Novel and known minor alleles of CNTNAP2 gene variants are associated with comorbidity of intellectual disability and epilepsy phenotypes: a case-control association study reveals potential biomarkers.

BACKGROUND: Neurodevelopmental disorders are heterogeneous due to underlying multiple shared genetic pathways and risk factors. Intellectual disability, epilepsy and autism spectrum disorder phenotypes overlap which indicates the diverse effects of common genes. Recent studies suggested the probable contribution of CNTNAP2 gene polymorphisms to the comorbidity of these neurological conditions. METHODS AND RESULTS: This study was conducted to investigate the role of CNTNAP2 polymorphisms rs147815978 (G>T) and rs2710102 (A>G) as a risk factor for comorbidity of intellectual disability and epilepsy in a group of 345 individuals including 170 patients and 175 healthy controls recruited from various ethnic groups of Pakistani population. Our case-control study group was genotyped by tetra primer ARMS-PCR technique and results were analysed to know the effects of CNTNAP2 rs147815978 (G>T) and rs2710102 (A>G) polymorphisms in the group. The frequency of risk allele T (rs147815978) and risk allele G (rs2710102) for homozygous recessive genotypes (TT/GG) in our study group was 36.47% while odds ratios for risk allele T (rs147815978) was 5.45 (3.90-7.61: 95% CI, P = 0.000) and that for risk allele G (rs2710102) was 2.39 (1.76-3.24: 95% CI, P = 0.0001). Homozygous recessive genotypes (TT/GG) appeared only in cases and not in control group which indicated these as suspected risk genotypes and the significant association (p < 0.05%) of CNTNAP2 gene polymorphisms rs147815978 (G>T) and rs2710102 (A>G) with co-occurrence of intellectual disability and epilepsy phenotypes in our study group which is in HWE (χ2 = 174, P < 0.0001). Logistic regression analysis shows additive (p < 0.0001) and multiplicative (p < 0.001) models which confirms significant association of both the polymorphisms in our data, which are closely located on same haplotype (D' = - 0.168). CONCLUSIONS: We propose that CNTNAP2 rs147815978 (G>T) and rs2710102 (A>G) polymorphisms are possible risk loci for overlapping neurodevelopmental disorders in Pakistani population. We propose the role of a previously reported common SNP rs2710102 (A>G) with a rarely reported novel SNP rs147815978 (G>T) for CNTNAP2 gene association with neurodevelopmental disorders in our data. Our study has expanded the knowledge of CNTNAP2 gene polymorphisms as probable biomarkers for susceptibility of co-occurrence of intellectual disability and epilepsy phenotypes in Pakistani population. We hope that our study will open new horizons of CNTNAP2 gene variants research to cure the neurological conditions in Pakistani population where consanguinity is a tradition and prevalence of neurodevelopmental disorders has increased from 1 to 2% during last 5 years.

Contributing risk factors of common psychiatric disorders in the Pakistani population.

With an increasing incidence of psychiatric disorders worldwide, there is a need for a better understanding of the population-specific contributing risk factors that are associated with common psychiatric conditions. This study aimed to assess the correlation between socioeconomic, environmental and clinical features associated with major depression (MDD n = 479), bipolar disorder (BD n = 222) and schizophrenia (SHZ n = 146), in the Pakistani population. Multinomial logistic regression and Pearson's correlation were applied to assess the association and correlation between demographic, socioeconomic, environmental, and clinical features of MDD, BD and SHZ. In the present study, MDD was found to be more prevalent than BD and SHZ. The average age at onset (AAO), was observed to be earlier in females with BD and SHZ, in addition, females with a positive family history of MDD, BD and SHZ also had an earlier AAO. The fitted multinomial logistic regression model indicated a significant association of; aggression, tobacco use, drugs abuse, history of head injuries and family history with BD as compared to MDD, while insomnia and suicidality were significantly associated with MDD. Strong positive correlations were observed mainly between age/AAO, AAO/tobacco use and aggression/insomnia in all three cohorts. In conclusion, the present study identifies possible contributing socio-demographic, biological and environmental factors that are correlated and associated with the psychiatric conditions in the Pakistani population.

Association of 11 variants of the dopaminergic and cognitive pathways genes with major depression, schizophrenia and bipolar disorder in the Pakistani population.

