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The primary hyperoxalurias are rare disorders of glyoxylate metabolism. Accurate diagnosis is essential for therapeutic and management strategies. We conducted a molecular study on patients suffering from recurrent calcium-oxalate stones and nephrocalcinosis and screened primary hyperoxaluria causing genes in a large cohort of early-onset cases. Disease-associated pathogenic-variants were defined as missense, nonsense, frameshift-indels, and splice-site variants with a reported minor allele frequency1% in controls. We found pathogenic-variants in 34% of the cases. Variants in the AGXT gene causing PH-I were identified in 81% of the mutation positive cases. PH-II-associated variants in the GRHPR gene are found in 15% of the pediatric PH-positive population. Only 3% of the PH-positive cases have pathogenic-variants in the HOGA1 gene, responsible to cause PH-III. A population-specific AGXT gene variant c.1049GA; p.Gly350Asp accounts for 22% of the PH-I-positive patients. Pathogenicity of the identified variants was evaluated by in-silico tools and ACMG guidelines. We have devised a rapid and low-cost approach for the screening of PH by using targeted-NGS highlighting the importance of an accurate and cost-effective screening platform. This is the largest study in Pakistani pediatric patients from South-Asian region that also expands the mutation spectrum of the three known genes.

Nephrolithiasis (NL) affects 1 in 11 individuals worldwide, leading to significant patient morbidity. NL is associated with nephrocalcinosis (NC), a risk factor for chronic kidney disease. Causative genetic variants are detected in 11-28% of NL and/or NC, suggesting additional NL/NC-associated genetic loci await discovery. Therefore, we employed genomic approaches to discover novel genetic forms of NL/NC.Exome sequencing and directed sequencing of the OXGR1 locus were performed in a worldwide NL/NC cohort. Putatively deleterious rare OXGR1 variants were functionally characterized.Exome sequencing revealed a heterozygous OXGR1 missense variant (c.371TG, p.L124R) co-segregating with calcium oxalate NL and/or NC disease in an autosomal dominant inheritance pattern within a multi-generational family with five affected individuals. OXGR1 encodes 2-oxoglutarate (α-ketoglutarate) receptor 1 in the distal nephron. In response to its ligand α-ketoglutarate (AKG), OXGR1 stimulates the chloride-bicarbonate exchanger Pendrin, which also regulates transepithelial calcium transport in cortical connecting tubules. Strong amino acid conservation in orthologues and paralogues, severe in silico prediction scores, and extreme rarity in exome population databases suggested the variant was deleterious. Interrogation of the OXGR1 locus in 1107 additional NL/NC families identified five additional deleterious dominant variants in five families with calcium oxalate NL/NC. Rare, potentially deleterious OXGR1 variants were enriched in NL/NC subjects relative to ExAC controls (ΧRare, dominant loss-of-function OXGR1 variants are associated with recurrent calcium oxalate NL/NC disease.

Objective: The objectives of this study were to find the correlation among the intelligence and academic achievement of MBBS students and to see if the correlation differs between boys and girls. Study Design: Cross Sectional Study. Setting: Ameer Ud Din Medical College / PGMI Lahore. Period: 1st January 2019 to 31st January 2019 Material & Methods: A total of 100 second year MBBS students volunteered to participate (46 males and 54 females). Modified Wechsler Adult Intelligence Scale (WAIS) was used to access the IQ of students. Previous academic records of the students before their admission in medical college and written and oral test marks of second year MBBS students during the year were used for measuring academic achievements of students. The results were analyzed by spearmans correlation, Independent t test and Mann Whitney U test. Results: A statistically significant difference (p = 0.04) between IQ of boys and girls was found with males having mean IQ of 97.4±22.7 as compared to females 89.3±15.5. No significant difference was observed in academic achievements of male and female students. Correlation of IQ with academic achievements was also non-significant. Conclusion: Although males have higher IQ but it has no relationship with academic achievement of medical students. Hard work of the students with average or low IQ could be the reason that there was no difference in the academic achievement when they were compared with the students having higher IQ.

The infection caused by Severe Acute Respiratory Syndrome-Corona Virus 2 (SARS-CoV-2) has rapidly emerged as a serious pandemic, causing substantial morbidity and sometimes mortality with a significant healthcare burden Unfortunately, Pakistan is among top twenty countries of the world affected by COVID-19 The clinical spectrum in COVID-19 is diverse, ranging from mild disease having flu-like symptoms to potentially fatal ARDS, cytokine storm, multiple organ failure and death Common risk factors associated with severe outcome in COVID-19 infection include male gender, older age and presence of comorbidities such as hypertension, diabetes and cardiovascular disease Here we reviewed the available literature and report that the underlying mechanisms that account for a broad range of symptoms during respiratory viral infections, that have been well studied in the case of influenza viruses, adenoviruses, SARS-CoV and MERS-CoV, suggest that host genetic and epigenetic factors may also play a significant role in determining susceptibility and clinical outcome in COVID-19 infection In this review we discuss the potential roles of host genetic factors including cellular receptors for COVID-19, HLA and inflammatory cytokine genes Based on the SARS-CoV-2 genome and protein-protein interactions map between host and viral proteins we also describe the potential roles of several viral proteins in epigenetic modulation of host inflammatory innate immune response by targeting different cellular pathways particularly NF-κB activation, which may lead to the inflammatory cytokine storm and a severe COVID-19 disease Investigations of these genetic and epigenetic mechanisms during COVID-19, especially in local settings, will be helpful in management of patients with higher risks and in the development of novel antiviral therapeutics [ABSTRACT FROM AUTHOR] Copyright of Biomedica is the property of Knowledge Bylanes and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use This abstract may be abridged No warranty is given about the accuracy of the copy Users should refer to the original published version of the material for the full abstract (Copyright applies to all Abstracts )

