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This study aims to clinically and genetically assess 30 unrelated consanguineous Pakistani families from various ethnic backgrounds, all exhibiting features of neurodevelopmental disorders (NDDs). We 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. WES identified likely disease-causing homozygous variants in 30 unrelated consanguineous families. Six families presented newly described variants in known NDD-related genes: (c.1439 T > G; p.Phe480Cys) [OMIM613163], (c.2865_2865insT; p.Glu955Asnfs*5) [OMIM 218000], (c.1305-3_1,305-2delTT; p.Gln29-_Gly305del) [OMIM 606232], (c.356_356insC; p.Gly119Alafs*24) [OMIM 614923], (c.2065G > T; p.Asp689Tyr) [OMIM 615033], (c.1255G > A; p.Glu419Lys) [OMIM 610756]. Additionally, 12 families had previously reported disease-causing variants associated with different types of NDDs: (c.109C > T; p.Arg37*) [OMIM 309580], [ ] (c.1423C > T; p.Arg475*) [OMIM 606854], (c.1694G > A; p.Arg565Gln) [OMIM 252920], (c.3G > A; p.Met1Ile) [OMIM 610768], (c.815C > T; p.Ser272Leu) [OMIM 616281], (c.607 + 1G > A; p.?) [OMIM 618492], (c.885delT; p.Leu295Phefs25*) [OMIM 606220], (c.869G > A; p.Arg290His) [OMIM 618103], and (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. In the present study, we observed a high frequency of 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.

Herein, we reported mutations in five DNA Damage Repair (DDR) i.e., TP53 , ATR , ATM , CHEK1 and CHEK2 involved in OSCC using NG-WES and their analysis using bioinformatics tools. Out of 42 identified mutations, 16.7% are reported for the 1st time. A total of 28 nonsynonymous SNVs are identified. TP53 harbored the highest number of mutations followed by ATM , ATR , CHEK1 and CHEK2 . Nine mutations ( TP53 p.R43H , TP53 p.L125Q , TP53 p.R116Q , TP53 p.C110Y , TP53 p.L62F , ATR p.H120Y , ATM p.P1054R , ATM p.D1853V , ATM p.T2934N ) were predicted highly pathogenic. SAAFEQ-SEQ predicted destabilizing effects for all mutations, while ISPRED-SEQ identified 09 IS mutations, 07 on TP53 , 01 in ATR and 01 in CHEK1 with no IS mutations predicted for ATM and CHEK2 . Among the IS mutations, only SNVs were used in MDS simulations. The gyration radius for all IS SNVs was larger for mutant as compared to the wild type indicating perturbed folding behavior of the mutant proteins. Structural deviations across the carbon back bone were noted by RMSD for mutant and wild type. The TP53 IS mutations include TP53 p.R116Q , TP53 p.C110Y , TP53 p.R43H , TP53 p.E214X , TP53 p.R210X , TP53 p.C110Afs*5 and TP53 p,S108Ffs*23 whereas ATR and CHEK1 IS mutations consist of ATR p.M1932T and CHEK1 p.E76Kfs*21 . ConSurf analysis revealed four SNVs with a high conservation score (9) on TP53 and ATM. TP53 p.P33R was predominantly associated with moderately differentiated tumors (84.60%), naswar users (86.60%) and positive family history of cancer (91.60%). The TP53 p.P33R , ATR p.M211T and CHEK1 p.I437V mutations were found recurrently in 21/27 (77.7%), 20/27 (74.04%), and 27/27 (100%) patients, suggesting its potential biomarker applications in local screening.

Primary Congenital Glaucoma (PCG) is a severe form of glaucoma that affects infants and young children that damage and causes vision impairment. Despite being a well-known condition, the genetic basis of PCG, particularly in highly consanguineous populations like the Pashtun community, still needs to be explored. Six consanguineous Pashtun families (PCG-01, PCG-02, PCG-03, PCG-04, PCG-05, & PCG-07) suffering from PCG were recruited for whole exome sequencing. A prioritization strategy was employed to identify variants in known PCG-related genes, primarily focusing on CYP1B1 . Sanger sequencing was carried out to validate candidate variants and perform segregation studies in affected individuals, siblings, parents, and controls. Whole exome sequencing revealed four pathogenic homozygous variants in six PCG families. Notably, a novel homozygous mutation, c.9delC (S4Afs9), was identified in the CYP1B1 gene in one family (PCG-07). Additionally, the previously unreported variant c.1168 C > A (p.R390S) was found in two families (PCG-2 and PCG-5). Known mutations, including c.868dupC (p. R290Pfs36) and c.1169G > A (p.R390H), were also detected in PCG-01 and PCG-04 families, respectively. Furthermore, a polymorphism, c.1294 C > G (p.L432V), was observed in family PCG-03. This study identifies novel pathogenic variants associated with PCG in consanguineous Pashtun families, highlighting the role of CYP1B1 mutations in PCG development. The findings contribute to a deeper understanding of the genetic basis of PCG and may aid in genetic counselling and early intervention strategies in affected populations.

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% confidence interval (CI). No significant difference was noticed in the expression of COX-2 in CIS compared with ESCC with p-value >.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.