Background: The dopaminergic pathways control neural signals that modulate mood and behaviour along and have a vital role in the aetiology of major depression (MDD), schizophrenia (SHZ) and bipolar disorder (BD). Genome-wide association studies (GWAS) have reported several dopaminergic and cognitive pathway genes association with these disorders however, no such comprehensive data was available regarding the Pakistani population.Objective: The present study was conducted to analyse the 11 genetic variants of dopaminergic and cognitive system genes in MDD, SHZ, and BD in the Pakistani population.Methods: A total of 1237 subjects [MDD n = 479; BD n = 222; SHZ n = 146; and controls n = 390], were screened for 11 genetic variants through polymerase chain reaction (PCR) techniques. Univariant followed by multivariant logistic regression analysis was applied to determine the genetic association.Results: Significant risk associations were observed for rs4532 and rs1799732 with MDD; and rs1006737 and rs2238056 with BD. However, after applying multiple test corrections rs4532 and rs1799732 association did not remain significant for MDD. Moreover, a protective association was found for three variants; DRD4-120bp, rs10033951 and rs2388334 in the current cohort.Conclusions: The present study revealed the risk association of single nucleotide polymorphisms (SNPs) rs1006737 and rs2238056 with BD and the protective effect of the DRD4-120bp variant in MDD and BD, of rs2388334 in BD and of rs10033951 in MDD, BD, and SHZ in the current Pakistani cohort. Thus, the study is valuable in understanding the genetic basis of MDD, BD and SHZ in the Pakistani population, which may pave the way for future functional studies.

Bi-allelic Variants in DYNC1I2 Cause Syndromic Microcephaly with Intellectual Disability, Cerebral Malformations, and Dysmorphic Facial Features.

Cargo transport along the cytoplasmic microtubular network is essential for neuronal function, and cytoplasmic dynein-1 is an established molecular motor that is critical for neurogenesis and homeostasis. We performed whole-exome sequencing, homozygosity mapping, and chromosomal microarray studies in five individuals from three independent pedigrees and identified likely-pathogenic variants in DYNC1I2 (Dynein Cytoplasmic 1 Intermediate Chain 2), encoding a component of the cytoplasmic dynein 1 complex. In a consanguineous Pakistani family with three affected individuals presenting with microcephaly, severe intellectual disability, simplification of cerebral gyration, corpus callosum hypoplasia, and dysmorphic facial features, we identified a homozygous splice donor site variant (GenBank: NM_001378.2:c.607+1G>A). We report two additional individuals who have similar neurodevelopmental deficits and craniofacial features and harbor deleterious variants; one individual bears a c.740A>G (p.Tyr247Cys) change in trans with a 374 kb deletion encompassing DYNC1I2, and an unrelated individual harbors the compound-heterozygous variants c.868C>T (p.Gln290∗) and c.740A>G (p.Tyr247Cys). Zebrafish larvae subjected to CRISPR-Cas9 gene disruption or transient suppression of dync1i2a displayed significantly altered craniofacial patterning with concomitant reduction in head size. We monitored cell death and cell cycle progression in dync1i2a zebrafish models and observed significantly increased apoptosis, likely due to prolonged mitosis caused by abnormal spindle morphology, and this finding offers initial insights into the cellular basis of microcephaly. Additionally, complementation studies in zebrafish demonstrate that p.Tyr247Cys attenuates gene function, consistent with protein structural analysis. Our genetic and functional data indicate that DYNC1I2 dysfunction probably causes an autosomal-recessive microcephaly syndrome and highlight further the critical roles of the dynein-1 complex in neurodevelopment.

KIF1A novel frameshift variant p.(Ser887Profs*64) exhibits clinical heterogeneity in a Pakistani family with hereditary sensory and autonomic neuropathy type IIC.

Background: Hereditary sensory and autonomic neuropathies (HSANs) are rare heterogeneous group of neurological disorders caused by peripheral nerve deterioration. The HSANs sub-clinical classes have clinical and genetic overlap which often lead to misdiagnosis. In the present study a Pakistani family with five affected members suffering from severe neuropathy were genetically analyzed to identify the disease causative element in the family.Methods: Genome wide high-density single nucleotide polymorphism (SNP) microarray analysis was carried out followed by whole exome sequencing of the affected proband and another affected sibling. Shared homozygous regions in all severely affected members were identified through homozygosity mapping approach.Results: The largest homozygous region of 14.1 Mb shared by the five severely affected members of the family was identified on chromosome 2. Subsequent exome sequencing identified a novel single nucleotide deletion c.2658del; p.(Ser887Profs*64) in KIF1A. Segregation analysis revealed that this mutation was homozygous in all five affected individuals of the family with severe clinical manifestation, while members of the family that were heterozygous carriers shared abnormal skin features (scaly skin) only with the homozygous affected members.Conclusions: A novel frameshift mutation p.(Ser887Profs*64) in KIF1A is the potential cause of severe HSANIIC in a Pakistani family along with incomplete penetrance in mutation carriers. We demonstrate that using a combination of different techniques not only strengthens the gene finding approach but also helps in proper sub-clinical characterization along with identification of mutated alleles exhibiting incomplete penetrance leading to intrafamilial clinical variability in HSAN group of inherited diseases.

