mean for normal individuals having same age and sex. Till date, 30 known loci have been reported for MCPH. METHODS: In the present study, Sanger sequencing was performed followed by linkage analysis to validate the mutation in ASPM gene of the consanguineous Pakistani clans. Bioinformatics tools were also used to confirm the pathogenicity of the diseased variant in the gene. MRI scan was used to compare the brain structure of both the affected individuals (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023). RESULTS: Our study described a consanguineous family with two patients with a known ASPM (MCPH5) variant c.8508_8509delGA causing a frameshift mutation in exon 18 which located in calmodulin-binding IQ domain of the ASPM protein. The salient feature of this study is that a single variant led to significantly distinct changes in the architecture of brain of both siblings which is further confirmed by MRI results. The computation analysis showed that the change in the conservation of this residue cause this variant highly pathogenic. Carrier screening and genetic counselling were also remarkable features of this study (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023). CONCLUSION: This study explores the extraordinary influence of a single ASPM variant on divergent brain structure in consanguineous siblings and enable us to reduce the incidence of further microcephalic cases in this Pakistani family (Aslam et al. in Kinnaird's 2nd International Conference on Science, Technology and Innovation, Lahore, 2023).

Congenital Microcephaly: A Debate on Diagnostic Challenges and Etiological Paradigm of the Shift from Isolated/Non-Syndromic to Syndromic Microcephaly.

Congenital microcephaly (CM) exhibits broad clinical and genetic heterogeneity and is thus categorized into several subtypes. However, the recent bloom of disease-gene discoveries has revealed more overlaps than differences in the underlying genetic architecture for these clinical sub-categories, complicating the differential diagnosis. Moreover, the mechanism of the paradigm shift from a brain-restricted to a multi-organ phenotype is only vaguely understood. This review article highlights the critical factors considered while defining CM subtypes. It also presents possible arguments on long-standing questions of the brain-specific nature of CM caused by a dysfunction of the ubiquitously expressed proteins. We argue that brain-specific splicing events and organ-restricted protein expression may contribute in part to disparate clinical manifestations. We also highlight the role of genetic modifiers and de novo variants in the multi-organ phenotype of CM and emphasize their consideration in molecular characterization. This review thus attempts to expand our understanding of the phenotypic and etiological variability in CM and invites the development of more comprehensive guidelines.

De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway.

CSNK2B encodes for casein kinase II subunit beta (CK2ß), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and ß-catenin with mutated CK2ß. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated ß-catenin and consequent absence of active ß-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear ß-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS.

Monoallelic and biallelic variants in LEF1 are associated with a new syndrome combining ectodermal dysplasia and limb malformations caused by altered WNT signaling.

PURPOSE: LEF1 encodes a transcription factor acting downstream of the WNT-ß-catenin signaling pathway. It was recently suspected as a candidate for ectodermal dysplasia in 2 individuals carrying 4q35 microdeletions. We report on 12 individuals harboring LEF1 variants. METHODS: High-throughput sequencing was employed to delineate the genetic underpinnings of the disease. Cellular consequences were characterized by immunofluorescence, immunoblotting, pulldown assays, and/or RNA sequencing. RESULTS: Monoallelic variants in LEF1 were detected in 11 affected individuals from 4 unrelated families, and a biallelic variant was detected in an affected individual from a consanguineous family. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia. Depending on the type and location of LEF1 variants, the inheritance of this novel Mendelian condition can be either autosomal dominant or recessive. Our functional data indicate that 2 molecular mechanisms are at play: haploinsufficiency or loss of DNA binding are responsible for a mild to moderate phenotype, whereas loss of ß-catenin binding caused by biallelic variants is associated with a severe phenotype. Transcriptomic studies reveal an alteration of WNT signaling. CONCLUSION: Our findings establish mono- and biallelic variants in LEF1 as a cause for a novel syndrome comprising limb malformations and ectodermal dysplasia.

