Genetic insights into crossbred dairy cattle of Pakistan: exploring allele frequency, linkage disequilibrium, and effective population size at a genome-wide scale.

Linkage disequilibrium (LD) affects genomic studies accuracy. High-density genotyping platforms identify SNPs across animal genomes, increasing LD evaluation resolution for accurate analysis. This study aimed to evaluate the decay and magnitude of LD in a cohort of 81 crossbred dairy cattle using the GGP_HDv3_C Bead Chip. After quality control, 116,710 Single Nucleotide Polymorphisms (SNPs) across 2520.241 Mb of autosomes were retained. LD extent was assessed between autosomal SNPs within a 10 Mb range using the r2 statistics. LD value declined as inter-marker distance increased. The average r2 value was 0.24 for SNP pairs < 10 kb apart, decreasing to 0.13 for 50-100 kb distances. Minor allele frequency (MAF) and sample size significantly impact LD. Lower MAF thresholds result in smaller r2 values, while higher thresholds show increased r2 values. Additionally, smaller sample sizes exhibit higher average r2 values, especially for larger physical distance intervals (> 50 kb) between SNP pairs. Effective population size and inbreeding coefficient were 150 and 0.028 for the present generation, indicating a decrease in genetic diversity over time. These findings imply that the utilization of high-density SNP panels and customized/breed-specific SNP panels represent a highly favorable approach for conducting genome-wide association studies (GWAS) and implementing genomic selection (GS) in the Bos indicus cattle breeds, whose genomes are still largely unexplored. Furthermore, it is imperative to devise a meticulous breeding strategy tailored to each herd, aiming to enhance desired traits while simultaneously preserving genetic diversity.

Correction: Mutational analysis in sodium-borate cotransporter SLC4A11 in consanguineous families from Punjab, Pakistan.

[This corrects the article DOI: 10.1371/journal.pone.0273685.].

Genetic Polymorphism and serum levels of Insulin like Growth Factor-1 (Igf-1) in patients of rheumatoid arthritis.

Objectives: Insulin like growth factor-1(IGF-1), is a modulator of immunity and inflammation, it promotes the anabolic role of growth hormone (GH) on bone and skeletal tissue. Genetic polymorphism in IGF-1 gene is reported to affect the transcriptional efficiency affecting its serum level. In this study we aim: 1) To study the presence of 192bp polymorphism of IGF-1 gene in patients of rheumatoid arthritis (RA), 2) To study the association of 192 bp polymorphism of IGF-1 gene with serum IGF-1 levels and disease severity in patients of RA. Methods: A cross-sectional study was carried out at University of Health Sciences (UHS), Lahore. Diagnosed RA cases who fulfilled the American College of Rheumatology (ACR) criteria were recruited from Fatima Memorial Hospital (FMH) and Behbud Rheumatology Clinics, Lahore during 2018-2019. Serum IGF-1 levels were determined by ELISA in blood samples of 200 RA patients and 200 healthy individuals. DNA was extracted and genetic polymorphism was determined. Results: The serum IGF-1 level in RA group was significantly lower compared to healthy group. Our study shows presence of 192bp allele of IGF-1in 77% of the studied population. Carriers of 192bp allele of IGF-1 had a significantly higher serum level of IGF-1 as compared to non-carriers in the RA patients. Rheumatoid factor (RF) positive patients had a higher number of 192bp carriers in comparison to RF negative patients. Significant difference was also seen in severity of disease between carrier and non-carriers of 192bp allele with the disease being more severe in male carriers. Conclusions: There is an association of IGF-1gene polymorphism with variation in serum IGF-1 levels and severity of RA.

Mutational analysis in sodium-borate cotransporter SLC4A11 in consanguineous families from Punjab, Pakistan.