Objective: To observe vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism in patients resistant to warfarin therapy, and to calculate the allele frequency of the polymorphism in local patients. Method: The cross-sectional case-control study was conducted at the Punjab Institute of Cardiology, Lahore, from 2013 to 2014, and comprised patients with heart valve replacement. Thy were divided into warfarin-resistant group 1 taking 10mg/day, 70mg/week, and control group 2 taking a standard dose of 5mg/day, 35mg/week. The vitamin K epoxide reductase complex subunit 1-1639 G>A polymorphism analysis was done by polymerase chain reaction, followed by restriction fragment length polymorphism technique. Data was analysed using SPSS 20. Results: Of the 146 patients, there were 73(50%) in each of the two groups. In group 1, there were 37(50.68%) males and 36(49.32%) were females with an overall mean age of 33±12 years, while group 2 had 36(49.32%) males and 37(50.68%) females with an overall mean age of 37±13 years. There were no significant differences in mean values of age, serum cholesterol, triglycerides and albumin levels between the groups (p>0.05). The G allele was the most frequently found in both groups, with 140(96%) in group-1 and 137(94%) in group-2. Overall, the homozygous GG genotype was significantly higher in the sample 132(90.4%) (pA polymorphism alone may not be the dominant genetic factor associated with warfarin response variability. Key Words: Warfarin, VKORC1 gene, Single nucleotide polymorphism, PCR-RFLP.

Purpose Deletion of Glutathione S-Transferase Theta 1 (GSTT1) encoding gene is implicated in breast cancer susceptibility, clinical outcomes, and survival. Contradictory results have been reported in different studies. The present investigation based on a representative Pakistani population evaluated the GSTT1-absent genotype in breast cancer risk and prognosis. Methods A prospective study comprising case-control analysis and case series analysis components was designed. Peripheral blood samples were collected from enrolled participants. After DNA extraction, GSTT1 genotyping was carried out by a multiplex PCR with β-globin as an amplification control. Association evaluation of GSTT1 genotypes with breast cancer risk, specific tumor characteristics, and survival were the primary endpoints. Results A total of 264 participants were enrolled in the molecular investigation (3 institutions). The study included 121 primary breast cancer patients as cases and 143 age-matched female subjects, with no history of any cancer, as controls. A significant genetic association between GSTT1-absent genotype and breast cancer susceptibility (p-value: 0.03; OR: 2.13; 95% CI: 1.08-4.29) was reported. The case-series analysis showed lack of association of GSTT1 genotypes with menopause (p-value: 0.86), tumor stage (p-value: 0.12), grade (p-value: 0.32), and size (p-value: 0.07). The survival analysis revealed that GSTT1-absent genotype cases had a statistically significant shorter overall survival (OS) than those with the GSTT1-present genotype cases (mean OS: 23 months vs 33 months). The HR (95% CI) for OS in patients carrying GSTT1-absent genotype was 8.13 (2.91-22.96) when compared with the GSTT1-present genotype. Conclusions The present study is the first report of an independent significant genetic association between GSTT1-absent genotype and breast cancer susceptibility in a Pakistani population. It is also the foremost report of the association of this genotype with OS in breast cancer cases. Upon further validation, GSTT1 variation may serve as a marker for devising better population-specific strategies. The information may have translational implications in the screening and treatment of breast cancers.

Vascular Endothelial Growth Factor (VEGF) has a potent role in tumorigenesis and metastasis. VEGF gene is highly polymorphic. Specific variants have been associated with risk and disease progression in disorders with microvascular involvement such as diabetes mellitus and cancer. However, no study has reported association analysis of common single nucleotide variants (SNVs) in the promoter and 3 untranslated region (3-UTR) with head and neck cancers (HNCs). The present study addresses this gap. It investigates the association of two SNVs, -2578 C/A (rs699947) in the promoter region and +936 C/T (rs3025039) in 3-UTR of the VEGF gene, with risk of HNCs and tumour characteristics. After the ethical approval and informed consent, 323 participants were enrolled in the study. In the case-control component of the study, there were 121 HNC patients and 202 controls. Demographic details and medical history were recorded. Peripheral blood samples (10ml) were collected in ACD-coated vacutainers. DNA isolation was carried out by organic-solvent method. PCR-RFLP methods were optimized for the genotyping of selected SNVs. The protocol was validated by Sanger sequencing. The susceptibility association was determined by using genetic models applying Hardy-Weinberg equilibrium. Statistical analyses included Chi-squared test of independence or Fishers Exact test with significance at p-value

COVID-19 displays marked variability in the clinical course as well as regional epidemiology. Abnormalities in RAAS system especially stemming from genetic variability in ACE and ACE2 expression (including ACE I/D polymorphism) have been proposed to explain underlying pathogenesis and variability in SARS-CoV-2 infection. In a meta-regression data set of 30 countries, we found significant associations of ACE I/D ratio and COVID-19 prevalence, deaths and recovery rate but not when adjusted for possible confounders. This ecological study suggests potential of ACE I/D data as predictive biomarker COVID-19 risk and severity in a population specific manner, subject to validation in large genetic epidemiological and functional studies.

Type III von Willebrand disease is present in the Punjab province of Pakistan along with other inherited bleeding disorders like hemophilia. Cousin marriages are very common in Pakistan so genetic studies help to establish protocols for screening, especially at the antenatal level. Factors behind the phenotypic variation of the severity of bleeding in type III vWD are largely unknown. The study was conducted to determine Mutations/genetic alterations in type III von Willebrand disease and also to determine the association of different mutations, methylation status, ITGA2B/B3 mutations and alloimmunization with the severity of type III vWD. After informed consent and detailed history of the patients, routine tests and DNA extraction from blood, mutational analysis was performed by Next Generation Sequencing on Ion Torrent PGM. DNA methylation status was also checked with the help of PCR. In our cohort, 55 cases were detected with pathogenic mutations. A total of 27 different mutations were identified in 55 solved cases; 16 (59.2%) were novel. The mean bleeding score in truncating mutations and essential splice site mutations was relatively higher than weak and strong missense mutations. The mean bleeding score showed insignificant variation for different DNA methylation statuses of the VWF gene at the cg23551979 CpG site. Mutations in exons 7,10, 25, 28, 31, 43, and intron 41 splice site account for 75% of the mutations.

