ATP7B Mutation Analysis: Wilson Disease, A Difficult to Diagnose Case.

Wilson's Disease (WD) is a common metabolic disorder predominantly involving liver, brain, and eyes. Pancreatic, renal, psychiatric, and cardiac involvement have also been described. No single investigation can be considered diagnostic of WD; therefore, diagnosis is based upon a series of tests best interpreted using Wilson disease diagnostic index (WDDI). We present a difficult-to-diagnose, 9-year girl of consanguineous parents, with chronic liver disease and portal hypertension. Initial workup was equivocal with significantly low serum ceruloplasmin, normal urinary copper excretion and absent Kaiyser-Fleischer (KF) rings. Diagnosis was established by ATP7B mutation analysis. The patient was found homozygous for c.3955C>T (p.Arg1319Ter) in exon 19, a rare mutation described in literature, which results in premature truncation of peptide chain. Key Words: ATP7B, Wilson disease, Copper, Mutations, Hepatolenticular degeneration.

Identification of two novel variants in GNPTAB underlying mucolipidosis II in a Pakistani family.

Background Mucolipidosis II is a rare inherited metabolic disorder characterized by multiple pathologies including coarse facial features, thickened skin, dysostosis multiplex, and skeletal abnormalities. The disorder results due to variants in GNPTAB leading to reduced activity of the enzyme GlcNAc-1-phosphotransferase (GlcNAc-PT). Methods In the present study, a consanguineous Pakistani family was diagnosed with MLII based on clinical and biochemical examination. Peripheral blood samples were collected and subjected to DNA sequencing of all coding exons along with exon-intron boundaries of GNPTAB. Results Molecular investigation of the family identified two novel variants c.25C > T: p.Gln9* (maternal allele) in exon 1 and c.1160C > T: p.Ala387Val (paternal allele) in exon 10 segregating in compound heterozygous form in the affected individuals. Conclusions The GNPTAB variant c.25C > T variant is highly plausible to undergo nonsense-mediated mRNA decay, while the GNPTAB variant c.1160C > T is located in a highly conserved domain, thus both the variants predict to lead to affect the enzyme activity. Two novel variants have been identified in GNPTAB as the underlying cause of ML-II in a Pakistani family. The study thus expands the available GNPTAB mutation spectrum.

Identification of two novel variants in GAA underlying infantile-onset Pompe disease in two Pakistani families.

Background Pompe disease (PD) is an autosomal recessive metabolic myopathy with an average incidence of one in 40,000 live births. It has a variable age of onset and can be diagnosed within the first 3 months. Heart involvement and muscle weakness are its primary manifestations. Case presentation We describe two families affected by PD with two rare, novel variants. To date, pathogenic variants in acid α-glucosidase (GAA) alone have accounted for all cases of the disease. Both families were screened for pathogenic sequence variations. This study presents the implications of regulatory or modifier sequences in the disease pathogenesis for the first time. A homozygous missense p.Arg854Gln variant in family A and a single heterozygous variant (p.Asn925His) in family B were found to be segregating according to the disease phenotype. The variants were not detected in our in-house database comprising 50 whole-exome sequences of healthy individuals from a local unrelated Pakistani population. In silico analyses predicted that the variants would have deleterious effects on the protein structure. Conclusions The variants likely underlie the infantile-onset PD (IOPD) in these Pakistani families. The study expands the mutation spectrum of GAA associated with IOPD and highlights the insufficiency of screening the GAA coding sequence to determine the cause of IOPD. The work should be helpful in carrier identification, improving genetic counselling, and prenatal diagnosis.

Genetic testing of two Pakistani patients affected with rare autosomal recessive Fanconi-Bickel syndrome and identification of a novel SLC2A2 splice site variant.