AIM: To identify the molecular basis of Congenital Hereditary Endothelial Dystrophy CHED caused by mutations in SLC4A11, in the consanguineous Pakistani families. METHODS: A total of 7 consanguineous families affected with Congenital Hereditary Endothelial Dystrophy were diagnosed and registered with the help of ophthalmologists. Blood samples were collected from affected and unaffected members of the enrolled families. Mutational analysis was carried out by DNA sequencing using both Sanger and Whole Exome Sequencing (WES). Probands of each pedigree from the 7 families were used for WES. Results were analyzed with the help of different bioinformatics tools. RESULTS: The sequencing results demonstrated three known homozygous mutations in gene SLC4A11 in probands of 7 families. These mutations p.Glu675Ala, p.Val824Met, and p.Arg158fs include 2 missense and 1 frameshift mutation. The mutations result in amino acids that were highly conserved in SLC4A11 across different species. The mutations were segregated with the disease phenotype in the families. CONCLUSION: This study reports 3 mutations in 7 families. One of the pathogenic mutations (p.R158fs) was identified for the first time in the Pakistani population. However, two mutations (p.Glu675Ala, p.Val824Met) were previously reported in two and one Pakistani family respectively. As these mutations segregate with the disease phenotype and bioinformatics tool also liable them as pathogenic, they are deemed as probable cause of underlying disease.

Delineating Novel and Known Pathogenic Variants in TYR, OCA2 and HPS-1 Genes in Eight Oculocutaneous Albinism (OCA) Pakistani Families.

Oculocutaneous albinism (OCA) is associated with a wide range of clinical presentations and has been categorized with syndromic and non-syndromic features. The most common causative genes in non-syndromic OCA are TYR and OCA2 and HSP1 is in the syndromic albinism. The objective of this study was to identify pathogenic variants in congenital OCA families from Pakistan. Eight consanguineous families were recruited, and clinical and ophthalmological examination was carried out to diagnose the disease. Whole blood was collected from the participating individuals, and genomic DNA was extracted for sequencing analysis. TruSight one-panel sequencing was carried out on one affected individual of each family, and termination Sanger sequencing was carried out to establish the co-segregation of the causative gene or genes. In silico analysis was conducted to predict the causative pathogenic variants. Two families were found to have novel genetic pathogenic variants, and six families harbored previously reported variants. One novel compound heterozygous pathogenic variant in the TYR gene, c.1002delA; p.Ala335LeufsTer20, a novel frameshift deletion pathogenic variant and c.832C>T; and p.Arg278Ter (a known pathogenic variant) were found in one family, whereas HPS1; c.437G>A; and p.Trp146Ter were detected in another family. The identification of new and previous pathogenic variants in TYR, OCA2, and HPS1 genes are causative of congenital OCA, and these findings are expanding the heterogeneity of OCA.

SLC4A11 mutations causative of congenital hereditary endothelial dystrophy (CHED) progressing to Harboyan syndrome in consanguineous Pakistani families.

BACKGROUND: Autosomal recessive corneal hereditary endothelial dystrophy (CHED) is a rare congenital disorder of cornea. Mutations in SLC4A11 gene are associated with CHED phenotype. CHED is also an early feature of Harboyan syndrome. The aim of the present study was to identify genetic mutations in the SLC4A11 gene in CHED cases belonging to inbred Pakistani families. Furthermore, all homozygous mutation carriers were investigated for hearing deficit. METHODS AND RESULTS: This study included consanguineous CHED families presented at Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan from June 2018 to September 2018. DNA was extracted from blood samples. Direct sequencing of SLC4A11 gene was performed. All identified variants were evaluated by in silico programs i.e., SIFT, PolyPhen-2, and MutationTaster. Pathogenicity of the two identified splice site variants was analyzed by Human Splicing Finder and MaxEnt Scan. Screening of five CHED families revealed a total of three previously un reported (p.Arg128Gly, c.2241-2A > T and c.1898-2A > C in family CHED19, CHED22 and CHED26 respectively) and two already reported homozygous disease causing variants (p.Arg869Cys and p.Val824Met in family CHED24 and CHED25 respectively) as predicted by mutation taster. All of these variants segregated with disease phenotype and were not detected in controls. CONCLUSION: Affected individuals of the five CHED families screened in this study had the disease due to SLC4A11 mutations and progressing to Harboyan syndrome. Identification of previously unreported mutations aid to heterogeneity of SLC4A11 and CHED pathogenesis as well as helped to provide genetic counseling to affected families.

