The Mutational Landscape Of Genetic Cholestatic Diseases In Pakistani Children.

Objectives: To report the mutational landscape of a clinically diagnosed cohort of paediatric patients with cholestasis liver diseases. METHODS: The retrospective study was conducted at the University of Child Health Sciences, The Children Hospital, Lahore, Pakistan, from December 10, 2021, to March 31, 2022, and comprised data collected from the Paediatric Gastroenterology and Hepatology unit on demographics, clinical and laboratory findings related to children of either gender aged <12 years and diagnosed with cholestatic liver disease from July 2018 to June 2021. The diagnosis was based on clinical and biochemical findings, with no evidence of biliary atresia and metabolic liver disease. Molecular characterisation was done through whole exome sequencing. RESULTS: Of the 171 children evaluated, 92(53.8%) were diagnosed with genetic cholestatic disorders. There were 52(56%) boys and 41(44%) girls. The median age at presentation was 19.5 months (interquartile range: 51 months). Consanguinity was found in 82(88.1%) cases, and positive family history with one or more affected siblings was noted in 60(64.5%). Exome sequencing identified pathogenic mutations in 13 genes underlying the hereditary cholestasis; ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, DCDC2, ACOX2, AKR1D1, HSD3B7, ABCC2, USP53, SLC10A1, and SLC51A. Of the 70 variants identified, 50(71.4%) were novel variants. The ABCB11-related hereditary cholestasis was the most frequent 27(29%), followed by ABCB4 (26(27.9%). Homozygosity was frequently seen in all except 8(8.6%) children, who had compound heterozygous pathogenic variants. There was no evidence of phenotypic expression in the carrier parents despite the severe nature of the respective mutations identified in the patients. CONCLUSIONS: Genetic heterogeneity of paediatric intrahepatic cholestasis showed recurrent and novel mutations.

Genetic Analysis of Tyrosinemia Type 1 and Fructose-1, 6 Bisphosphatase Deficiency Affected in Pakistani Cohorts.

Background: Inborn errors of metabolism are inherited disorders that present in early childhood and are usually caused by monogenic recessive mutations in specific enzymes that metabolize dietary components. Distinct mutations are present in specific populations.Objective: To determine which genomic variants are present in Pakistani cohorts with hepatorenal tyrosinemia type 1 (HT1) and fructose 1,6-bisphosphatase deficiency (FBPD).Materials and Methods: We sequenced the fumaryl acetoacetate hydrolase encoding gene (FAH) including flanking regions in four unrelated HT1 cohorts and the fructose 1,6-bisphosphatase gene (FBP1) in eight FBPD cohorts.Results: We mapped two recessive mutations in FAH gene for HT1; c.1062 + 5G > A(IVS12 + 5G > A) in three families and c.974C > T(pT325M) in one. We identified three mutations in FBP1 gene; c.841G > A(p.E281K) in five FBPD families, c.472C > T(p.R158W) in two families and c.778G > A(p.G260R) in one.Conclusion: Knowledge of common variants for HTI and FBDP in our study population can be used in the future to build a diagnostic algorithm.

Clinical and Genetic Description of Hereditary Chronic Pancreatitis in Pakistani Children.

BACKGROUND/AIMS: The purpose of this study was to identify the spectrum and frequency of pathogenic variants as well as the clinical and genetic insight of hereditary chronic pancreatitis in Pakistani children. MATERIALS AND METHODS: The deoxyribonucleic acid of affected probands of 44 unrelated Pakistani families, having hereditary chronic pancreatitis-affected children, were subjected to massive parallel sequencing for candidate reported genes (SPINK1, PRSS1, CFTR, CPA1, CTRC, CBS, AGL, PHKB, and LPL). Data were analyzed using different bioinformatics tools for the variants and in-silico analysis. All the identified variants were validated by direct sequencing of the targeted exons in the probands and their parents. RESULTS: There were 50 patients included in this study with confirmed hereditary chronic pancreatitis. Nine known mutations in SPINK1, PRSS1, CFTR, CTRC, CBS, and AGL genes, and 10 novel variants in LPL, CFTR, CTR, and PHKB genes were identified. The identified variants were found in heterozygous, compound heterozygous, and trans-heterozygous forms, with rare allele frequency in the normal population. The novel variants were [c.378C>T(p.Lys126Asn) and c.719G>A(p.Arg240Gln) in CTRC, c.586-3C>A and c.763A>G(p.Arg255Gly) in CPA1, c.1160_1161insT(p.Lys387Asnfs*26), c.784C>T(p.Gln262*), c.1139+1G>A, c.175G>A(p.Gly59Arg) in LPL, c.388C>G(p.leu130val) in CFTR, and c.2327G>A(p.Arg776His in PHKB)]. The phenotypic characteristics were variable and correlated with the relevant variant. CONCLUSIONS: The genetic composition plays a significant role in the predisposition of hereditary chronic pancreatitis. The clinical presentation varies with the genetic determinant involved. This information would help in building up a diagnostic algorithm for our population that can be used for genetic screening services in affected cohorts.