A retrospective study was conducted for prenatal diagnosis and selective foetal reduction, which included 41 beta thalassemia carrier couples with twin pregnancy. Chorionic villi sampling was carried out at 12-14 weeks of gestation. Thirty-three couples presented with dizygotic while eight had monozygotic twins. Molecular analysis for beta thalassemia revealed similar results in both the twins in 27 cases, while different results were obtained in 14 cases. Selective foetal reduction was offered to couples with discordant results. One miscarriage occurred due to the procedure. The results of prenatal molecular analysis were confirmed by postnatal molecular analysis. This series of trans-abdominal Chorionic villi sampling in twin pregnancies with the option of selective foetal reduction of the affected foetus was found to be useful as well as acceptable by the at-risk couples.

Background Urolithiasis is a worldwide urological problem with significant contribution of genetic factors. Pakistan, which resides within the Afro-Asian stone belt, has a high reported prevalence (12%) of urolithiasis. Osteopontin (SPP1) is a urinary macromolecule with a suggested critical role in modulating renal stone formation, genetic polymorphisms of which may determine individual risk of developing urolithiasis. However, results of previous studies regarding SPP1 polymorphisms and susceptibility to urolithiasis have apparent inconsistencies with no data available for local population. Methods A total of 235 urolithiasis patients and 243 healthy controls, all of Pakistani ancestry, underwent genotyping for six SPP1 genetic polymorphisms in an effort to investigate potential association with urolithiasis using indigenous candidate gene association study design. Further, a comprehensive meta-analysis following a systematic literature search was also done to ascertain an evidence based account of any existent association regarding SPP1 promoter polymorphisms and risk of developing urolithiasis. Results Three SPP1 promoter polymorphisms, rs2853744:G > T, rs11730582:T > C and rs11439060:delG>G, were found to be significantly associated with risk of urolithiasis in indigenous genetic association study (OR = 3.14; p = 0.006, OR = 1.78; p = 0.006 and OR = 1.60; p = 0.012, respectively). We also observed a 1.68-fold positive association of a tri-allelic haplotype of these SPP1 promoter polymorphisms (G-C-dG) with risk of urolithiasis (OR = 1.68; p = 0.0079). However, no association was evident when data were stratified according to gender, age at first presentation, stone recurrence, stone multiplicity, parental consanguinity and family history of urolithiasis. The overall results from meta-analysis, which included 4 studies, suggested a significant association of SPP1 rs2853744:G > T polymorphism with susceptibility of urolithiasis (OR = 1.37; p = 0.004), but not for other SPP1 polymorphic variants analyzed. Conclusions In conclusion, we report significant association of 3 SPP1 polymorphisms with urolithiasis for the first time from South Asia, however, this association persisted only for SPP1 rs2853744:G > T polymorphism after meta-analysis of pooled studies. Further studies with a larger sample size will be required to validate this association and assess any potential usefulness in diagnosis and prognosis of renal stone disease.

BACKGROUND: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide and causes significant patient morbidity. We previously demonstrated a genetic cause of NL can be identified in 11–29% of pre-dominantly American and European stone formers. Pakistan, which resides within the Afro-Asian stone belt, has a high prevalence of nephrolithiasis (12%) as well as high rate of consanguinity (>50%). METHODS: We recruited 235 Pakistani subjects hospitalized for nephrolithiasis from 5 tertiary hospitals in the Punjab province of Pakistan. Subjects were surveyed for age of onset, NL recurrence, and family history. We conducted high-throughput exon sequencing of 30 NL disease genes and variant analysis to identify monogenic causative mutations in each subject. RESULTS: We detected likely causative mutations in 4 of 30 disease genes, yielding a likely molecular diagnosis in 7% (17 of 235) of NL families. Only 1 of 17 causative mutations was identified in an autosomal recessive disease gene. 10 of the 12 detected mutations were novel mutations (83%). SLC34A1 was most frequently mutated (12 of 17 solved families). We observed a higher frequency of causative mutations in subjects with a positive NL family history (13/109, 12%) versus those with a negative family history (4/120, 3%). Five missense SLC34A1 variants identified through genetic analysis demonstrated defective phosphate transport. CONCLUSIONS: We examined the monogenic causes of NL in a novel geographic cohort and most frequently identified dominant mutations in the sodium-phosphate transporter SLC34A1 with functional validation.

BackgroundThe Parsis, one of the smallest religious community in the world, reside in South Asia. Previous genetic studies on them, although based on low resolution markers, reported both Iranian and Indian ancestries. To understand the population structure and demographic history of this group in more detail, we analyzed Indian and Pakistani Parsi populations using high-resolution autosomal and uniparental (Y-chromosomal and mitochondrial DNA) markers. Additionally, we also assayed 108 mitochondrial DNA markers among 21 ancient Parsi DNA samples excavated from Sanjan, in present day Gujarat, the place of their original settlement in India.ResultsOur extensive analyses indicated that among present-day populations, the Parsis are genetically closest to Middle Eastern (Iranian and the Caucasus) populations rather than their South Asian neighbors. They also share the highest number of haplotypes with present-day Iranians and we estimate that the admixture of the Parsis with Indian populations occurred ∼1,200 years ago. Enriched homozygosity in the Parsi reflects their recent isolation and inbreeding. We also observed 48% South-Asian-specific mitochondrial lineages among the ancient samples, which might have resulted from the assimilation of local females during the initial settlement.ConclusionsWe show that the Parsis are genetically closest to the Neolithic Iranians, followed by present-day Middle Eastern populations rather than those in South Asia and provide evidence of sex-specific admixture from South Asians to the Parsis. Our results are consistent with the historically-recorded migration of the Parsi populations to South Asia in the 7thcentury and in agreement with their assimilation into the Indian sub-continent’s population and cultural milieu “like sugar in milk”. Moreover, in a wider context our results suggest a major demographic transition in West Asia due to Islamic-conquest.