A novel missense variant of SCN4A co-segregates with congenital essential tremor in a consanguineous Kurdish family.

Essential tremor (ET) is a neurological disorder characterized by bilateral and symmetric postural, isometric, and kinetic tremors of forelimbs produced during voluntary movements. To date, only a single SCN4A variant has been suggested to cause ET. In continuation of the previous report on the association between SCN4A and ET in a family from Spain, we validated the pathogenicity of a novel SCN4A variant and its involvement in ET in a second family affected by this disease. We recruited a Kurdish family with four affected members manifesting congenital tremor. Using whole-exome sequencing, we identified a novel missense variant in SCN4A, NM_000334.4:c.4679C>T; p.(Pro1560Leu), thus corroborating SCN4A's role in ET. The residue is highly conserved across vertebrates and the substitution is predicted to be pathogenic by various in silico tools. Western blotting and immunocytochemistry performed in cells derived from one of the patients showed reduced immunoreactivity of SCN4A as compared to control cells. The study provides supportive evidence for the role of SCN4A in the etiology of ET and expands the phenotypic spectrum of channelopathies to this neurological disorder.

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.

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.

Biallelic SYNE2 Missense Mutations Leading to Nesprin-2 Giant Hypo-Expression Are Associated with Intellectual Disability and Autism.

Autism spectrum disorder (ASD) is a group of neurological and developmental disabilities characterised by clinical and genetic heterogeneity. The current study aimed to expand ASD genotyping by investigating potential associations with SYNE2 mutations. Specifically, the disease-causing variants of SYNE2 in 410 trios manifesting neurodevelopmental disorders using whole-exome sequencing were explored. The consequences of the identified variants were studied at the transcript level using quantitative polymerase chain reaction (qPCR). For validation, immunofluorescence and immunoblotting were performed to analyse mutational effects at the protein level. The compound heterozygous variants of SYNE2 (NM_182914.3:c.2483T>G; p.(Val828Gly) and NM_182914.3:c.2362G>A; p.(Glu788Lys)) were identified in a 4.5-year-old male, clinically diagnosed with autism spectrum disorder, developmental delay and intellectual disability. Both variants reside within the nesprin-2 giant spectrin repeat (SR5) domain and are predicted to be highly damaging using in silico tools. Specifically, a significant reduction of nesprin-2 giant protein levels is revealed in patient cells. SYNE2 transcription and the nuclear envelope localisation of the mutant proteins was however unaffected as compared to parental control cells. Collectively, these data provide novel insights into the cardinal role of the nesprin-2 giant in neurodevelopment and suggest that the biallelic hypomorphic SYNE2 mutations may be a new cause of intellectual disability and ASD.

A 24-generation-old founder mutation impairs splicing of RBBP8 in Pakistani families affected with Jawad syndrome.

Jawad syndrome is a multiple congenital anomaly and intellectual disability syndrome with mutation in RBBP8 reported only in two families. Here, we report on two new families from Pakistan and identified a previously reported variant in RBBP8, NM_002894.3:c.1808-1809delTA. We could show that this mutation impairs splicing resulting in two different abnormal transcripts. Finally, we could verify a shared haplotype among all four families and estimate the founder event to have occurred some 24 generations ago.

Modifier Genes in Microcephaly: A Report on WDR62, CEP63, RAD50 and PCNT Variants Exacerbating Disease Caused by Biallelic Mutations of ASPM and CENPJ.

