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ObjectiveThis study aims to clinically and genetically assess 30 unrelated consanguineous Pakistani families from various ethnic backgrounds, all exhibiting features of neurodevelopmental disorders (NDDs).MethodsWe conducted clinical, genetic, biochemical, and molecular analyses on 30 consanguineous families with NDDs enrolled from various regions of Pakistan. The likely molecular causes of primary microcephaly and NDDs were identified. Detailed clinical investigations and molecular diagnoses were performed using whole exome sequencing (WES) of the proband, followed by Sanger sequencing for validation and segregation in the available family members of the affected families.ResultsWES identified likely disease-causing homozygous variants in 30 unrelated consanguineous families. Six families presented newly described variants in known NDD-related genes: ABAT (c.1439 T > G; p.Phe480Cys) [OMIM613163], SLC12A6 (c.2865_2865insT; p.Glu955Asnfs*5) [OMIM 218000], SHANK3 (c.1305-3_1,305-2delTT; p.Gln29-_Gly305del) [OMIM 606232], BCKDK (c.356_356insC; p.Gly119Alafs*24) [OMIM 614923], DDHD2 (c.2065G > T; p.Asp689Tyr) [OMIM 615033], ERCC2 (c.1255G > A; p.Glu419Lys) [OMIM 610756]. Additionally, 12 families had previously reported disease-causing variants associated with different types of NDDs: ATRX (c.109C > T; p.Arg37*) [OMIM 309580], GPR56 [ADGRG1] (c.1423C > T; p.Arg475*) [OMIM 606854], NAGLU (c.1694G > A; p.Arg565Gln) [OMIM 252920], DOLK (c.3G > A; p.Met1Ile) [OMIM 610768], GPT2 (c.815C > T; p.Ser272Leu) [OMIM 616281], DYNC1I2 (c.607 + 1G > A; p.?) [OMIM 618492], FBXL3 (c.885delT; p.Leu295Phefs25*) [OMIM 606220], LINGO1 (c.869G > A; p.Arg290His) [OMIM 618103], and ASPM (c.3978G > A; Trp1326*, c.9557C > G; p.Ser3186*, c.6994C > T; p.Arg2332*) [OMIM 608716]. All the identified variants showed segregation compatible with autosomal recessive inheritance.ConclusionIn the present study, we observed a high frequency of ASPM variants in the genetic analysis of 30 consanguineous families exhibiting features of NDDs, particularly those associated with autosomal recessive primary microcephaly. These findings contribute to studies on genotype–phenotype correlation, genetic counseling for families, and a deeper understanding of human brain function and development.

We report the genetic analysis of two consanguineous pedigrees of Pakistani ancestry in which two siblings in each family exhibited developmental delay, epilepsy, intellectual disability and aggressive behavior. Whole-genome sequencing was performed in Family 1, and we identified ~80,000 variants located in regions of homozygosity. Of these, 615 variants had a minor allele frequency ≤ 0.001, and 21 variants had CADD scores ≥ 15. Four homozygous exonic variants were identified in both affected siblings: PDZD7 (c.1348_1350delGAG, p.Glu450del), ALG6 (c.1033G>C, p.Glu345Gln), RBM20 (c.1587C>G, p.Ser529Arg), and CNTNAP2 (c.785G>A, p.Gly228Arg). Sanger sequencing revealed co-segregation of the PDZD7, RBM20, and CNTNAP2 variants with disease in Family 1. Pathogenic variants in PDZD7 and RBM20 are associated with autosomal recessive non-syndromic hearing loss and autosomal dominant dilated cardiomyopathy, respectively, suggesting that these variants are unlikely likely to contribute to the clinical presentation. Gene panel analysis was performed on the two affected siblings in Family 2, and they were found to also be homozygous for the p.Gly228Arg CNTNAP2 variant. Together these families provide a LOD score 2.9 toward p.Gly228Arg CNTNAP2 being a completely penetrant recessive cause of this disease. The clinical presentation of the affected siblings in both families is also consistent with previous reports from individuals with homozygous CNTNAP2 variants where at least one allele was a nonsense variant, frameshift or small deletion. Our data suggests that homozygous CNTNAP2 missense variants can also contribute to disease, thereby expanding the genetic landscape of CNTNAP2 dysfunction.

Esophageal squamous cell carcinoma (ESCC) is one of the aggressive malignancies and mechanisms underlying its pathogenesis remain unclear. Cyclooxygenase-2 (COX-2) enzyme system plays a crucial role in many gastrointestinal malignancies and is an important regulator of cell growth, proliferation, apoptosis, differentiation and transformation. More precise outcome of COX-2 in ESCC is less investigated. In this study we investigated the risk factors of ESCC and expression of COX-2 in Carcinoma in situ (CIS) and ESCC compared to normal esophageal mucosa. ESCC relationship to clinico-pathological parameters using immunohistochemistry was also part of this investigation. Current study was conducted in the Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan. A total of 69 diagnosed patients of ESCC, both Pakistanis and Afghans were enrolled. Various risk factors associated with ESCC were recorded. Mean age at the time of diagnosis was 55 years. Out of 69 patients of ESCC 46 (67%) were users of dipping tobacco (Naswar). Expression of COX-2 was determined in normal esophageal mucosa, CIS and invasive ESCC using Immunohistochemistry (IHC). Differences of mean were computed using ANOVA followed by applying Post Hoc test. Patients were categorized as positive with high expression or negative with low to nil expression. ANOVA showed large differences in expression of COX-2 in normal healthy mucosa compared with CIS and ESCC with the mean difference of -9.529 and -7.370 respectively, p-value being .05 at 95% CI. Our complete cohort (23-85 years) showed statistically significant difference in the expression of COX-2 gene in ESCC and CIS tissue samples compared with normal healthy mucosa. Results of this study indicate that over-expression of COX-2 is positively associated with ESCC.