Mutations in the NPHS1, NPHS2, LAMB2, and the WT1 genes are responsible for causing nephrotic syndrome (NS) in two third of the early onset cases. This study was carried out to assess the frequencies of mutations in these genes in a cohort of pediatric NS patients. A total of 64 pediatric familial or sporadic SRNS cases were recruited. Among these, 74% had a disease onset of up to 3 years of age. We found one homozygous frameshift mutation in the NPHS1 gene in one CNS case and two homozygous mutations in the NPHS2 gene. Six mutations in four cases in the LAMB2 gene were also identified. No mutation was detected in the WT1 gene in isolated SRNS cases. LAMB2 gene missense mutations were segregating in NS cases with no extra-renal abnormalities. Analysis of the population genomic data (1000 genome and gnomAD databases) for the prevalence estimation revealed that NS is more prevalent than previously determined from clinical cohorts especially in Asian population compared with overall world populations (prevalence worldwide was 1in 189036 and in South-Asian was 1in 56689). Our results reiterated a low prevalence of mutations in the NPHS1, NPHS2, LAMB2, and WT1 genes in the studied population from Pakistan as compared to some European population that showed a high prevalence of mutations in these genes. This is a comprehensive screening of the genes causing early onset NS in sporadic and familial NS cases suggesting a more systematic and robust approach for mutation identification in all the 45 disease-causing genes in NS in our population is required.

To investigate TP53 gene codon 72 polymorphism in women with endometriosis and compare it with healthy samples.This case-control study was carried out at Jinnah Hospital, Services Hospital and Sheikh Zayed Hospital, Lahore, Pakistan, from 2014 to 2016, and comprised patients with endometriosis and healthy controls. SPSS 21 was used for statistical analysis.Of the 176 participants, 88(50%) were healthy controls and 88(50%) were endometriosis patients. The observed genotype frequencies for controls and patients were 14(15.9%) and 31(35.3%) for proline/proline, 46(52.3%) and 35(39.8%) for proline/arginine, and 28(31.8%) and 22(25%) for arginine/arginine, respectively. The association of different genotypes was not significant in patients with moderate-to-severe endometriosis (p=0.574). The presence of pro/pro genotype enhanced the chances/odds of getting the disease (p0.05). However, the risk further increased with the advancement of age, particularly in the 27-46 age group (p0.05).In Pakistani women the association of TP53 gene codon 72 arginine/proline polymorphism was present..

Supplementary text explaining the archeological details of ancient samples; isolation of ancient DNA, genotyping, statistical analyses and peopling of South Asia and Parsi chapters. 12 figures and 10 tables are also incorporated in this file. (PDF 13264 kb)

To compare the pattern of Vitamin D receptor (VDR) polymorphisms (Apa I and Fok I) in Type I Diabetes mellitus (T1DM) as cases vs healthy population as control and to investigate the association of VDR polymorphism with vitamin D levels in cases and controls.The hypothesis of the study was "VDR gene polymorphisms (Fok 1 and Apa 1) and vitamin D levels are associated with the T1DM". The case-control study was carried out on 44 cases and 44 controls. Clinically diagnosed unrelated cases were recruited from the Diabetic Clinic of Jinnah Hospital, Lahore during Aug. 2012 to Jan 2013. Unrelated controls with normal glucose levels and no first-degree family history of T1DM were selected by convenient sampling. Vitamin D levels of both cases and controls were measured using Enzyme Linked Immunosorbant Assay (ELISA). Genotyping was performed by Restriction Fragment Length Polymorphism (RFLP)-PCR method and the data were analyzed statistically with IBM-SPSS 21.Our results demonstrated suboptimal vitamin D levels in whole of our sample population, whether control or cases (p = 0.529). There was no statistically significant difference in 25-Hydroxyvitamin D3 levels between cases (11.351 ± 5.92) and controls (12.335 ± 6.64). VDR polymorphism was not associated with susceptibility to T1DM in our sample population. Similarly, no association between VDR polymorphism and vitamin D levels was observed i.e. FokI p=0.507 and p=0.543 and ApaI p=0.986 and p=0.307 for cases and controls respectively.There is an overall deficiency of Vitamin D levels in cases and control subjects while SNPs association studies suggested that in our sample population there was no association of VDR gene polymorphisms Fok I and Apa I with TIDM.