Clinical profile and screening of exon 6 and 14 of ABCB4 gene in obstetric cholestasis patients at a tertiary care hospital in Rawalpindi, Pakistan.

OBJECTIVE: Intrahepatic Cholestasis of Pregnancy (ICP) is a rare pregnancy specific disorder. Genetic variants of ABCB4 gene increase ICP risk. This study was conducted to determine frequency of ICP cases presented at a tertiary care hospital in Rawalpindi, Pakistan and to screen for genetic variants of exon 6 and 14 of ABCB4 gene in ICP cases. METHODS: This analytical study included ICP patients presenting at Department of Gynaecology and Obstetrics, Holy Family Hospital Rawalpindi, from February 2017 to May 2017. Sanger's sequencing was performed using genomic DNA extracted from blood samples of patients and controls. RESULTS: Twenty pregnant women out of 1150 (1.74%) had ICP and were enrolled during study period. Overall (19/20) 95% patients had pruritus and among them (8/20) 40%, (4/20) 20% and (2/20) 10% had a history of miscarriages, stillbirths and familial ICP respectively. Genetic analysis revealed an already reported variant i.e., c.504C>T in exon 6 in thirteen patients and a novel variant i.e., c.1686A>G in exon 14 in five patients. Both variants were not present in controls. In silico analysis suggested that both variants might affect pre-mRNA splicing of ABCB4 transcript. CONCLUSIONS: ICP had a frequency of 1.74% among pregnant women. Identification of a novel heterozygous variant in five patients and an already reported variant in thirteen patients reaffirms genetic heterogeneity and role of ABCB4 in ICP etiology.

Association of Interleukin 10 (IL-10) Gene with Type 2 Diabetes Mellitus by Single Nucleotide Polymorphism of Its Promotor Region G/A 1082.

Interleukin 10 (IL-10) is an anti-inflammatory cytokine with lower circulating levels in patients with type 2 diabetes mellitus (T2DM). Cytokines such as IL-10 downregulate the production of pro-inflammatory cytokines, which impair proper function of insulin. So any mutation in the IL-10 gene results in increased production of proinflammatory cytokines, which in turn affect insulin action and cause T2DM. In this study, a polymorphism (rs1800896) in the gene (IL-10) and its association with T2DM was determined with the amplification-refractory mutation system with PCR (ARMS-PCR). Study subjects were divided in two groups, control and T2DM. DNA was extracted from blood, and ARMS-PCR was performed by using specific primers for the promoter region. An amplified product was obtained at 258 base pairs (bp). In the control group, heterozygous bands with both alleles (A/G) were present, whereas AA and GG homozygosity was observed in the T2DM group. This polymorphism (rs1800896) in the IL-10 gene is associated with T2DM. Physical measurements were also obtained, and significant differences between the groups were determined with an independent t-test. Significance was based on a p-value level of 0.05 or less, and highly significant was based on a p-value of 0.01 or less at 95% confidence interval.

Genetic mapping of autosomal recessive microspherophakia to chromosome 14q24.3 in a consanguineous Pakistani family and screening of exon 36 of LTBP2 gene.

Latent transforming growth factor beta binding protein 2 (LTBP2) plays a critical role in the development of connective tissue structure and function. Mutations in gene encoding LTBP2 are known to cause syndromic and a non-syndromic microspherophakia. Here, we present a 'first' report of genetic linkage of microspherophakia (MSP) to LTBP2 locus in a large consanguineous Pakistani family with four affected individuals in three loops. Using polymorphic microsatellite markers, haplotypes and linkage analysis, the diseased phenotype in MSP001 family was mapped to the LTBP2 gene. A maximum two point Logarithm of the odds (LOD) score of 4.16 was obtained with marker D14S284 at θ =0. Mutational analysis of exon 36 of LTBP2 using Sanger's sequencing did not reveal any previously reported mutations. Further analysis of the remaining exons are required to identify the causative variant.

