A novel mutation in SETX and ATM causes ataxia in consanguineous Pakistani families.

Background & Objectives: Ataxia is usually caused by cerebellar pathology or a decrease in vestibular or proprioceptive afferent input to the cerebellum. It is characterized by uncoordinated walking, truncal instability, body or head tremors, uncontrolled coordination of the hands, dysarthria, and aberrant eye movements. The objective of the current investigation was to identify the underlying genetic cause of the hereditary ataxia that affects the Pakistani population. Methods: We studied numerous consanguineous Pakistani families whose members had ataxia-related clinical symptoms to varying degrees. The families were chosen from the Punjab province, and the neurophysician conducted a clinical examination. Peripheral blood samples from both sick and healthy members of the family were taken after obtaining informed consent. Genomic DNA was used to find potential variations in probands using whole exome sequencing. The study was carried out at the University Hospital of Tübingen, Germany, and Government College University in Faisalabad, Pakistan, during 2018-2023. Results: The molecular analysis of these families identified different variants including SGCB: c.902C>T, c.668G>A, ATM: c.6196_6197insGAA, SPG11: c.5769del, SETX c.5525_5533del, and ATM: c.7969A>T. A noteworthy mutation in ATM and SETX was observed among them, and its symptoms were shown to cause ataxia in these families. Conclusion: The current study broadens the mutation spectrum of several hereditary ataxia types and suggests the next generation sequencing in conjunction with clinical research for a more accurate diagnosis of overlapping phenotypes of this disorder in the Pakistani population.

Biallelic EPB41L3 variants underlie a developmental disorder with seizures and myelination defects.

Erythrocyte Membrane Protein Band 4.1 Like 3 (EPB41L3: NM_012307.5), also known as DAL-1, encodes the ubiquitously expressed, neuronally enriched 4.1B protein, part of the 4.1 superfamily of membrane-cytoskeleton adaptors. 4.1B plays key roles in cell spreading, migration, and cytoskeletal scaffolding that support oligodendrocyte axon adhesions essential for proper myelination. We herein describe six individuals from five unrelated families with global developmental delay, intellectual disability, seizures, hypotonia, neuroregression, and delayed myelination. Exome sequencing identified biallelic variants in EPB41L3 in all affected individuals: two nonsense (c.466C>T, p.(R156*); c.2776C>T, p.(R926*)) and three frameshift (c.666delT, p.(F222Lfs*46); c.2289dupC, p.(V764Rfs*19); c.948_949delTG, p.(A317Kfs*33)). Quantitative-real time PCR and Western blot analysis in human fibroblasts harbouring EPB41L3:c.666delT, p.(F222Lfs*46) indicate ablation of EPB41L3 mRNA and 4.1B protein expression. Inhibition of the nonsense mediated decay (NMD) pathway led to an upregulation of EPB41L3:c.666delT transcripts, supporting NMD as a pathogenic mechanism. Epb41l3-deficient mouse oligodendroglia cells showed significant reduction in mRNA expression of key myelin genes, reduced branching, and increased apoptosis. Our report provides the first clinical description of an autosomal recessive disorder associated with variants in EPB41L3, which we refer to as EPB41L3-associated developmental disorder (EADD). Moreover, our functional studies substantiate the pathogenicity of EPB41L3 hypothesized loss-of-function variants.

Molecular genetics, neuroimaging outcomes, and structural analyses of novel and recurrent variants of WDR62 gene in two consanguineous Pakistani families with autosomal recessive primary microcephaly.

BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental and genetically heterogeneous disorder, characterized by small cranium size (> - 3 SD below mean) and often results in varying degree of intellectual disability. Thirty genes have been identified for the etiology of this disorder due to its clinical and genetic heterogeneity. METHODS AND RESULTS: Here, we report two consanguineous Pakistani families affected with MCPH exhibiting mutation in WDR62 gene. The investigation approach involved Next Generation Sequencing (NGS) gene panel sequencing coupled with linkage analysis followed by validation of identified variants through automated Sanger sequencing and Barcode-Tagged (BT) sequencing. The molecular genetic analysis revealed one novel splice site variant (NM_001083961.2(WDR62):c.1372-1del) in Family A and one known exonic variant NM_001083961.2(WDR62):c.3936dup (p.Val1313Argfs*18) in Family B. Magnetic Resonance Imaging (MRI) scans were also employed to gain insights into the structural architecture of affected individuals. Neurological assessments showed the reduced gyral and sulcal patterns along with normal corpus callosum in affected individuals harboring novel variant. In silico assessments of the identified variants were conducted using different tools to confirm the pathogenicity of these variants. Through In silico analyses, both variants were identified as disease causing and protein modeling of exonic variant indicates subtle conformational alterations in prophesied protein structure. CONCLUSION: This study identifies a novel variant (c.1372-1del) and a recurrent pathogenic variant c.3936dup (p.Val1313Argfs*18) in the WDR62 gene among the Pakistani population, expanding the mutation spectrum for MCPH. These findings emphasize the importance of genetic counseling and awareness to reduce consanguinity and address the burden of this disorder.

Whole exome sequencing identified a homozygous novel variant in DOP1A gene in the Pakistan family with neurodevelopmental disabilities: case report and literature review.

Background: Hereditary neurodevelopmental disorders (NDDs) are prevalent in poorly prognostic pediatric diseases, but the pathogenesis of NDDs is still unclear. Irregular myelination could be one of the possible causes of NDDs. Case presentation: Here, whole exome sequencing was carried out for a consanguineous Pakistani family with NDDs to identify disease-associated variants. The co-segregation of candidate variants in the family was validated using Sanger sequencing. The potential impact of the gene on NDDs has been supported by conservation analysis, protein prediction, and expression analysis. A novel homozygous variant DOP1A(NM_001385863.1):c.2561A>G was identified. It was concluded that the missense variant might affect the protein-protein binding sites of the critical MEC interaction region of DOP1A, and DOP1A-MON2 may cause stability deficits in Golgi-endosome protein traffic. Proteolipid protein (PLP) and myelin-associate glycoprotein (MAG) could be targets of the DOP1A-MON2 Golgi-endosome traffic complex, especially during the fetal stage and the early developmental stages. This further supports the perspective that disorganized myelinogenesis due to congenital DOP1A deficiency might cause neurodevelopmental disorders (NDDs). Conclusion: Our case study revealed the potential pathway of myelinogenesis-relevant NDDs and identified DOP1A as a potential NDDs-relevant gene in humans.

Clinical and neurogenetic characterisation of autosomal recessive RBL2-associated progressive neurodevelopmental disorder.

Retinoblastoma (RB) proteins are highly conserved transcriptional regulators that play important roles during development by regulating cell-cycle gene expression. RBL2 dysfunction has been linked to a severe neurodevelopmental disorder. However, to date, clinical features have only been described in six individuals carrying five biallelic predicted loss of function (pLOF) variants. To define the phenotypic effects of RBL2 mutations in detail, we identified and clinically characterized a cohort of 28 patients from 18 families carrying LOF variants in RBL2 , including fourteen new variants that substantially broaden the molecular spectrum. The clinical presentation of affected individuals is characterized by a range of neurological and developmental abnormalities. Global developmental delay and intellectual disability were uniformly observed, ranging from moderate to profound and involving lack of acquisition of key motor and speech milestones in most patients. Frequent features included postnatal microcephaly, infantile hypotonia, aggressive behaviour, stereotypic movements and non-specific dysmorphic features. Common neuroimaging features were cerebral atrophy, white matter volume loss, corpus callosum hypoplasia and cerebellar atrophy. In parallel, we used the fruit fly, Drosophila melanogaster , to investigate how disruption of the conserved RBL2 orthologueue Rbf impacts nervous system function and development. We found that Drosophila Rbf LOF mutants recapitulate several features of patients harboring RBL2 variants, including alterations in the head and brain morphology reminiscent of microcephaly, and perturbed locomotor behaviour. Surprisingly, in addition to its known role in controlling tissue growth during development, we find that continued Rbf expression is also required in fully differentiated post-mitotic neurons for normal locomotion in Drosophila , and that adult-stage neuronal re-expression of Rbf is sufficient to rescue Rbf mutant locomotor defects. Taken together, this study provides a clinical and experimental basis to understand genotype-phenotype correlations in an RBL2 -linked neurodevelopmental disorder and suggests that restoring RBL2 expression through gene therapy approaches may ameliorate aspects of RBL2 LOF patient symptoms.