Background Gene polymorphisms of the chemokine receptors CCR2 and CCR5 (CCR2V64I, CCR5-59029G>A and CCR5Δ32) have been shown to be associated with renal allograft rejection. The aim of this study was to investigate the association of these polymorphisms with allograft rejection among Pakistani transplant patients. Method A total of 606 renal transplant patients and an equal number of their donors were included in this study. DNA samples were used to amplify polymorphic regions of CCR2V64I, CCR5-59029G>A and CCR5Δ32 by polymerase chain reaction using sequence specific primers. The amplified products of CCRV64I and CCR5-59029G>A were digested with restriction enzymes (BsaB1 and Bsp12861) respectively. The CCR5Δ32 genotypes were determined by sizing the PCR amplicons. The association of these polymorphisms with the biopsy proven rejection and other clinical parameters was evaluated using the statistical software SPSS v.17. Results In this study, the G/G genotype of CCR2V64I was associated with a high frequency of allograft rejection (p = 0.009; OR = 2.14; 95% CI = 1.2–3.7). Rejection episode(s) in the GA + AA genotypes were found to be significantly lower as compared to the GG genotype (p = 0.009; OR = 0.4; 95% CI = 0.2–0.8). The Kaplan–Meier curve also indicated a reduced overall allograft survival for patients with the G/G genotype of CCR2V64I (59.2 ± 1.4 weeks, log p = 0.008). There was a significant association with rejection by female donors possessing the CCR2 GG genotype (p = 0.02; OR = 2.6; CI = 1.1–6.3) and male donors with the CCR5-59029 GG genotype (p = 0.004; OR = 1.7; CI = 1.03–3.01). Conclusion This study shows an association of the CCR2V64I (G/G) genotype with renal allograft rejection. However, no such association was found for the CCR5 gene polymorphisms. Therapeutic interventions such as blocking the CCR2 receptor (especially G polymorphism) may yield better survival of renal allograft in this patient group. Further, chemokine receptors may be added to the spectrum of the immunogenetic factors that are known to be associated with renal allograft rejection.

Nephrotic syndrome is a common pediatric glomerular disease associated with heavy proteinuria. Since, the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is a putative genetic risk factor for NS, in this study, ACE (I/D) polymorphism was analyzed in 268 NS and 223 control samples by a PCR-based method. The genotypic and allelic frequencies were determined and the association between ACE I/D polymorphism and NS was evaluated. The frequency distribution of the II, ID and DD genotypes was 82 (30.6%), 128 (47.8%) and 58 (21.6%) in the NS patients and 9 (4.0%), 171 (76.7%) and 43 (19.3%) in the control samples respectively. In the Pakistani pediatric NS population, the II genotypic and allelic frequencies were found to be significantly associated with the disease (OR = 6.755; C.I = 3–14.9). No significant association was found between this polymorphism and the response to standard steroid therapy. Thus, in contrast to reports from other parts of the world, the II genotype was found to be significantly associated with NS in the Indian and Malay populations and in the Pakistani population described here. To our knowledge, this is the first report from Pakistan describing the association of the ACE I/D polymorphism with pediatric NS. On the basis of these results, it is suggested that analysis of the ACE (I/D) polymorphism should be performed for the early diagnosis in the high risk NS patients in South Asia.

Cytochrome P450 2E1, gene symbol CYP2E1, is one of a family of enzymes with a central role in activating and detoxifying xenobiotics and endogenous compounds. Genetic variation at this gene has been reported in different human populations, and some association studies have reported increased risk for cancers and other diseases. To the best of our knowledge, multi-single-nucleotide polymorphism haplotypes and linkage disequilibrium (LD) have not been systematically studied for CYP2E1 in multiple populations. Haplotypes can greatly increase the power both to identify patterns of genetic variation relevant for gene expression as well as to detect disease-related susceptibility mutations. We present frequency and LD data and analyses for 11 polymorphisms and their haplotypes that we have studied on over 2600 individuals from 50 human population samples representing the major geographical regions of the world. The diverse patterns of haplotype variation found in the different populations we have studied show that ethnicity may be an important variable helping to explain inconsistencies that have been reported by association studies. More studies clearly are needed of the variants we have studied, especially those in the 5' region, such as the variable number of tandem repeats, as well as studies of additional polymorphisms known for this gene to establish evidence relating any systematic differences in gene expression that exist to the haplotypes at this gene.

Background/aim Fanconi anemia (FA) is an autosomal recessive disease determined by mutations in at least 16 genes, with distinct distributions in different populations. To the best of our knowledge, there are no reports regarding the molecular basis of the disease in FA patients in Pakistan. The current study aimed to determine the frequency of FANCC gene mutations, i.e. IVS4+4A>T, del322G, and R548X, in FA patients. Materials and methods Genomic DNA was obtained from 36 FA patients. All samples were analyzed by polymerase chain reaction and restriction fragment length polymorphism techniques. Results Mutation IVS4+4A>T was identified in 26 (72.2%) patients. It was homozygous in 6 and heterozygous in 20 patients. Del322G and R548X were found with the following prevalences: del322G, 5.6%, and R548X, 5.6%. Patients with these two mutations were compound heterozygotes having concomitant IVS4+4A>T mutation. Conclusion These results suggest that mutation IVS4+4A>T is the most prevalent mutation in our group of patients. This analysis of Pakistani patients also suggests that there is no significant difference between IVS4+4A>T homozygotes and the rest of the patients with regard to severity of clinical phenotype.

Cone-rod retinal dystrophy (CORD) characteristically leads to early impairment of vision due to the simultaneous involvement of both cone and rod photoreceptor cells. Several loci/genes have been identified for CORD, including the cone-rod dystrophy (CORD8) locus [OMIM#605549] identified for a Pakistani family. All members of this family underwent detailed clinical re-examination to determine the nature of the dystrophy. All affected individuals suffered from bilateral CORD8 with an autosomal recessive mode of inheritance. The CORD8 locus, mapped on chromosome 1q12-q24, consisted of a very large critical disease region of 21 cM. Analysis with more recently available microsatellite markers within the reported region showed heterozygosity with some of the new markers, and the crossovers lead to a refinement of the disease region from 21 to 11.53 cM. Mutation screening has excluded some of the candidate genes in the region. The disease phenotype of this family could be due to a mutation in a novel gene located within the refined CORD8 locus.