Congenital microcephaly is the clinical presentation of significantly reduced head circumference at birth. It manifests as both non-syndromic-microcephaly primary hereditary (MCPH)-and syndromic forms and shows considerable inter- and intrafamilial variability. It has been hypothesized that additional genetic variants may be responsible for this variability, but data are sparse. We have conducted deep phenotyping and genotyping of five Pakistani multiplex families with either MCPH (n = 3) or Seckel syndrome (n = 2). In addition to homozygous causal variants in ASPM or CENPJ, we discovered additional heterozygous modifier variants in WDR62, CEP63, RAD50 and PCNT-genes already known to be associated with neurological disorders. MCPH patients carrying an additional heterozygous modifier variant showed more severe phenotypic features. Likewise, the phenotype of Seckel syndrome caused by a novel CENPJ variant was aggravated to microcephalic osteodysplastic primordial dwarfism type II (MOPDII) in conjunction with an additional PCNT variant. We show that the CENPJ missense variant impairs splicing and decreases protein expression. We also observed centrosome amplification errors in patient cells, which were twofold higher in MOPDII as compared to Seckel cells. Taken together, these observations advocate for consideration of additional variants in related genes for their role in modifying the expressivity of the phenotype and need to be considered in genetic counseling and risk assessment.

An update of pathogenic variants in ASPM, WDR62, CDK5RAP2, STIL, CENPJ, and CEP135 underlying autosomal recessive primary microcephaly in 32 consanguineous families from Pakistan.

BACKGROUND: Primary microcephaly (MCPH) is a congenital neurodevelopmental disorder manifesting as small brain and intellectual disability. It underlies isolated reduction of the cerebral cortex that is reminiscent of early hominids which makes it suitable model disease to study the hominin-specific volumetric expansion of brain. Mutations in 25 genes have been reported to cause this disorder. Although majority of these genes were discovered in the Pakistani population, still a significant proportion of these families remains uninvestigated. METHODS: We studied a cohort of 32 MCPH families from different regions of Pakistan. For disease gene identification, genome-wide linkage analysis, Sanger sequencing, gene panel, and whole-exome sequencing were performed. RESULTS: By employing these techniques individually or in combination, we were able to discern relevant disease-causing DNA variants. Collectively, 15 novel mutations were observed in five different MCPH genes; ASPM (10), WDR62 (1), CDK5RAP2 (1), STIL (2), and CEP135 (1). In addition, 16 known mutations were also verified. We reviewed the literature and documented the published mutations in six MCPH genes. Intriguingly, our cohort also revealed a recurrent mutation, c.7782_7783delGA;p.(Lys2595Serfs*6), of ASPM reported worldwide. Drawing from this collective data, we propose two founder mutations, ASPM:c.9557C>G;p.(Ser3186*) and CENPJ:c.18delC;p.(Ser7Profs*2), in the Pakistani population. CONCLUSIONS: We discovered novel DNA variants, impairing the function of genes indispensable to build a proper functioning brain. Our study expands the mutational spectra of known MCPH genes and also provides supporting evidence to the pathogenicity of previously reported mutations. These novel DNA variants will be helpful for the clinicians and geneticists for establishing reliable diagnostic strategies for MCPH families.

Nesprin-1 impact on tumorigenic cell phenotypes.

The largest protein of the nuclear envelope (NE) is Nesprin-1 which forms a network along the NE interacting with actin, Emerin, Lamin, and SUN proteins. Mutations in the SYNE1 gene and reduction in Nesprin-1 protein levels have been reported to correlate with several age related diseases and cancer. In the present study, we tested whether Nesprin-1 overexpression can reverse the malignant phenotype of Huh7 cells, a human liver cancer cell line, which carries a mutation in the SYNE1 gene resulting in reduced Nesprin-1 protein levels, has altered nuclear shape, altered amounts and localization of NE components, centrosome localization and genome stability. Ectopic expression of a mini-Nesprin-1 led to an improvement of the nuclear shape, corrected the mislocalization of NE proteins, the centrosome positioning, and the alterations in the DNA damage response network. Additionally, Nesprin-1 had a profound effect on cellular senescence. These findings suggest that Nesprin-1 may be effective in tumorigenic cell phenotype correction of human liver cancer.