Clinical and Molecular Spectrum of Autosomal Recessive CA8-Related Cerebellar Ataxia.

BACKGROUND: Based on a limited number of reported families, biallelic CA8 variants have currently been associated with a recessive neurological disorder named, cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ-3). OBJECTIVES: We aim to comprehensively investigate CA8-related disorders (CA8-RD) by reviewing existing literature and exploring neurological, neuroradiological, and molecular observations in a cohort of newly identified patients. METHODS: We analyzed the phenotype of 27 affected individuals from 14 families with biallelic CA8 variants (including data from 15 newly identified patients from eight families), ages 4 to 35 years. Clinical, genetic, and radiological assessments were performed, and zebrafish models with ca8 knockout were used for functional analysis. RESULTS: Patients exhibited varying degrees of neurodevelopmental disorders (NDD), along with predominantly progressive cerebellar ataxia and pyramidal signs and variable bradykinesia, dystonia, and sensory impairment. Quadrupedal gait was present in only 10 of 27 patients. Progressive selective cerebellar atrophy, predominantly affecting the superior vermis, was a key diagnostic finding in all patients. Seven novel homozygous CA8 variants were identified. Zebrafish models demonstrated impaired early neurodevelopment and motor behavior on ca8 knockout. CONCLUSION: Our comprehensive analysis of phenotypic features indicates that CA8-RD exhibits a wide range of clinical manifestations, setting it apart from other subtypes within the category of CAMRQ. CA8-RD is characterized by cerebellar atrophy and should be recognized as part of the autosomal-recessive cerebellar ataxias associated with NDD. Notably, the presence of progressive superior vermis atrophy serves as a valuable diagnostic indicator. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

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).

The interleukin-11 receptor variant p.W307R results in craniosynostosis in humans.

Craniosynostosis is characterized by the premature fusion and ossification of one or more of the sutures of the calvaria, often resulting in abnormal features of the face and the skull. In cases in which growth of the brain supersedes available space within the skull, developmental delay or cognitive impairment can occur. A complex interplay of different cell types and multiple signaling pathways are required for correct craniofacial development. In this study, we report on two siblings with craniosynostosis and a homozygous missense pathogenic variant within the IL11RA gene (c.919 T > C; p.W307R). The patients present with craniosynostosis, exophthalmos, delayed tooth eruption, mild platybasia, and a basilar invagination. The p.W307R variant is located within the arginine-tryptophan-zipper within the D3 domain of the IL-11R, a structural element known to be important for the stability of the cytokine receptor. Expression of IL-11R-W307R in cells shows impaired maturation of the IL-11R, no transport to the cell surface and intracellular retention. Accordingly, cells stably expressing IL-11R-W307R do not respond when stimulated with IL-11, arguing for a loss-of-function mutation. In summary, the IL-11R-W307R variant, reported here for the first time to our knowledge, is most likely the causative variant underlying craniosynostosis in these patients.

Genetic Investigation of Consanguineous Pakistani Families Segregating Rare Spinocerebellar Disorders.

Spinocerebellar disorders are a vast group of rare neurogenetic conditions, generally characterized by overlapping clinical symptoms including progressive cerebellar ataxia, spastic paraparesis, cognitive deficiencies, skeletal/muscular and ocular abnormalities. The objective of the present study is to identify the underlying genetic causes of the rare spinocerebellar disorders in the Pakistani population. Herein, nine consanguineous families presenting different spinocerebellar phenotypes have been investigated using whole exome sequencing. Sanger sequencing was performed for segregation analysis in all the available individuals of each family. The molecular analysis of these families identified six novel pathogenic/likely pathogenic variants; ZFYVE26: c.1093del, SACS: c.1201C>T, BICD2: c.2156A>T, ALS2: c.2171-3T>G, ALS2: c.3145T>A, and B4GALNT1: c.334_335dup, and three already reported pathogenic variants; FA2H: c.159_176del, APTX: c.689T>G, and SETX: c.5308_5311del. The clinical features of all patients in each family are concurrent with the already reported cases. Hence, the current study expands the mutation spectrum of rare spinocerebellar disorders and implies the usefulness of next-generation sequencing in combination with clinical investigation for better diagnosis of these overlapping phenotypes.