Sequencing the CaSR locus in Pakistani stone formers reveals a novel loss-of-function variant atypically associated with nephrolithiasis.

BACKGROUND: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide and causes significant morbidity and cost. Common variants in the calcium sensing receptor gene (CaSR) have been associated with NL. Rare inactivating CaSR variants classically cause hyperparathyroidism, hypercalcemia and hypocalciuria. However, NL and familial hypercalciuria have been paradoxically associated with select inactivating CaSR variants in three kindreds from Europe and Australia. METHODS: To discover novel NL-associated CaSR variants from a geographically distinct cohort, 57 Pakistani families presenting with pediatric onset NL were recruited. The CaSR locus was analyzed by directed or exome sequencing. RESULTS: We detected a heterozygous and likely pathogenic splice variant (GRCh37 Chr3:122000958A>G; GRCh38 Chr3:12228211A>G; NM_000388:c.1609-2A>G) in CaSR in one family with recurrent calcium oxalate stones. This variant would be predicted to cause exon skipping and premature termination (p.Val537Metfs*49). Moreover, a splice variant of unknown significance in an alternative CaSR transcript (GRCh37 Chr3:122000929G>C; GRCh38 Chr3:122282082G >C NM_000388:c.1609-31G >C NM_001178065:c.1609-1G >C) was identified in two additional families. CONCLUSIONS: Sequencing of the CaSR locus in Pakistani stone formers reveals a novel loss-of-function variant, expanding the connection between the CaSR locus and nephrolithiasis.

Variability of clinical and biochemical phenotype in liver phosphorylase kinase deficiency with variants in the phosphorylase kinase (PHKG2) gene.

Background PHKG2-related liver phosphorylase kinase deficiency is inherited in autosomal recessive pattern and is a rare type of liver glycogenosis. We demonstrated the clinical presentation and genetic determinants involved in children with PHKG2- related liver phosphorylase kinase deficiency. Methodology Ten Pakistani children with liver phosphorylase kinase from seven different families, were enrolled over a period of 18 months. All regions of the PHKG2 gene spanning exons and splicing sites were evaluated through targeted exome sequencing. Variants were analyzed using different bioinformatics tools. Novel variants were reconfirmed by direct sequencing. Results Seven different variants were identified in PHKG2 gene including five novel variants: three stop codons (c.226C>T [p.R76*], c.454C>T [p.R152*] and c.958C>T [p.R320*]), one missense variant c.107C>T (p.S36F) and one splice site variant (c.557-3C>G). All five novel variants were predicted to be damaging by in Silico analysis. The variants are being transmitted through recessive pattern of inheritance except one family (two siblings) has compound heterozygotes. Laboratory data revealed elevated transaminases and triglycerides, normal creatinine phosphokinase and uric acid levels but with glycogen loaded hepatocytes on liver histology. Conclusion PHKG2 related liver phosphorylase kinase deficiency can mimic both liver glycogenosis type I (glucose-6-phosphatase deficiency) & III(amylo-1,6 glucosidase) and characterized by early childhood onset of hepatomegaly, growth restriction, elevated liver enzymes and triglycerides. Molecular analysis would be helpful in accurate diagnosis and proper treatment. The symptoms and biochemical abnormalities in liver glycogenosis due phosphorylase kinase deficiency tend to improve with proper dietary restrictions but need to be monitored for long-term complications such as liver fibrosis and cirrhosis.

Mapping of IDUA gene variants in Pakistani patients with mucopolysaccharidosis type 1.