Fanconi-Bickel syndrome (FBS) is a rare autosomal recessive carbohydrate metabolism disorder caused by mutations in SLC2A2 encoding the glucose transporter 2 (GLUT2) protein. The clinical manifestations include hepatomegaly, conditional hypo/hyperglycemia, rickets, short stature and proximal renal tubular dysfunction. GLUT2 regulates monosaccharide homeostasis through sugar sensing and transmembrane transportation during high/low glucose levels. In the current study, we present two siblings suffering from FBS. The patients presented with doll-like facies, failure to gain weight and height, abdominal distension and firm hepatomegaly. The family had a history of deaths of twin male siblings in the neonatal period and twin female siblings at ages 10 months and 2.5 years, respectively. Clinical presentation and biochemical investigations including a complete blood count, electrolytes, liver and renal function tests suggested FBS. Mutation screening of SLC2A2 confirmed the diagnosis with identification of a novel homozygous splice site variant predicting an in-frame deletion [p.(Gly166-S169del)] in the GLUT2 protein. The in-silico analysis predicted the variant to affect the three-dimensional conformation of the fourth transmembrane helix of the encoded protein, rendering the non-functionality of GLUT2 in both patients of the family under study.

Spectrum of UGT1A1 variants in Pakistani children affected with inherited unconjugated hyperbilirubinemias.

Inherited unconjugated hyperbilirubinemias are a group of disorders characterized by increased levels of serum unconjugated bilirubin and arise because of the imbalance between its production and elimination from the body. It includes Crigler-Najjar syndrome and Gilbert syndrome. Crigler-Najjar syndrome type 1 represents the extreme severe end of the spectrum with complete absence of hepatic bilirubin uridine diphosphoglucuronate glucuronosyltransferase (UGT1A1). Crigler-Najjar syndrome type 2 patients have intermediate levels of bilirubin owing to incomplete deficiency of UGT1A1, and Gilbert syndrome lies at the extreme mild end of the spectrum with only slightly raised bilirubin level. Here, we present spectrum of UGT1A1 genetic variants in 25 Pakistani children from 23 unrelated families affected with persistent unconjugated hyperbilirubinemias. The promoter region, coding exons and splice junctions of the UGT1A1 were PCR amplified and subjected to Sanger sequencing. Eleven sequence variants were identified underlying disease phenotype including a novel c.582delC variant. Overall, c.622_625dupCAGC was the most frequent variant followed by c.1021C>T found in Crigler-Najjar syndrome type 1 patients. The evaluation of promoter polymorphism A(TA)nTAA in the affected children and their families further supported the body of evidence that the A(TA)7TAA allele could enhance the effect of other structural variants in Crigler-Najjar syndrome patients. To our knowledge, this is the first comprehensive study on molecular genetics of persistent unconjugated hyperbilirubinemias from Pakistan. This study expands the spectrum of UGT1A1 variants and should help in improved clinical diagnosis, genetic counseling and prenatal diagnosis of the affected families.

Novel IDS Variants Identified in Three Unrelated Pakistani Patients Affected with Mucopolysaccharidosis Type II (Hunter Syndrome).

INTRODUCTION: Mucopolysaccharidosis type II (MPS-II) or Hunter syndrome is a rare X-linked recessive disorder caused by genetic lesions in the IDS gene, encoding the iduronate-2-sulfatase (IDS) enzyme, disrupting the metabolism of certain sulfate components of the extracellular matrix. Thus, the undegraded components, also known as glycosaminoglycans, accumulate in multiple tissues resulting in multisystemic abnormalities. OBJECTIVE: To uncover causative genetic lesions in probands of three unrelated Pakistani families affected with rare X-linked recessive Hunter syndrome. METHODS: Screening of the IDS gene was performed in six individuals (three patients and their mothers) through whole genomic DNA extraction from peripheral blood followed by PCR and Sanger sequencing. MutationTaster, PROVEAN, Human Splicing Finder, Swiss-Model, and SwissPdbViewer were used for in silico analysis of identified variants. RESULTS: All probands were presented with coarse facies, recurrent respiratory tract infection, and reduced IDS activity. Molecular screening of IDS identified three different pathogenic variants including a novel duplication variant c.114_117dupCGTT, a novel splice site variant c.1006 + 1G>C, and a nonsense variant c.1165C>T. In silico analysis unanimously revealed the pathogenic nature of the variants due to their deleterious effects upon the encoded enzyme. CONCLUSION: Identified variants predictably lead to either the expression of a nonfunctional enzyme due to partial loss of SD1 and complete loss of SD2 subdomains or a complete lack of the IDS enzyme as a result of nonsense-mediated mRNA decay. Our study provides the first genetic depiction of MPS-II in Pakistan, expands the global IDS mutation spectrum, may provide insights into the three-dimensional structure of IDS, and should benefit the affected families in genetic counseling and prenatal diagnosis.