Exome sequencing in a Romanian Bardet-Biedl syndrome cohort revealed an overabundance of causal BBS12 variants.

Bardet-Biedl syndrome (BBS), is an emblematic ciliopathy hallmarked by pleiotropy, phenotype variability, and extensive genetic heterogeneity. BBS is a rare (~1/140,000 to ~1/160,000 in Europe) autosomal recessive pediatric disorder characterized by retinal degeneration, truncal obesity, polydactyly, cognitive impairment, renal dysfunction, and hypogonadism. Twenty-eight genes involved in ciliary structure or function have been implicated in BBS, and explain the molecular basis for ~75%-80% of individuals. To investigate the mutational spectrum of BBS in Romania, we ascertained a cohort of 24 individuals in 23 families. Following informed consent, we performed proband exome sequencing (ES). We detected 17 different putative disease-causing single nucleotide variants or small insertion-deletions and two pathogenic exon disruptive copy number variants in known BBS genes in 17 pedigrees. The most frequently impacted genes were BBS12 (35%), followed by BBS4, BBS7, and BBS10 (9% each) and BBS1, BBS2, and BBS5 (4% each). Homozygous BBS12 p.Arg355* variants were present in seven pedigrees of both Eastern European and Romani origin. Our data show that although the diagnostic rate of BBS in Romania is likely consistent with other worldwide cohorts (74%), we observed a unique distribution of causal BBS genes, including overrepresentation of BBS12 due to a recurrent nonsense variant, that has implications for regional diagnostics.

NGS-driven molecular diagnosis of heterogeneous hereditary neurological disorders reveals novel and known variants in disease-causing genes.

Hereditary neurological disorders (HNDs) are a clinically and genetically heterogeneous group of disorders. These disorders arise from the impaired function of the central or peripheral nervous system due to aberrant electrical impulses. More than 600 various neurological disorders, exhibiting a wide spectrum of overlapping clinical presentations depending on the organ(s) involved, have been documented. Owing to this clinical heterogeneity, diagnosing these disorders has been a challenge for both clinicians and geneticists and a large number of patients are either misdiagnosed or remain entirely undiagnosed. Contribution of genetics to neurological disorders has been recognized since long; however, the complete picture of the underlying molecular bases are under-explored. The aim of this study was to accurately diagnose 11 unrelated Pakistani families with various HNDs deploying NGS as a first step approach. Using exome sequencing and gene panel sequencing, we successfully identified disease-causing genomic variants these families. We report four novel variants, one each in, ECEL1, NALCN, TBR1 and PIGP in four of the pedigrees. In the rest of the seven families, we found five previously reported pathogenic variants in POGZ, FA2H, PLA2G6 and CYP27A1. Of these, three families segregate a homozygous 18 bp in-frame deletion of FA2H, indicating a likely founder mutation segregating in Pakistani population. Genotyping for this mutation can help low-cost population wide screening in the corresponding regions of the country. Our findings not only expand the existing repertoire of mutational spectrum underlying neurological disorders but will also help in genetic testing of individuals with HNDs in other populations.

Whole exome sequencing identifies a novel variant causing cockayne syndrome type I in a consanguineous Pakistani family.