Semaphorins are a large family of transmembrane proteins. The gene for SEMA4A encodes a transmembrane protein comprising 760 amino acids. To investigate its association with human retinal degeneration, mutation screening of the SEMA4A gene was carried out on 190 unrelated patients suffering from a variety of eye diseases. We report the first observation of the involvement of SEMA4A gene mutations causing retinitis pigmentosa (RP) and cone rod dystrophy (CRD). We screened the DNA of 135 patients with RP, 25 patients with CRD, and 30 with LCA using SSCP and direct DNA sequencing for mutations in the SEMA4A gene. Two mutations, p.D345H and p.F350C, were observed only in affected patients; they were not observed in any of the normal members or the 100 control subjects. Both mutations identified occur in the conserved semaphorin domain. Multiple sequence alignments using Clustal analysis showed that R713Q is a conserved substitution and D345H is a semi‐conserved substitution. We conclude that these mutations are a cause of various retinal degenerations.

Isolated (non-syndromic) congenital cataract may be inherited as an autosomal dominant, autosomal recessive, or X-linked recessive trait. Considerable progress has been made in identifying genes and loci for dominantly inherited cataract, but the molecular basis for autosomal recessive disease is less well defined. Hence we undertook genetic linkage studies in four consanguineous Pakistani families with non-syndromic autosomal recessive congenital cataracts. In two families linkage to a 38 cM region 9q13-q22 was detected. Although a locus for recessive congenital cataracts had not been mapped previously to this region, the target interval encompasses the candidate region autosomal recessive adult-onset pulverulent cataracts (CAAR). The CAAR was mapped previously to 9q13-q22, and may therefore be allelic to non-syndromic autosomal recessive congenital cataracts. The other two families did not demonstrate linkage to 9q, but both had a region of homozygosity at 16q22 containing the heat shock transcription factor 4 (HSF4) gene. The HSF4 mutations have been reported in four families with autosomal dominant cataracts and, recently, in a single kindred with autosomal recessive congenital cataract. Mutation analysis of HSF4 revealed homozygous mutations (p.Arg175Pro and c.595_599delGGGCC, respectively) in the two families. These findings confirm that mutations in HSF4 may result in both autosomal dominant and autosomal recessive congenital cataract, and highlight the locus heterogeneity in autosomal recessive congenital cataract.

The vertebrate basic helix-loop-helix (bHLH) transcription factor ATOH7 (Math5) is specifically expressed in the embryonic neural retina and is required for the genesis of retinal ganglion cells (RGCs) and optic nerves. In Atoh7 mutant mice, the absence of trophic factors secreted by RGCs prevents the development of the intrinsic retinal vasculature and the regression of fetal blood vessels, causing persistent hyperplasia of the primary vitreous (PHPV). We therefore screened patients with hereditary PHPV, as well as bilateral optic nerve aplasia (ONA) or hypoplasia (ONH), for mutations in ATOH7. We identified a homozygous ATOH7 mutation (N46H) in a large family with an autosomal recessive PHPV disease trait linked to 10q21, and a heterozygous variant (R65G, p.Arg65Gly) in one of five sporadic ONA patients. High-density single-nucleotide polymorphism analysis also revealed a CNTN4 duplication and an OTX2 deletion in the ONA cohort. Functional analysis of ATOH7 bHLH domain substitutions, by electrophoretic mobility shift and luciferase cotransfection assays, revealed that the N46H variant cannot bind DNA or activate transcription, consistent with structural modeling. The N46H variant also failed to rescue RGC development in mouse Atoh7-/- retinal explants. The R65G variant retains all of these activities, similar to wild-type human ATOH7. Our results strongly suggest that autosomal recessive persistent hyperplastic primary vitreous is caused by N46H and is etiologically related to nonsyndromic congenital retinal nonattachment. The R65G allele, however, cannot explain the ONA phenotype. Our study firmly establishes ATOH7 as a retinal disease gene and provides a functional basis to analyze new coding variants.

Background Mutations in the NPHS1 and NPHS2 genes are among the main causes of early-onset and familial steroid resistant nephrotic syndrome respectively. This study was carried out to assess the frequencies of mutations in these two genes in a cohort of Pakistani pediatric NS patients. Methods Mutation analysis was carried out by direct sequencing of the NPHS1 and NPHS2 genes in 145 nephrotic syndrome (NS) patients. This cohort included 36 samples of congenital or infantile onset NS cases and 39 samples of familial cases obtained from 30 families. Results A total of 7 homozygous (6 novel) mutations were found in the NPHS1 gene and 4 homozygous mutations in the NPHS2 gene. All mutations in the NPHS1 gene were found in the early onset cases. Of these, one patient has a family history of NS. Homozygous p.R229Q mutation in the NPHS2 gene was found in two children with childhood-onset NS. Conclusions Our results show a low prevalence of disease causing mutations in the NPHS1 (22% early onset, 5.5% overall) and NPHS2 (3.3% early onset and 3.4% overall) genes in the Pakistani NS children as compared to the European populations. In contrast to the high frequency of the NPHS2 gene mutations reported for familial SRNS in Europe, no mutation was found in the familial Pakistani cases. To our knowledge, this is the first comprehensive screening of the NPHS1 and NPHS2 gene mutations in sporadic and familial NS cases from South Asia.

Autosomal recessive congenital ichthyosis (ARCI) is a rare genetically heterogeneous disorder characterized by hyperkeratosis in addition to dry, scaly skin. There are six genes currently known to be associated with the disease. Exome sequencing data for two affected individuals with ichthyosis from two apparently unrelated consanguineous Pakistani families was analysed. Potential candidate mutations were analysed in additional family members to determine if the putative mutation segregated with disease status. A novel mutation (c.G4676T, p.Gly1559Val) inABCA12occurred at a highly conserved residue, segregated with disease status in both families, and was not detected in 143 control chromosomes. Genotyping with microsatellite markers demonstrated a partial common haplotype in the two families, and a common founder mutation could not be excluded. Comparison to previously reported cases was consistent with the hypothesis that severe loss of functionABCA12mutations are associated with Harlequin Ichthyosis and missense mutations are preferentially associated with milder phenotypes. In addition to identifying a possible founder mutation, this paper illustrates how advances in genome sequencing technologies could be utilised to rapidly elucidate the molecular basis of inherited skin diseases which can be caused by mutations in multiple disease genes.