Autosomal recessive congenital cataracts linked to HSF4 in a consanguineous Pakistani family.

PURPOSE: To investigate the genetic basis of autosomal recessive congenital cataracts (arCC) in a large consanguineous Pakistani family. METHODS: All participating members of family, PKCC074 underwent an ophthalmic examination. Slit-lamp photographs were ascertained for affected individuals that have not been operated for the removal of the cataractous lens. A small aliquot of the blood sample was collected from all participating individuals and genomic DNAs were extracted. A genome-wide scan was performed with polymorphic short tandem repeat (STR) markers and the logarithm of odds (LOD) scores were calculated. All coding exons and exon-intron boundaries of HSF4 were sequenced and expression of Hsf4 in mouse ocular lens was investigated. The C-terminal FLAG-tagged wild-type and mutant HSF4b constructs were prepared to examine the nuclear localization pattern of the mutant protein. RESULTS: The ophthalmological examinations suggested that nuclear cataracts are present in affected individuals. Genome-wide linkage analyses localized the critical interval to a 10.95 cM (14.17 Mb) interval on chromosome 16q with a maximum two-point LOD score of 4.51 at θ = 0. Sanger sequencing identified a novel missense mutation: c.433G>C (p.Ala145Pro) that segregated with the disease phenotype in the family and was not present in ethnically matched controls. Real-time PCR analysis identified the expression of HSF4 in mouse lens as early as embryonic day 15 with a steady level of expression thereafter. The immunofluorescence tracking confirmed that both wild-type and mutant HSF4 (p.Ala145Pro) proteins localized to the nucleus. CONCLUSION: Here, we report a novel missense mutation in HSF4 associated with arCC in a familial case of Pakistani descent.

Cytochrome 2C19 and paraoxonase-1 polymorphisms and clopidogrel resistance in ischemic heart disease patients.

Aim: Polymorphisms in cytochrome P450 (CYP) 2C19 and paraoxonase-1 (PON-1) genes are thought to be involved in clopidogrel high on treatment reactivity in ischemic heart disease (IHD) patients. Methods: A total of 240 patients with IHD were screened for CYP2C19 loss-of-function alleles (LOF; *2, *3) and PON-1 Q192R. Patients were classified as responders and nonresponders to clopidogrel based upon platelet aggregation studies. Genotyping of the CYP2C19 and PON-1 allele was carried out by PCR-RFLP. Results: Results showed that 14.3% of the patients were nonresponders, whereas 85.7% were responders to the clopidogrel therapy. CYP2C19*3 allele showed significant association with clopidogrel high on treatment reactivity in IHD patients. Conclusion: Result of our study demonstrate that IHD patients with CYP2C19*3 allele can face the problem of clopidogrel high on treatment reactivity in Punjabi Pakistani population.

Mutational analysis of the CYP1B1 gene in Pakistani primary congenital glaucoma patients: Identification of four known and a novel causative variant at the 3' splice acceptor site of intron 2.

Primary congenital glaucoma (PCG) causes blindness in early age. It has an autosomal recessive pattern of inheritance, hence is more prevalent in populations with frequent consanguineous marriages that occur in the Pakistani population. Mutations in the CYP1B1 gene are commonly associated with PCG. The aim of the present study was to identify genetic mutations in the CYP1B1 gene in PCG cases belonging to 38 Pakistani families. DNA was extracted using blood samples collected from all enrolled patients, their available unaffected family members and controls. Direct sequencing of the CYP1B1 gene revealed a novel 3' splice acceptor site causative variant segregating in an autosomal recessive manner in a large consanguineous family with four PCG-affected individuals. The novel variant was not detected in 93 ethnically matched controls. Furthermore, four already reported mutations, including p.G61E, p.R355X, p.R368H, and p.R390H were also detected in patients belonging to nine different families. All identified causative variants were evaluated by computational programs, that is, SIFT, PolyPhen-2, and MutationTaster. Pathogenicity of the novel splice site variant identified in this study was analyzed by Human Splicing Finder and MaxEntScan. Ten out of 38 families with PCG had the disease due to CYP1B1 mutations, suggesting CYP1B1 was contributing to PCG in these Pakistani patients. Identification of this novel 3' splice acceptor site variant in intron 2 is the first report for the CYP1B1 gene contributing to genetic heterogeneity of disease.