BACKGROUND: Cockayne syndrome (CS) is a rare neurodegenerative disorder characterized by impaired neurological functions, cachectic dwarfism, microcephaly and photosensitivity. Complementation assays identify two groups of this disorder, CS type I (CSA) and CS type II (CSB), caused by mutations in ERCC8 and ERCC6, respectively. OBJECTIVES: This study aimed to investigate the genetic basis of a consanguineous Pakistani family with three affected individuals presenting with typical clinical symptoms of CS. METHODS: We employed whole exome sequencing of the proband and then Sanger sequenced all the family members to confirm its segregation in the family. Different bioinformatics tools were used to predict pathogenicity of this variant. RESULTS: Variants were filtered according to the pedigree structure. We identified a novel homozygous variant (c.202A>T; p.Ile68Phe) in ERCC8 gene in the proband. The variant was found to segregate in the family. CONCLUSIONS: These findings add to the genetic heterogeneity of ERCC8 and expands the mutation spectrum. Also, identification of this variant can facilitate prenatal diagnosis/genetic counselling set ups in Pakistan where this disease largely remains undiagnosed.

Identification of Pathogenic Mutations in Primary Microcephaly- (MCPH-) Related Three Genes CENPJ, CASK, and MCPH1 in Consanguineous Pakistani Families.

Microcephaly (MCPH) is a developmental anomaly of the brain known by reduced cerebral cortex and underdeveloped intellectual disability without additional clinical symptoms. It is a genetically and clinically heterogenous disorder. Twenty-five genes (involved in spindle positioning, Wnt signaling, centriole biogenesis, DNA repair, microtubule dynamics, cell cycle checkpoints, and transcriptional regulation) causing MCPH have been identified so far. Pakistani population has contributed in the identification of many MCPH genes. WES of three large consanguineous families revealed three pathogenic variants of MCPH1, CENPJ, and CASK. One novel (c.1254delT) deletion variant of MCPH1 and one known (c.18delC) deletion variant of CENPJ were identified in family 1 and 2, respectively. In addition to this, we also identified a missense variant (c.1289G>A) of CASK in males individuals in family 3. Missense mutation in the CASK gene is frequent in the boys with intellectual disability and autistic traits which are the common features that are associated with FG Syndrome 4. The study reports novel and reported mutant alleles disrupting the working of genes vital for normal brain functioning. The findings of this study enhance our understanding about the genetic architecture of primary microcephaly in our local pedigrees and add to the allelic heterogeneity of 3 known MCPH genes. The data generated will help to develop specific strategies to reduce the high incidence rate of MCPH in Pakistani population.

Whole exome sequencing identifies a novel mutation in ASPM and ultra-rare mutation in CDK5RAP2 causing Primary microcephaly in consanguineous Pakistani families.

BACKGROUND & OBJECTIVES: Primary Microcephaly (MCPH) is a rare neurogenetic disease, manifesting congenitally reduced head circumference and non-progressive intellectual disability (ID). To date, twenty-eight genes with biallelic mutations have been reported for this disorder. The study aimed for molecular genetic characterization of Pakistani families segregating MCPH. METHODS: We studied two unrelated consanguineous families (family A and B) presenting >2 patients with diagnostic symptoms of MCPH, born to asymptomatic parents. We employed whole-exome sequencing (WES) of probands to find putative causal mutations. The candidate variants were further confirmed and analyzed for co-segregation by Sanger sequencing of all available members of each family. This study was conducted at Government College University, Faisalabad, Pakistan, and Cologne Center for Genomics (CCG), University of Cologne, Germany; during 2017-2020. RESULTS: We identified a novel homozygous variant c.10097_10098delGA, p.(Gly3366Glufs*19) in exon 26 of ASPM gene in family A which presents with moderate intellectual disability, speech impairment, visual abnormalities, seizures, and ptyalism. Family B was found to segregate nonsense, homozygous variant c.448C>T p.(Arg150*) in CDK5RAP2. The patients also exhibited mild to severe seizures without ptyalism that has not been previously reported in patients with mutations in the CDK5RAP2 gene. CONCLUSION: We report a novel mutation in ASPM and ultra-rare mutation in the CDK5RAP2 gene, both causing primary microcephaly. The study expands the mutational spectrum of the ASPM gene to 212, and also adds to the clinical spectrum of CDK5RAP2 mutations. It also demonstrated the utility of WES in the investigation and genetic diagnosis of genetically heterogeneous disorders like MCPH. These findings would aid in diagnostic and preventive strategies including carrier screening, cascade testing, and genetic counselling.

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.