To identify the disease causing gene in a four generation consanguineous family in which eleven family members were suffering from Woolly hair/hypotrichosis phenotype.Linkage analysis was carried out to identify the disease-causing gene in this family. Genomic DNA of all the available family members was genotyped for the microsatellite markers for all the known woolly hair/hypotrichosis loci.Automated DNA sequencing of the candidate gene was performed to identify the disease-causing mutation.By using homozygosity linkage analysis we have mapped the family on chromosome 3q27.3 with a two point LOD score of 4.04, Mutation screening of the LIPH gene revealed a homozygous c.659_660delTA deletion mutation segregating with the disease phenotype.The results indicate that the c.659_660delTA mutation in the LIPH gene cause autosomal recessive WH/hypotrichosis phenotype in this family. This mutation has been reported in several Pakistani and Guyanese families suggesting a founder mutation in the LIPH gene in Indo-Pak sub-continent.

To identify the gene causing inherited hypertrophic cardiomyopathy (HCM) in a Pakistani family.Cross-sectional, observational study.Department of Cardiology, Shifa International Hospital and Biomedical and Genetic Engineering Laboratories, Islamabad, from 2005 to 2007.A large family of 17 individuals was included in this study. In the family 6 members were suffering from hypertrophic cardiomyopathy. Linkage analysis was carried out to map the disease-causing gene. Genomic DNA from each individual of the whole family was genotyped for microsatellite markers for all the known HCM loci followed by a whole genome search. Automated DNA sequencing was done for mutation identification in the candidate genes.Linkage analysis of 17 family members showed a maximum two point Lod score of 3.97 with marker D1S1660 at chromosome 1q 32.2. A disease region of 4.16cM was defined by proximal and distal cross-overs with markers GATA135F02 and D1S3715 respectively. This region contained the candidate genes TNNT2 (cardiac troponin T) and TNNI1 (troponin I 1). Direct sequencing of these genes for the whole family containing 17 members showed no diseaseassociated mutation in either of these genes.Through linkage analysis, a disease locus for HCM family was mapped within a region of 4.16cM at chromosome 1q31.3-q32.1. So far no disease-associated mutation has been found in the candidate genes.

Summary Mitochondrial aldehyde dehydrogenase (ALDH2) is one of the most important enzymes in human alcohol metabolism. The oriental ALDH2*504Lys variant functions as a dominant negative, greatly reducing activity in heterozygotes and abolishing activity in homozygotes. This allele is associated with serious disorders such as alcohol liver disease, late onset Alzheimer disease, colorectal cancer, and esophageal cancer, and is best known for protection against alcoholism. Many hundreds of papers in various languages have been published on this variant, providing allele frequency data for many different populations. To develop a highly refined global geographic distribution of ALDH2*504Lys, we have collected new data on 4,091 individuals from 86 population samples and assembled published data on a total of 80,691 individuals from 366 population samples. The allele is essentially absent in all parts of the world except East Asia. The ALDH2*504Lys allele has its highest frequency in Southeast China, and occurs in most areas of China, Japan, Korea, Mongolia, and Indochina with frequencies gradually declining radially from Southeast China. As the indigenous populations in South China have much lower frequencies than the southern Han migrants from Central China, we conclude that ALDH2*504Lys was carried by Han Chinese as they spread throughout East Asia. Esophageal cancer, with its highest incidence in East Asia, may be associated with ALDH2*504Lys because of a toxic effect of increased acetaldehyde in the tissue where ingested ethanol has its highest concentration. While the distributions of esophageal cancer and ALDH2*504Lys do not precisely correlate, that does not disprove the hypothesis. In general the study of fine scale geographic distributions of ALDH2*504Lys and diseases may help in understanding the multiple relationships among genes, diseases, environments, and cultures.

Three Pakistani populations residing in northern Pakistan, the Burusho, Kalash and Pathan claim descent from Greek soldiers associated with Alexander's invasion of southwest Asia. Earlier studies have excluded a substantial Greek genetic input into these populations, but left open the question of a smaller contribution. We have now typed 90 binary polymorphisms and 16 multiallelic, short-tandem-repeat (STR) loci mapping to the male-specific portion of the human Y chromosome in 952 males, including 77 Greeks in order to re-investigate this question. In pairwise comparisons between the Greeks and the three Pakistani populations using genetic distance measures sensitive to recent events, the lowest distances were observed between the Greeks and the Pathans. Clade E3b1 lineages, which were frequent in the Greeks but not in Pakistan, were nevertheless observed in two Pathan individuals, one of whom shared a 16 Y-STR haplotype with the Greeks. The worldwide distribution of a shortened (9 Y-STR) version of this haplotype, determined from database information, was concentrated in Macedonia and Greece, suggesting an origin there. Although based on only a few unrelated descendants, this provides strong evidence for a European origin for a small proportion of the Pathan Y chromosomes.

The C-type lectin DC-SIGN is involved in early interactions between human innate immune cells and a variety of pathogens. Here we sought to evaluate whether DC-SIGN interacts with the leprosy bacillus, Mycobacterium leprae, and whether DC-SIGN genetic variation influences the susceptibility and/or pathogenesis of the disease. A case-control study conducted in a cohort of 272 individuals revealed no association between DC-SIGN variation and leprosy. However, our results clearly show that DC-SIGN recognizes M. leprae, indicating that mycobacteria recognition by this lectin is not as narrowly restricted to the Mycobacterium tuberculosis complex as previously thought. Altogether, our results provide further elucidation of M. leprae interactions with the host innate immune cells and emphasize the importance of DC-SIGN in the early interactions between the human host and the infectious agents.