Unilateral L4-dorsal root ganglion stimulation evokes pain relief in chronic neuropathic postsurgical knee pain and changes of inflammatory markers: part II whole transcriptome profiling.

BACKGROUND: In our recent clinical trial, increased peripheral concentrations of pro-inflammatory molecular mediators were determined in complex regional pain syndrome (CRPS) patients. After 3 months adjunctive unilateral, selective L4 dorsal root ganglion stimulation (L4-DRGSTIM), significantly decreased serum IL-10 and increased saliva oxytocin levels were assessed along with an improved pain and functional state. The current study extended molecular profiling towards gene expression analysis of genes known to be involved in the gonadotropin releasing hormone receptor and neuroinflammatory (cytokines/chemokines) signaling pathways. METHODS: Blood samples were collected from 12 CRPS patients for whole-transcriptome profiling in order to assay 18,845 inflammation-associated genes from frozen blood at baseline and after 3 months L4-DRGSTIM using PANTHER™ pathway enrichment analysis tool. RESULTS: Pathway enrichment analyses tools (GOrilla™ and PANTHER™) showed predominant involvement of inflammation mediated by chemokines/cytokines and gonadotropin releasing hormone receptor pathways. Further, screening of differentially regulated genes showed changes in innate immune response related genes. Transcriptomic analysis showed that 21 genes (predominantly immunoinflammatory) were significantly changed after L4-DRGSTIM. Seven genes including TLR1, FFAR2, IL1RAP, ILRN, C5, PKB and IL18 were down regulated and fourteen genes including CXCL2, CCL11, IL36G, CRP, SCGB1A1, IL-17F, TNFRSF4, PLA2G2A, CREB3L3, ADAMTS12, IL1F10, NOX1, CHIA and BDKRB1 were upregulated. CONCLUSIONS: In our sub-group analysis of L4-DRGSTIM treated CRPS patients, we found either upregulated or downregulated genes involved in immunoinflammatory circuits relevant for the pathophysiology of CRPS indicating a possible relation. However, large biobank-based approaches are recommended to establish genetic phenotyping as a quantitative outcome measure in CRPS patients. Trial registration The study protocol was registered at the 15.11.2016 on German Register for Clinical Trials (DRKS ID 00011267). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00011267.

Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.

Altered paracellular cation permeability due to a rare CLDN10B variant causes anhidrosis and kidney damage.

Claudins constitute the major component of tight junctions and regulate paracellular permeability of epithelia. Claudin-10 occurs in two major isoforms that form paracellular channels with ion selectivity. We report on two families segregating an autosomal recessive disorder characterized by generalized anhidrosis, severe heat intolerance and mild kidney failure. All affected individuals carry a rare homozygous missense mutation c.144C>G, p.(N48K) specific for the claudin-10b isoform. Immunostaining of sweat glands from patients suggested that the disease is associated with reduced levels of claudin-10b in the plasma membranes and in canaliculi of the secretory portion. Expression of claudin-10b N48K in a 3D cell model of sweat secretion indicated perturbed paracellular Na+ transport. Analysis of paracellular permeability revealed that claudin-10b N48K maintained cation over anion selectivity but with a reduced general ion conductance. Furthermore, freeze fracture electron microscopy showed that claudin-10b N48K was associated with impaired tight junction strand formation and altered cis-oligomer formation. These data suggest that claudin-10b N48K causes anhidrosis and our findings are consistent with a combined effect from perturbed TJ function and increased degradation of claudin-10b N48K in the sweat glands. Furthermore, affected individuals present with Mg2+ retention, secondary hyperparathyroidism and mild kidney failure that suggest a disturbed reabsorption of cations in the kidneys. These renal-derived features recapitulate several phenotypic aspects detected in mice with kidney specific loss of both claudin-10 isoforms. Our study adds to the spectrum of phenotypes caused by tight junction proteins and demonstrates a pivotal role for claudin-10b in maintaining paracellular Na+ permeability for sweat production and kidney function.