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 five affected individuals from three unrelated families manifesting global developmental delay, postnatal microcephaly, and hypotonia. We employed 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 two unrelated families segregated two 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 two 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.

Whole exome sequencing identifies variable expressivity of CLN6 variants in Progressive myoclonic epilepsy affected families.

Progressive myoclonic epilepsies (PMEs) are a group of neurodegenerative disorders, predominantly affecting adolescents and, characterized by generalized worsening myoclonus epilepsies, ataxia, cognitive deficits, and dementia. To date, several genes, having implications in diverse phenotypic expressions associated with PMEs, have been identified. Genetic diagnosis is available for most of the adolescence-onset myoclonic epilepsies. This study aimed to elucidate the genetic basis of PMEs in three multiplex Pakistani families exhibiting clinically variable phenotypes. Causative variant(s) in the studied families, and mode of segregation were identified by Whole Exome Sequencing (WES) of the probands, followed by bi-directional Sanger sequencing for final validation. We identified homozygous recessive CLN6 missense variant c.768 C>G (p.Asp256Glu) in Family 1, and c.889 C>A (p.Pro297Thr) variant in Family 2. While in Family 3, we found a homozygous variant (c.316dup) that caused a frameshift mutation, leading to a premature stop codon in the CLN6 protein, resulting in a truncated protein (p.Arg106ProfsTer26). Though CLN6 is previously identified to underlie late infantile and adolescent onset neuronal ceroid lipofuscinosis, this study supports and expands the phenotypic spectrum of CLN6 mutations and signifies diagnositc potential CLN6 variants for PMEs. Diverse pathological effects of variant c .768 C>G were observed in Family 1, with same genotypes, suggesting clinical heterogeneity and/or variable expressivity that might be the implication of pleiotropic effects of the gene in these cases.

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.

Whole-Exome sequencing identifies GYS2 biallelic variants in individuals with suspected epilepsy.

BACKGROUND: Adequate glucose supply is essential for brain function, therefore hypoglycemic states may lead to seizures. Since blood glucose supply for brain is buffered by liver glycogen, an impairment of liver glycogen synthesis by mutations in the liver glycogen synthase gene (GYS2) might result in a substantial neurological involvement. Here, we describe the phenotypes of affected siblings of two families harboring biallelic mutations in GYS2. METHODS: Two suspected families - a multiplex Pakistani family (family A) with three affected siblings and a family of Moroccan origin (family B) with a single affected child who presented with seizures and reduced fasting blood glucose levels were genetically characterized. Whole exome sequencing (WES) was performed on the index patients, followed by Sanger sequencing-based segregation analyses on all available members of both families. RESULTS: The variant prioritization of WES and later Sanger sequencing confirmed three mutations in the GYS2 gene (12p12.1) consistent with an autosomal recessive pattern of inheritance. A homozygous splice acceptor site variant (NM_021957.3, c. 1646 -2A>G) segregated in family A. Two novel compound heterozygous variants (NM_021957.3: c.343G>A; p.Val115Met and NM_021957.3: c.875A>T; p.Glu292Val) were detected in family B, suggesting glycogen storage disorder. A special diet designed to avoid hypoglycemia, in addition to change of the anti-seizure medication led to reduction in seizure frequency. CONCLUSIONS: This study suggests that the seizures in patients initially diagnosed with epilepsy might be directly caused, or influenced by hypoglycemia due to pathogenic variants in the GYS2 gene.

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.

Biallelic MED27 variants lead to variable ponto-cerebello-lental degeneration with movement disorders.

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'.

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.

A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family.

Autosomal-recessive cerebellar ataxias (ARCAs) are heterogeneous rare disorders mainly affecting the cerebellum and manifest as movement disorders in children and young adults. To date, ARCA causing mutations have been identified in nearly 100 genes; however, they account for less than 50% of all cases. We studied a multiplex, consanguineous Pakistani family presenting with a slowly progressive gait ataxia, body imbalance, and dysarthria. Cerebellar atrophy was identified by magnetic resonance imaging of brain. Using whole exome sequencing, a novel homozygous missense mutation ERCC8:c.176T>C (p.M59T) was identified that co-segregated with the disease. Previous studies have identified homozygous mutations in ERCC8 as causal for Cockayne Syndrome type A (CSA), a UV light-sensitive syndrome, and several ARCAs. ERCC8 plays critical roles in the nucleotide excision repair complex. The p.M59T, a substitution mutation, is located in a highly conserved WD1 beta-transducin repeat motif. In silico modeling showed that the structure of this protein is significantly affected by the p.M59T mutation, likely impairing complex formation and protein-protein interactions. In cultured cells, the p.M59T mutation significantly lowered protein stability compared to wildtype ERCC8 protein. These findings expand the role of ERCC8 mutations in ARCAs and indicate that ERCC8-related mutations should be considered in the differential diagnosis of ARCAs.

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.

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 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.

Identification of a novel variant in GPR56/ADGRG1 gene through whole exome sequencing in a consanguineous Pakistani family.

GPR56 gene is best known for its pivotal role in cerebral cortical development. Mutations inGPR56give rise to cobblestone-like brain malformation, white matter changes and cerebellar dysplasia. This study aimed to identify causative variant in a consanguineous family having five individuals affected with developmental delay, mild to severe intellectual disability, speech impairment, strabismus and seizures. Whole exome sequencing was performed to identify mutation in affected individuals. Variants were filtered and further validated by Sanger sequencing and segregation analysis. A novel frameshift variant c.1601dupT leading to p.Ala535GlyfsTer17) was identified in GPR56 gene by whole exome sequencing and subsequent filtering. All five affected individuals were homozygous for the mutant allele while four asymptomatic individuals carried the variant in heterozygous state. Radiological findings of a representative patient presented features of GPR56-associated cobblestone like brain malformation. MRI findings suggested paucity of sulci, dilated ventricular system and brainstem atrophy. The microgyria were observed in a simplified gyral pattern (cobblestone). This single bp insertion, and the consequent frameshift, results in the truncation of GPR56 protein. This could result in a malformed cortex giving the brain a cobblestone like shape. Our study identified a 7th novel frameshift variant from Pakistani population in GPR56 gene, thus broadening mutation spectrum.

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 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.

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.

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.