To elucidate the novel molecular cause in two unrelated consanguineous families with autosomal recessive intellectual disability. A combination of homozygosity mapping and exome sequencing was used to locate the plausible genetic defect in family F162, while only exome sequencing was followed in the family PKMR65. The protein 3D structure was visualized with the University of California–San Francisco Chimera software. All five patients from both families presented with severe intellectual disability, aggressive behavior, and speech and motor delay. Four of the five patients had microcephaly. We identified homozygous missense variants in LINGO1, p.(Arg290His) in family F162 and p.(Tyr288Cys) in family PKMR65. Both variants were predicted to be pathogenic, and segregated with the phenotype in the respective families. Molecular modeling of LINGO1 suggests that both variants interfere with the glycosylation of the protein. LINGO1 is a transmembrane receptor, predominantly found in the central nervous system. Published loss-of-function studies in mouse and zebrafish have established a crucial role of LINGO1 in normal neuronal development and central nervous system myelination by negatively regulating oligodendrocyte differentiation and neuronal survival. Taken together, our results indicate that biallelic LINGO1 missense variants cause autosomal recessive intellectual disability in humans.

Background Hepatitis B virus (HBV) infection is a serious health problem in the developing countries including Pakistan. Various risk factors are responsible for the spread of this infectious disease. Prevalence of HBV infection in apparently suspected individual of Punjab province of Pakistan was analyzed during January 2008 to December 2010. Current study was aimed to investigate the epidemiology and risk factors of HBV infection. Methodology Four thousand eight hundred and ninety patients suffering from chronic liver disease were screened for the presence of HBV DNA using qualitative Real Time PCR methodology to confirm their status of infection. A predesigned standard questionnaire was filled for all the patients that included information about the possible risk factors. Results A total of 4890 ELISA positive patients were screened for Hepatitis B virus infection. Of these 3143 were positive for HBV, includes 68.15% males and 31.85% females. Male were observed to be more frequently infected as compared to the female with a positivity ratio of 2.14: 1. The rate of infection increases with the passage of time in the course of three years. Highest frequency of infection was found in the age of 21-30 was 34.93% followed by 23.83% in 31-40. Only (13.39%) were belonging to the age group 11-20 year. The rate of infection declines with increasing age as shown by age groups 41-50 (16.13%) and 51-60 (7.09%). While children aged 0-10 and very old >60 age groups were very less frequently 1.49% and 1.65% infected respectively. Important risk factors contributing to HBV spread include barber risk (23.60%), blood transfusion (4.04%), History of injection 26.19%, Reuse of syringes 26.60%, dental risk (11.20%) and surgical procedure (4.26%). Among the entire respondents trend sharing personal items was very common. History of injection, barber risk, surgery and dental procedure and reuse of syringes appear as major risk factors for the transmission. Conclusion Male were more frequently exposed to the risk factors as compared to female. Similarly the younger age group had high rate of infection as compared to the children's and the older age groups. Reuse of syringes', barber risk and History of injection were main risk identified during the present study. To lower HBV transmission rate Government should take aggressive steps towards massive awareness and vaccination programs to decrease the burden of HBV from the Punjab province of Pakistan.

HIV (Human immunodeficiency virus) transmission has been reduced by protected sex and screening of blood products and other body fluids in the developed countries. It has been reported that Pakistan is at high risk of HIV/AIDS infection but presently the prevalence rate is considerably low. The number of reported cases of HIV/AIDS in Pakistan has been continuously increasing since 1987. By 2010 the total number of registered cases has reached to 6000 and this figure is on the rise with the passage of time. Some serious strategies must be implemented to control this deadly disease.

Background Hepatitis C virus (HCV) is one of the major health concerns globally, with genotype 3a as the most prevalent in Pakistan. Lack of efficient HCV genotype 3a small animal models as well as genomic replicons has hampered the complete understanding of its life cycle, pathogenesis and therapeutic options. In this study we aimed to develop a persistent HCV genotype 3a infectious cell culture model. Methods We inoculated Huh-7 cells with HCV genotype 3a serum. Cells and media supernatant were collected at different time periods up to 40 th day post infection. Culture media supernatant was also collected to find out its ability to infect naive Huh-7 cells. Results HCV replication was confirmed at both RNA and protein level through Real Time RCR and western blot using HCV core as marker. In order to validate the persistence of our model for HCV genotype 3a replication we inhibited the HCV replication through core specific siRNAs. The HCV RNA was detected intracellularly from the day one post infection up till 40 th day, while HCV core protein was detected from the second day up to 40 th day consistently. In culture media supernatant HCV RNA was also actively detected conferring its ability to infect the naive Huh-7 cells. Furthermore, core specific siRNA showed significant inhibition at 24 th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3 rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA. Conclusion Finally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.

Background Hepatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection. Results This study was design to assess or explore the silencing effect of small interference RNAs (siRNAs) against full length HCV particles of genotype 1a. In the present study six 21-bp siRNAs were designed against different regions of HCV structural genes (Core, E1 and E2). Selected siRNAs were labeled as Csi 301, Csi 29, E1si 52, E1si 192, E2si 86 and E2si 493. Our results demonstrated that siRNAs directed against HCV core gene showed 70% reduction in viral titer in HCV infected liver cells. Moreover, siRNAs against E1 and E2 envelop genes showed a dramatic reduction in HCV viral RNA, E2si 86 exhibited 93% inhibition, while E1si 192, E2si 493 and E1si 52 showed 87%, 80%, and 66% inhibition respectively. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggested that siRNAs targeted against HCV structural genes efficiently silence full length HCV particles and provide an effective therapeutic option against HCV infection.

Background and Aims Erythropoietin (EPO) is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO) cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7) to produce recombinant EPO. Materials and methods Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV) promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression ( P < 0.05) of EPO was observed in the medium from Huh-7 cell line. Conclusion Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.