A de novo microdeletion in NRXN1 in a Dutch patient with mild intellectual disability, microcephaly and gonadal dysgenesis.

This report is regarding a Dutch female with microcephaly, mild intellectual disability (ID), gonadal dysgenesis and dysmorphic facial features with synophrys. Upon genotyping, an ~455 kb de novo deletion encompassing the first exon of NRXN1 was found. Bidirectional sequencing of the coding exons of the NRXN1 alpha isoform was subsequently performed to investigate the possibility of a pathogenic mutation on the other allele, but we could not find any other mutation. Previously, many heterozygous mutations as well as microdeletions in NRXN1 were shown to be associated with ID, autism, schizophrenia, and other psychiatric and psychotic disorders. Our results are in agreement with other reports that show that NRXN1 deletions can lead to ID, microcephaly and mild dysmorphic features. However, this is the first report of gonadal dysgenesis being associated with such deletions. It is not clear whether there is a causal relationship between the NRXN1 deletion and gonadal dysgenesis, but it is of interest that the FSHR gene, which encodes the follicle-stimulating hormone receptor causative correlation that is mutated in ovarian dysgenesis, is located proximal to the NRXN1 gene. Given that most of the females carrying NRXN1 deletions have been diagnosed at a prepubertal age, gynecologic screening of female carriers of a NRXN1 deletion is warranted.

Microcephaly, Short Stature, Intellectual Disability, Speech Absence and Cataract Are Associated with Novel Bi-Allelic Missense Variant in RTTN Gene: A Seckel Syndrome Case Report.

The RTTN gene encodes centriole biogenesis, replication, symmetry and cohesion, basal body organization and has recently been associated with the appearance of microcephaly syndromes. RTTN-related neurological defects including microcephaly, intellectual disability, congenital dwarfism, ophthalmic manifestations, and epilepsy are mainly due to abnormal brain development pathways and loss-of-function protein mutations. We present a consanguineous Pakistani family clinically suspected of Seckel syndrome with severe microcephaly, severe intellectual disability, short stature, absence of speech, pointed nose, narrow face and bilateral cataract in two siblings residing in the suburbs of Islamabad. Forty cases of Seckel syndrome have been reported to date in the literature due to mutations in the ATR, TRAIP, RBBP8, NSMCE2, NIN, CENPJ, DNA2, CEP152 and CEP63 genes. The objective of the study was to perform a clinical diagnosis, genetic analysis, and pathophysiology of Seckel syndrome in the proband. Whole-exome sequencing discovered NM_173630.4: c.57G > T(pGlu19Asp) missense variant in exon 2 of the RTTN gene that co-segregates in the family. This novel variant, to the best of our knowledge, is pathogenic and with autosomal recessive inheritance expressed as Seckel syndrome in the affected members of the family. The present study has expanded the genetic knowledge of novel RTTN gene variants associated with Seckel syndrome and has broadened its phenotype spectrum in the Pakistani population, which comprises diverse ethnicities. We hope that our study will open new horizons for individual molecular diagnosis and therapeutics to improve the life of patients with this congenital syndrome.

Genome-wide CNV analysis uncovers novel pathogenic regions in cohort of five multiplex families with neurodevelopmental disorders.