Retinitis pigmentosa (RP) is the most prevalent hereditary retinal degenerative disease. To date, approximately 40 loci and mutations in more than 25 genes have been identified as the cause of various types of RP.1 The gene for human oxygen regulated photoreceptor protein ( RP1 ) encodes a protein of 2156 amino acids that is localised in the connecting cilia of both rod and cone photoreceptors.2 The RP1 protein is required for the morphogenesis of the outer segments of the photoreceptor cells.3,4 Several laboratories have found mutations in the RP1 gene to be the cause of autosomal dominant retinitis pigmentosa (adRP).5–7 However, to our knowledge, association of the RP1 gene with recessive RP has never been reported. The aim of the present study was to map the disease locus for three consanguineous Pakistani families suffering from RP. We present mapping of these autosomal recessive RP families to the 8q11 locus. The results show, for the first time, that in these families a form of arRP is caused by homozygous mutations of the RP1 gene. Although these mutations were found in the parents and some of the siblings who had normal vision (carriers) in a heterozygous state, they were not found in any of a panel of 100 normal controls. We studied three consanguineous Pakistani families (442RP, 452RP, and 336RP) suffering from autosomal recessive RP (fig 1). The patients had night blindness since early childhood and progressive deterioration of vision with age. All the patients were completely blind by the age of 12–15 years in the case of the 442RP and 452RP families and 17–18 years in the 336RP family. Fundoscopic examination and electroretinographic (ERG) analyses were carried out on all the patients, their heterozygous parents and siblings, and unaffected normal siblings. Figure 1 Pedigrees of autosomal recessive RP families …

Objectives: To describe the phenotype of Leber congenital amaurosis (LCA) in 26 probands with mutations in aryl hydrocarbon receptor interacting protein-like 1 protein (AIPL1) and compare it with phenotypes of other LCA-related genes. To describe the electroretinogram (ERG) in heterozygote carriers. Methods: Patients with AIPL1-related LCA were identified in a cohort of 303 patients with LCA by polymerase chain reaction single-strand confirmational polymorphism mutation screening and/or direct sequencing. Phenotypic characterization included clinical and ERG evaluation. Seven heterozygous carrier parents also underwent ERG testing. Results: Seventeen homozygotes and 9 compound heterozygotes were identified. The W278X mutation was most frequent (48% of alleles). Visual acuities ranged from light perception to 20/400. Variable retinal appearances, ranging from near normal to varying degrees of chorioretinal atrophy and intraretinal pigment migration, were noted. Atrophic and/or pigmentary macular changes were present in 16 (80%) of 20 probands. Keratoconus and cataracts were identified in 5 (26%) of 19 patients, all of whom were homozygotes. The ERG of a parent heterozygote carrier revealed significantly reduced rod function, while ERGs for 6 other carrier parents were normal. Conclusions: The phenotype of LCA in patients with AIPL1 mutations is relatively severe, with a maculopathy in most patients and keratoconus and cataract in a large subset. Rod ERG abnormalities may be present in heterozygous carriers of AIPL1 mutations. Clinical Relevance: Understanding and recognizing the phenotype of LCA may help in defining the course and severity of the disease. Identifying the gene defect is the first step in preparation for therapy since molecular diagnosis in LCA will mandate the choice of treatment.

Cone-rod dystrophies (CRD) are forms of inherited retinal dystrophy which characteristically lead to early impairment of vision. An initial loss of colour vision (cone mediated functions) and of visual acuity, usually from the first or second decade of life, is followed by night blindness (largely rod mediated) and loss of peripheral visual fields.1 CRD patients suffer from severe photophobia and show reduced ERG responses. In later life, vision may be reduced to a bare perception of light. CRD is a milder condition compared to Leber congenital amaurosis (LCA) which is the most severe form of all the inherited retinal dystrophies and is diagnosed as bilateral congenital blindness, with a diminished or absent electroretinogram (ERG). Cone-rod dystrophy loci have been mapped to chromosomes 17q,2 19q,3 18q,4 17p13,5,6 6q,7 1q12,8 and 8p11.9 Mutations in the peripherin/RDS ,10 CRX ,11,12 and RetGC-I 13,14 genes have been shown to cause autosomal dominant CRD. Mutations in the ATP binding cassette transporter rim protein ( ABCR) gene have been shown to be associated with autosomal recessive CRD.15 Mutations in the CNGA3 gene encoding the α-subunit of the cone photoreceptor cGMP gated channel have also been reported to cause cone photoreceptor disorders.16 The RPGRIP1 protein (retinitis pigmentosa GTPase regulator interacting protein 1, MIM 605446) is encoded by the gene located on chromosome 14q11. It consists of 24 exons and the predicted size of its protein product is 1259 amino acids. It is expressed specifically in the rod and cone photoreceptors and is a structural component of the ciliary axoneme. One of its functions is to anchor the RPGR protein within the photoreceptor connecting cilium.17 Recently, in an in vivo investigation of RPGRIP1 function and its physical interaction, it has been shown that …

Three populations from northern Pakistan, the Burusho, Kalash, and Pathan, claim descent from soldiers left behind by Alexander the Great after his invasion of the Indo-Pak subcontinent. In order to investigate their genetic relationships, we analyzed nine Alu insertion polymorphisms and 113 autosomal microsatellites in the extant Pakistani and Greek populations. Principal component, phylogenetic, and structure analyses show that the Kalash are genetically distinct, and that the Burusho and Pathan populations are genetically close to each other and the Greek population. Admixture estimates suggest a small Greek contribution to the genetic pool of the Burusho and Pathan and demonstrate that these two northern Pakistani populations share a common Indo-European gene pool that probably predates Alexander's invasion. The genetically isolated Kalash population may represent the genetic pool of ancestral Eurasian populations of Central Asia or early Indo-European nomadic pastoral tribes that became sequestered in the valleys of the Hindu Kush Mountains.