Prevalence of stromal corneal dystrophies in Lahore.

To determine the prevalence of Stromal Corneal Dystrophies (SCDs) in patient from Lahore hospitals. The study was performed between November, 2014 to July 2015 at the Layton Rahmatullah Benevolent Trust Hospital, Mughal Hospital, Mayo Hospital and General Hospital, Lahore. For the clinical evaluation of SCD by ophthalmologists examination of cornea was done by biomicroscopy, specular microscopy, topography, keratometry, orbscan and far visual acuity. Fifty cases of SCDs were recognized from Lahore, matching to hospital prevalence of 0.4%. The variables examined were age, gender, main complaint, corneal thickness, intraocular pressure and far visual acuity. SCDs are predominant in age group of 40-50 years. SCDs are more in male (n=30) as compared to females (n=20). Careful clinical evaluation, genotyping, governmental approval and subsequent development of human clinical trials of possible therapies and treatments should be taken to continue making improvement and effective control of SCDs.

In silico analysis of five missense mutations in CYP1B1 gene in Pakistani families affected with primary congenital glaucoma.

PURPOSE: The purpose of this study was to characterize the five missense mutations in CYP1B1 gene identified in Pakistani families affected with primary congenital glaucoma (PCG) using various bioinformatics and protein modeling tools. METHODS: We previously reported four novel missense mutations in CYP1B1 gene segregating in consanguineous Pakistani families. These mutations were identified by direct sequencing of all coding exons, the exon-intron boundaries and the 5' untranslated region of CYP1B1 using genomic DNA from affected and unaffected family members. In order to understand the effect of CYP1B1 mutations on protein structure and function, we used bioinformatics tools to investigate five mutations including four novels (W434R, D374E, L487P and L177R) and one known (E229K) mutation previously reported by our group in four Pakistani PCG-affected families. RESULTS: In silico analysis of the missense mutations using the computational algorithms SNAP, I-Mutant 2.0 IUPred, PrDOS and PASTA predicted pathogenic effects on stability and function of protein. CONCLUSION: In silico analysis of identified mutations confirmed their molecular pathogenicity. Similar analysis will be helpful in understanding of the biological role of CYP1B1 and the effect of mutations on the regulatory and enzymatic functions of CYP1B1 that result in PCG.

Fine mapping of chromosome 9 locus associated with congenital cataract.

PURPOSE: The purpose of this study was to study the molecular basis of inherited autosomal recessive cataracts in Pakistan population and to identify the molecular defect segregating with the disease phenotype. METHODS: Families having two or more affected individuals were identified through hospital, blood samples were collected and DNA was extracted. We employed the traditional strategy of linkage analysis using M13-labeled primers to map the already known genes for autosomal recessive cataract. Statistically, the data were evaluated through LOD score. RESULTS: Ten families affected with autosomal receive congenital cataract were enrolled for this study. Overall, three families were linked to reported loci for autosomal recessive congenital cataract. Out of these, one family Bl05 was linked to a cataract locus at 9q13. Fine mapping of the chromosome 9 locus considerably delimited the previously reported linkage interval from 13.99 to 7.99 cM in this study. CONCLUSION: Our results reduced the linkage interval of previously reported cataract locus on chromosome 9, thus considerably reducing the number of candidate genes.

Interleukin 6 Receptor (IL6-R) Gene Polymorphisms Underlie Susceptibility to Rheumatoid Arthritis.