Identification of a novel GLB1 mutation in a consanguineous Pakistani family affected by rare infantile GM1 gangliosidosis.

Monosialotetrahexosylganglioside (GM1) is a rare lysosomal storage disorder caused by the deficiency of beta-galactosidase (ß-Gal) encoded by galactose beta 1 (GLB1). It is clinically characterized by developmental delay attributed to multifold accumulation of GM1 gangliosides in nerve cells. In this study, we present a case of infantile GM1 gangliosidosis in a consanguineous Pakistani family. The child was presented with developmental delay, hepatosplenomegaly and recurrent chest infections at 7.5 months of age. Radiological and biochemical investigations including magnetic resonance imaging (MRI), bonemarrow biopsy and urine oligosaccharide analyses suggested lysosomal storage disorder. Significantly low levels of ß-Gal enzyme confirmed the diagnosis of GM1 gangliosidosis. DNA sequencing of GLB1 identified a homozygous 2-bp deletion c.881-882delAT (p.Tyr294Terfs) in exon 8. In silico analysis supported the deleterious effect of the variant. This study extends GLB1 mutation spectrum and should benefit genetic counselling and prenatal diagnosis of the affected family.

Genetic Testing of a Large Consanguineous Pakistani Family Affected with Mucolipidosis III Gamma Through Next-Generation Sequencing.

BACKGROUND: Mucolipidosis III gamma (MLIIIγ) is a rare autosomal recessive disorder characterized by radiographic evidence of mild-to-moderate dysostosis multiplex, progressive joint stiffness and pain, scoliosis, and normal to mildly impaired cognitive development. Cardiac valve involvement and respiratory complications can be significant. MLIIIγ is caused by mutations in the GNPTG, which encodes the γ subunit of the enzyme N-acetylglucosamine-1-phosphotransferase. OBJECTIVE: Clinical and genetic study of seven individuals of a consanguineous Pakistani family affected with mucolipidosis phenotype who never pursued medical care. METHODS: Genome-wide homozygosity mapping was performed using Affymetrix Human SNP Array 6.0 followed by whole exome and Sanger sequencing. RESULTS: The affected individuals showed characteristic clinical features of MLIIIγ. Whole-genome single nucleotide polymorphism genotyping identified a region of homozygosity shared by affected individuals of the family on chromosome 16p13.3. Whole exome sequencing identified a novel 4-bp deletion in the GNPTG segregating in the family in agreement with autosomal recessive pattern. CONCLUSIONS: We identified a novel mutation in the GNPTG gene as the underlying cause of MLIIIγ in a Pakistani family. This study supports the role of next-generation sequencing technologies for the molecular diagnosis of rare inherited disorders.

UGT1A1 gene mutations in Pakistani children suffering from inherited nonhemolytic unconjugated hyperbilirubinemias.

Two inherited unconjugated hyperbilirubinemias, Crigler-Najjar syndrome and Gilbert syndrome, arise due to deficiency of UGT1A1 enzyme activity. Crigler-Najjar syndrome type 1 (CN1) lies at the extreme severe end of the spectrum of UGT1A1 activity characterized by complete absence, followed by the less severe Crigler-Najjar syndrome type 2 (CN2). Gilbert syndrome is the mild form having only partial loss of UGT1A1 activity. The present study aimed to identify molecular genetic defects underlying unconjugated hyperbilirubinemias in children from six consanguineous Pakistani families. The patients were clinically diagnosed by exclusion of other unconjugated hyperbilirubinemias. Differential diagnosis of CN1 and CN2 was made on the basis of patient's response to phenobarbitone. The promoter region, coding exons, and adjacent splice sites of the UGT1A1 gene were PCR amplified from genomic DNA of all patients and their families, and were sequenced. DNA sequence analysis identified five different homozygous mutations: two novel missense mutations p.Y230C (proband A) and p.D36N (proband B), a 4-bp insertion c.622-625dupCAGC/p.Q208QfsX50 (probands C and E), a nonsense mutation p.R341X (proband D), and a TA insertion A(TA)7TAA in the promoter region (proband F). The present study extends the spectrum of UGT1A1 gene mutations and may be helpful in the diagnosis of Crigler-Najjar syndrome and Gilbert syndrome.