Structural reorganization of chromosomes by genomic duplications and/or deletions are known as copy number variations (CNVs). Pathogenic and disease susceptible CNVs alter gene dosage and its phenotypic expression that often leads to human genetic diseases including Neurological disorders. CNVs affecting same common genes in multiple neurodevelopmental disorders can better explain the shared clinical and genetic aetiology across brain diseases. Our study presents the novel copy number variations in a cohort of five multiplex consanguineous families with intellectual disability, microcephaly, ASD, epilepsy, and neurological syndromic features. Cytoscan HD microarray suite has revealed genome wide deletions, duplications and LOH regions which are co-segregating in the family members for the rare neurodevelopmental syndromic phenotypes. This study identifies 1q21.1 microduplication, 16p11.2 microduplication, Xp11.22 microduplication, 4p12 microdeletion and Xq21.1 microdeletion that significantly contribute to primary disease onset and its progression for the first time in Pakistani families. Our study has potential impact on the understanding of pathogenic genetic predisposition for appearance of complex and heterogeneous neurodevelopmental disorders with otherwise unexplained syndromic features. Identification of altered gene dosage across the genome is helpful in improved diagnosis, better disease management in day-to-day life activities of patients with cognitive impairment and genetic counselling of families where consanguinity is a tradition. Our study will contribute to expand the knowledge of genotype-phenotype expression and future gateways in therapeutics and precision medicine research will be open in Pakistan.

A common missense variant rs874881 of PADI4 gene and rheumatoid arthritis: Genetic association study and in-silico analysis.

The peptidylarginine-deiminase 4 (PADI4) is involved in the post-translational catalytic conversion of arginine into citrulline. The autoantibodies including anti-citrullinated protein antibodies (ACPAs) produced in response to hypercitrullinated proteins are a hallmark of rheumatoid arthritis (RA) autoimmunity. Therefore, the role of a missense variant rs874881 (Gly112Ala) of PADI4 in RA susceptibility was analyzed, along with in-silico analysis of structural and functional impacts of this substitution. We did a case-control association study and in-silico analysis. For the case-control study, confirmed RA cases and healthy controls were recruited. Genotyping for rs874881 (n = 750) was performed through polymerase chain reaction-restriction fragment length polymorphism. Multivariate logistic regression analysis was employed to determine association. The in-silico analysis was carried out through HOPE, VarMap, MutationAssessor, MutPred2, SIFT, PolyPhen, CADD, REVEL and MetaLR. In the case-control study, the rs874881 exhibited a strong association with increased RA susceptibility (G vs C odds ratio = 3.85, 95 % confidence interval = 2.81-5.27). Interaction analysis revealed significant interaction of genotype with smoking and gender (p < 0.05). Significant results (p < 0.05) were also obtained in stratified analysis by presence/absence of comorbidities and radiographic damage. According to in-silico pathogenicity prediction analysis, this Gly112Ala substitution does not exert a major effect on protein structure and function including its enzymatic activity. We report a significant association of PADI4 rs874881 with overall RA susceptibility. To our knowledge, this is the first study to do the interaction and stratified analyses on the PADI4 rs874881 in RA. Similar detailed studies should also be performed in other populations.

Role of 19 SNPs in 10 genes with type 2 diabetes in the Pakistani population.

Meta-GWAS report numerous variants associated with type 2 diabetes (T2D), however, for diabetic retinopathy (DR) no loci achieved genome-wide significance. There are limited candidate gene analyses for T2D and/or DR reported from the Pakistani population. Therefore, the current study was designed to evaluate the genetic association of 10 loci with T2D, non-proliferative DR (NPDR), and proliferative DR (PDR). In total 375 T2D cases and 205 controls were collected. The T2D cases included diabetic no retinopathy (n = 196), NPDR (n = 95), and PDR (n = 84). Genomic DNA was isolated, and 19 SNPs were genotyped. To determine association of SNPs with T2D, logistic regression analyses were performed adjusting for age and sex. Moreover, for association of SNPs with NPDR and PDR logistic regression analyses adjusting for diabetes duration and age of T2D onset were performed. In multivariate analysis, the minor alleles of rs1043618 [G > C, odds ratio (OR) 95% confidence interval (CI) = 1.45 (1.13-1.87), p = 4.00E-3], rs3807987 [G > A, 1.87 (1.22-2.94), p = 0.01], rs12672038 [G > A, 1.53 (1.04-2.30), p = 0.03] and rs2055858 [G > C, 1.70 (1.20-2.43), p = 3.00E-3] were associated with higher risk while rs1801133 (C > T, 0.59 (0.42-0.83), p = 2.28E-3) was associated with a lower risk of T2D. Moreover, minor alleles of rs2055858 [G > C, 1.77 (1.17-2.68), p = 0.02], and rs3759890 [C > G, 2.17 (1.39-3.39), p = 4.00E-3] showed an association with PDR when compared with DNR. However, only the association of rs1801133 survived multiple test correction. Hence, we report that rs1801133 is associated with T2D in the Pakistani population. In addition, out of studied 10 genes 8 proteins had higher interactions among themselves that are predicted to be partially biologically connected, as a group.