BACKGROUND: Increased production of interleukin 6 (IL-6) is associated with rheumatoid arthritis that acts through its receptor, IL-6R (interleukin 6 receptor). Various single nucleotide polymorphisms in the IL6R gene conferring susceptibility to rheumatoid arthritis have been identified in various populations yet these associations have not been fully established. The present study was pursued with the aim to evaluate a possible association between three single nucleotide polymorphisms (rs2228145, rs4537545, rs4845617) of the IL6R gene and rheumatoid arthritis in Pakistani patients. METHODS: For this purpose, we recruited 60 patients diagnosed with rheumatoid arthritis and 60 healthy age and gender matched controls. Blood samples were collected and DNA was extracted. Sanger DNA sequencing was performed to evaluate the SNPs in IL6R and the data were statistically evaluated using chi-square test. RESULTS: Results of our study indicated that rs2228145 and rs4845617 were significantly associated with rheumatoid arthritis in Pakistani population. However, no association could be established between IL6R (rs4537545) and rheumatoid arthritis in Pakistani population. CONCLUSIONS: This study reports a possible genetic association of IL6R (rs2228145 and rs4845617) to the genetic susceptibility of rheumatoid arthritis.

Determination of Serum Trace Elements (Zn, Cu, and Fe) in Pakistani Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, which mainly involves the joints. RA is prevalent worldwide with increasing prevalence in elderly people. The mechanism of RA pathogenesis is still undefined, and it is interplaying between genetic susceptibility and environmental factors. Although risk factors for RA are not fully established, various studies have focused on the role of trace elements in association with RA. Trace elements act as co-factors for most of the enzymes, and their deficiency is associated with many untoward effects on human health. The homeostatic alterations in the metabolism of trace elements may partly be due to inflammatory response in RA. The objective of the present study was to determine the serum concentrations and correlation of zinc, copper, and iron in RA patients and healthy controls. The study comprised of 61 RA patients and 61 age- and sex-related healthy individuals of Pakistani population. Serum levels of Zn, Cu, and Fe were measured in all the participants by atomic absorption spectrophotometer. Serum Zn and Fe were significantly reduced in the RA patients than those in the healthy controls. Serum Cu concentrations were found elevated in the RA patients. Correlation studies of trace elements determine that there was negative correlation between Zn and Cu in the RA patients and no correlation in the control group. It is very important to explore the deficiency of essential trace metals in biological samples of the RA patients in different populations which may be helpful for diagnosis and supplementary management of rheumatoid arthritis patients.

Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts.

PURPOSE: To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family. METHODS: All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model. RESULTS: Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15) increased after birth to a level that was sustained through the postnatal time points. CONCLUSION: A novel missense mutation in LIM2 is responsible for autosomal recessive congenital cataracts.

Missense mutation in SLC4A11 in two Pakistani families affected with congenital hereditary endothelial dystrophy (CHED2).

BACKGROUND: Autosomal recessive congenital hereditary endothelial dystrophy (CHED2) is a recessively inherited eye disorder that is more common in consanguineous populations. METHODS: Two families affected with CHED2 were recruited from the Punjab province of Pakistan to identify the underlying genetic defect. Blood samples from both the families, designated as CH01 and CH02, were collected. Genomic DNA was isolated. Initially, linkage analysis using microsatellite markers was carried out to confirm the linkage to the SLC4A11 gene, previously reported to be implicated in the pathology of the disease. Later on, sequencing was carried out to find the pathogenic mutation in the enrolled families. Identified variation was further confirmed by typing 50 ethnically matched normal control samples. RESULTS: The results of linkage analysis indicated the putative linkage to SLAC4A11 gene, located at the CHED2 locus on chromosome 20p13-p12 in both families. Mutational analysis revealed an unidentified homozygous mutation c.2024A>C (p.E675A) in the affected members of both the families. Haplotype analysis of both the families showed that the affected members carry the same haplotype, thereby indicating a possibility of a common ancestral mutation. Use of bioinformatic tools including PolyPhen and SIFT suggested that a single amino acid change of E to A at position 675 affected the function of the protein. CONCLUSION: This study reports a newly identified mutation (c.2024A>C) in the SLC4A11 gene segregating with the diseased haplotype in two consanguineous Pakistani families.