A Homozygous AKNA Frameshift Variant Is Associated with Microcephaly in a Pakistani Family.

Primary microcephaly (MCPH) is a prenatal condition of small brain size with a varying degree of intellectual disability. It is a heterogeneous genetic disorder with 28 associated genes reported so far. Most of these genes encode centrosomal proteins. Recently, AKNA was recognized as a novel centrosomal protein that regulates neurogenesis via microtubule organization, making AKNA a likely candidate gene for MCPH. Using linkage analysis and whole-exome sequencing, we found a frameshift variant in exon 12 of AKNA (NM_030767.4: c.2737delG) that cosegregates with microcephaly, mild intellectual disability and speech impairment in a consanguineous family from Pakistan. This variant is predicted to result in a protein with a truncated C-terminus (p.(Glu913Argfs*42)), which has been shown to be indispensable to AKNA's localization to the centrosome and a normal brain development. Moreover, the amino acid sequence is altered from the beginning of the second of the two PEST domains, which are rich in proline (P), glutamic acid (E), serine (S), and threonine (T) and common to rapidly degraded proteins. An impaired function of the PEST domains may affect the intracellular half-life of the protein. Our genetic findings compellingly substantiate the predicted candidacy, based on its newly ascribed functional features, of the multifaceted protein AKNA for association with MCPH.

A Novel Missense Mutation in TNNI3K Causes Recessively Inherited Cardiac Conduction Disease in a Consanguineous Pakistani Family.

Cardiac conduction disease (CCD), which causes altered electrical impulse propagation in the heart, is a life-threatening condition with high morbidity and mortality. It exhibits genetic and clinical heterogeneity with diverse pathomechanisms, but in most cases, it disrupts the synchronous activity of impulse-generating nodes and impulse-conduction underlying the normal heartbeat. In this study, we investigated a consanguineous Pakistani family comprised of four patients with CCD. We applied whole exome sequencing (WES) and co-segregation analysis, which identified a novel homozygous missense mutation (c.1531T>C;(p.Ser511Pro)) in the highly conserved kinase domain of the cardiac troponin I-interacting kinase (TNNI3K) encoding gene. The behaviors of mutant and native TNNI3K were compared by performing all-atom long-term molecular dynamics simulations, which revealed changes at the protein surface and in the hydrogen bond network. Furthermore, intra and intermolecular interaction analyses revealed that p.Ser511Pro causes structural variation in the ATP-binding pocket and the homodimer interface. These findings suggest p.Ser511Pro to be a pathogenic variant. Our study provides insights into how the variant perturbs the TNNI3K structure-function relationship, leading to a disease state. This is the first report of a recessive mutation in TNNI3K and the first mutation in this gene identified in the Pakistani population.

Biallelic variants in PCDHGC4 cause a novel neurodevelopmental syndrome with progressive microcephaly, seizures, and joint anomalies.

PURPOSE: We aimed to define a novel autosomal recessive neurodevelopmental disorder, characterize its clinical features, and identify the underlying genetic cause for this condition. METHODS: We performed a detailed clinical characterization of 19 individuals from nine unrelated, consanguineous families with a neurodevelopmental disorder. We used genome/exome sequencing approaches, linkage and cosegregation analyses to identify disease-causing variants, and we performed three-dimensional molecular in silico analysis to predict causality of variants where applicable. RESULTS: In all affected individuals who presented with a neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability we identified biallelic disease-causing variants in Protocadherin-gamma-C4 (PCDHGC4). Five variants were predicted to induce premature protein truncation leading to a loss of PCDHGC4 function. The three detected missense variants were located in extracellular cadherin (EC) domains EC5 and EC6 of PCDHGC4, and in silico analysis of the affected residues showed that two of these substitutions were predicted to influence the Ca2+-binding affinity, which is essential for multimerization of the protein, whereas the third missense variant directly influenced the cis-dimerization interface of PCDHGC4. CONCLUSION: We show that biallelic variants in PCDHGC4 are causing a novel autosomal recessive neurodevelopmental disorder and link PCDHGC4 as a member of the clustered PCDH family to a Mendelian disorder in humans.

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.