A complex microcephaly syndrome in a Pakistani family associated with a novel missense mutation in RBBP8 and a heterozygous deletion in NRXN1.

We report on a consanguineous Pakistani family with a severe congenital microcephaly syndrome resembling the Seckel syndrome and Jawad syndrome. The affected individuals in this family were born to consanguineous parents of whom the mother presented with mild intellectual disability (ID), epilepsy and diabetes mellitus. The two living affected brothers presented with microcephaly, white matter disease of the brain, hyponychia, dysmorphic facial features with synophrys, epilepsy, diabetes mellitus and ID. Genotyping with a 250K SNP array in both affected brothers revealed an 18 MB homozygous region on chromosome 18 p11.21-q12.1 encompassing the SCKL2 locus of the Seckel and Jawad syndromes. Sequencing of the RBBP8 gene, underlying the Seckel and Jawad syndromes, identified the novel mutation c.919A>G, p.Arg307Gly, segregating in a recessive manner in the family. In addition, in the two affected brothers and their mother we have also found a heterozygous 607kb deletion, encompassing exons 13-19 of NRXN1. Bidirectional sequencing of the coding exons of NRXN1 did not reveal any other mutation on the other allele. It thus appears that the phenotype of the mildly affected mother can be explained by the NRXN1 deletion, whereas the more severe and complex microcephalic phenotype of the two affected brothers is due to the simultaneous deletion in NRXN1 and the homozygous missense mutation affecting RBBP8.

Exome sequencing identifies three novel candidate genes implicated in intellectual disability.

Intellectual disability (ID) is a major health problem mostly with an unknown etiology. Recently exome sequencing of individuals with ID identified novel genes implicated in the disease. Therefore the purpose of the present study was to identify the genetic cause of ID in one syndromic and two non-syndromic Pakistani families. Whole exome of three ID probands was sequenced. Missense variations in two plausible novel genes implicated in autosomal recessive ID were identified: lysine (K)-specific methyltransferase 2B (KMT2B), zinc finger protein 589 (ZNF589), as well as hedgehog acyltransferase (HHAT) with a de novo mutation with autosomal dominant mode of inheritance. The KMT2B recessive variant is the first report of recessive Kleefstra syndrome-like phenotype. Identification of plausible causative mutations for two recessive and a dominant type of ID, in genes not previously implicated in disease, underscores the large genetic heterogeneity of ID. These results also support the viewpoint that large number of ID genes converge on limited number of common networks i.e. ZNF589 belongs to KRAB-domain zinc-finger proteins previously implicated in ID, HHAT is predicted to affect sonic hedgehog, which is involved in several disorders with ID, KMT2B associated with syndromic ID fits the epigenetic module underlying the Kleefstra syndromic spectrum. The association of these novel genes in three different Pakistani ID families highlights the importance of screening these genes in more families with similar phenotypes from different populations to confirm the involvement of these genes in pathogenesis of ID.

A novel homozygous 10 nucleotide deletion in BBS10 causes Bardet-Biedl syndrome in a Pakistani family.

Bardet-Biedl Syndrome is a multisystem autosomal recessive disorder characterized by central obesity, polydactyly, hypogonadism, learning difficulties, rod-cone dystrophy and renal dysplasia. Bardet-Biedl Syndrome has a prevalence rate ranging from 1 in 100,000 to 1 in 160,000 births although there are communities where Bardet-Biedl Syndrome is found at a higher frequency due to consanguinity. We report here a Pakistani consanguineous family with two affected sons with typical clinical features of Bardet-Biedl Syndrome, in addition to abnormal liver functioning and bilateral basal ganglia calcification, the latter feature being typical of Fahr's disease. Homozygous regions obtained from SNP array depicted three known genes BBS10, BBS14 and BBS2. Bidirectional sequencing of all coding exons by traditional sequencing of all these three genes showed a homozygous deletion of 10 nucleotides (c.1958_1967del), in BBS10 in both affected brothers. The segregation analysis revealed that the parents, paternal grandfather, maternal grandmother and an unaffected sister were heterozygous for the deletion. Such a large deletion in BBS10 has not been reported previously in any population and is likely to be contributing to the phenotype of Bardet-Biedl Syndrome in this family.