Background Mucopolysaccharidosis type 1 (MPS1) is a rare debilitating multisystem lysosomal disorder resulting due to the deficiency of α-L-iduronidase enzyme (IDUA), caused by recessive mutations in the IDUA gene. Lack or improper amount of the IDUA enzyme results in the improper metabolism of mucopolysaccharides or glycosaminoglycans (GAGs). These large sugar molecules accumulate in lysosomes within cells leading to different systemic complications. The estimated global incidence of MPS1 is 1:100,000 live births for the Hurler and 1:800,000 for the Scheie phenotypes. Methods Thirteen MPS1-affected children from 12 unrelated cohorts were enrolled. All coding and flanking regions of the IDUA gene were sequenced. Bioinformatics tools were used for data analysis and protein prediction for clinical correlations. Results Six IDUA gene mutations were mapped co-segregating with the recessive pattern of inheritance including a novel variant. A novel missense variant c.908T > C (p.L303P) was mapped in two affected siblings in a cohort in the homozygous form. The variant c.1469T > C (p.L490P) was mapped in five unrelated patients and c.784delC (p.H262Tfs*55) was mapped in three unrelated patients, while mutations c.1598C > G (p.P533R), c.314G > A (p.R105Q) and c.1277ins9 (p.[A394-L395-L396]) were mapped in a single patient each. Conclusions Multisystem disorders and a wide range of clinical presentation impede the evaluation of patients as well as make it difficult to differentiate between different phenotypes of MPS. Early and accurate diagnosis is crucial for the disease management and implementation of an expanded new-born genetic screening program for inborn errors of metabolism including MPS1. We recommend c.784delC (p.H262Tfs*55) and c.1469T > C (p.L490P) as first-line genetic markers for the molecular diagnosis of MPS1 in Pakistan.

Genetic analysis of fructose-1,6-bisphosphatase (FBPase) deficiency in nine consanguineous Pakistani families.

BACKGROUND: Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inherited metabolic disorder characterized by recurrent episodes of hypoglycemia, ketosis and lactic acidosis. FBPase is encoded by FBP1 gene and catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate in the last step of gluconeogenesis. We report here FBP1 mutations in nine consanguineous Pakistani families affected with FBPase deficiency. METHODS: Nine families having one or two individuals affected with FBPase deficiency were enrolled over a period of 3 years. All FBP1 exonic regions including splicing sites were PCR-amplified and sequenced bidirectionally. Familial cosegregation of mutations with disease was confirmed by direct sequencing and PCR-RFLP analysis. RESULTS: Three different FBP1 mutations were identified. Each of two previously reported mutations (c.472C>T (p.Arg158Trp) and c.841G>A (p.Glu281Lys)) was carried by four different families. The ninth family carried a novel 4-bp deletion (c.609_612delAAAA), which is predicted to result in frameshift (p.Lys204Argfs*72) and loss of FBPase function. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. CONCLUSIONS: FBPase deficiency is often fatal in the infancy and early childhood. Early diagnosis and prompt treatment is therefore crucial to preventing early mortality. We recommend the use of c.472C>T and c.841G>A mutations as first choice genetic markers for molecular diagnosis of FBPase deficiency in Pakistan.

Direct sequencing of FAH gene in Pakistani tyrosinemia type 1 families reveals a novel mutation.

BACKGROUND: Hereditary tyrosinemia type 1 (HT1) is a rare inborn error of tyrosine catabolism with a worldwide prevalence of one out of 100,000 live births. HT1 is clinically characterized by hepatic and renal dysfunction resulting from the deficiency of fumarylacetoacetate hydrolase (FAH) enzyme, caused by recessive mutations in the FAH gene. We present here the first report on identification of FAH mutations in HT1 patients from Pakistan with a novel one. METHODS: Three Pakistani families, each having one child affected with HT1, were enrolled over a period of 1.5 years. Two of the affected children had died as they were presented late with acute form. All regions of the FAH gene spanning exons and splicing sites were amplified by polymerase chain reaction (PCR) and mutation analysis was carried out by direct sequencing. Results of sequencing were confirmed by restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: Three different FAH mutations, one in each family, were found to co-segregate with the disease phenotype. Two of these FAH mutations have been known (c.192G>T and c.1062+5G>A [IVS12+5G>A]), while c.67T>C (p.Ser23Pro) was a novel mutation. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. CONCLUSIONS: Most of the HT1 patients die before they present to hospitals in Pakistan, as is indicated by enrollment of only three families in 1.5 years. Most of those with late clinical presentation do not survive due to delayed diagnosis followed by untimely treatment. This tragic condition advocates the establishment of expanded newborn screening program for HT1 within Pakistan.