Ramanath Majumdar

A clinical and genetic study of 56 Saudi Wilson disease patients: identification of Saudi‐specific mutations

Wilson disease (WD) is a hereditary disorder, with recessive transmission and genetic heterogeneity. Several mutations of ATP7B, the gene underlying WD, were reported in many ethnic groups. In this study, mutation screening in ATP7B of 56 Saudi Arabian WD patients was undertaken. The clinical data of all patients were recorded. The entire ATP7B coding sequence, including intron–exon boundaries were screened for mutation by the polymerase chain reaction (PCR)‐based mutation detection technique and DNA sequencing. Thirty‐nine patients were symptomatic at presentation and 17 subjects were pre‐symptomatic siblings of affected patients. Fourteen patients had neurological, 11 patients had mixed (hepatic and neurological), and 14 patients had hepatic presentations. Family history suggestive of WD was present in 72% of cases and 68% had consanguineous parents. Genetic analysis showed disease‐causing mutations in three exons (exons 8, 19 and 21) of the ATP7B gene in 28 patients (50%). Mutations in exons 21 (18 cases) and 19 (one case) were unique for Saudis. This large series of Saudi patients with WD has shown wide variability in the genomic substrate of WD. There is no correlation between genotype and clinical presentation.

A novel deletion mutation within the carboxyl terminus of the copper-transporting ATPase gene causes Wilson disease

In patients with Wilson disease (WD), an autosomal recessive disorder, toxic accumulation of copper results in fatal liver disease and irreversible neuronal degeneration. ATP7B, the gene mutated in WD, contains 21 exons and encodes a copper-transporting ATPase. In this study, all exons of the ATP7B gene of nine WD patients were screened for alterations by conventional mutation detection enhancement (MDE) heteroduplex analysis, followed by direct sequencing of the regions that showed heteroduplex formation. For the first time, a novel deletion mutation (4193delC) in exon 21, causing a frameshift leading to premature truncation of the protein was detected in four of nine patients. The 4193delC removes several signals within the carboxyl terminal domain that may disrupt trafficking of ATP7B protein through trans-Golgi network at the cellular level.

Molecular analysis of the SMN and NAIP genes in Saudi spinal muscular atrophy patients

In this study we examined the deletion of the SMN and NAIP genes in 14 Saudi families (16 patients and 38 relatives of the patients, including parents and siblings) and six healthy Saudi volunteers. The homozygous deletions of exons 7 and 8 of the telomeric SMN gene and exon 5 of the NAIP gene were found in seven out of eight spinal muscular atrophy (SMA) type-I patients. In seven SMA type-II patients, exons 7 and 8 of telomeric SMN were deleted in six cases and exon 5 of NAIP was deleted in three cases. Three patients with SMA diagnosis did not show either of the above deletions. All control Saudi volunteers and all but two family members of the patients had both normal SMN and NAIP genes. Our results show that the incidence of NAIP deletion is higher in the more severe SMA cases and the dual deletions of the SMN and NAIP genes are more common in Saudi SMA type-I patients compared to patients of other ethnic groups.

Spinal Muscular Atrophy Carrier Screening by Multiplex Polymerase Chain Reaction using Dried Blood Spot on Filter Paper: SMA Carrier Detection using Dried Blood Spot

Spinal muscular atrophy (SMA) is a common, often fetal, autosomal recessively inherited disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. The SMA‐determining gene, called the survival of motor neuron gene (SMN), is present on 5q13 in two nearly identical copies, telomeric SMN (SMN1) and centromeric SMN (SMN2). It has been established that SMA is caused by mutations in SMN1 whereas homozygous deletion of SMN2 has apparently no pathological consequences. The aim of this study is to develop an easy and inexpensive method for the isolation of high‐quality template DNA from blood samples for SMA carrier screening by multiplex polymerase chain reaction. We have developed a protocol that optimizes detection of the SMN1 copy number in the human genome, producing a specific and sensitive assay using DNA extracted from a dried blood spot on IsoCode™ paper.

4193delC, a common mutation causing Wilson's disease in Saudi Arabia: rapid molecular screening of patients and carriers.

Background: In patients with Wilson’s disease (WD), an autosomal recessive disorder, toxic accumulation of copper results in fatal liver disease and irreversible neuronal degeneration. ATP7B, the gene mutated in WD, contains 21 exons and encodes a copper transporting ATPase. A novel disease causing mutation (4193delC) in exon 21 of the ATP7B gene has previously been detected by heteroduplex analysis and DNA sequencing. Aims: To screen for the above mutation in patients with WD and carriers using an amplification refractory mutation system (ARMS). Methods: ARMS was used to screen for the 4193delC mutation in 30 patients with WD and their relatives. Results: A homozygous mutation was detected in 16 of 30 patients with WD. Conclusions: This polymerase chain reaction based method, which has been known for years, is a simple, inexpensive, and rapid method for screening common and specific mutations in patients with WD and carriers.

A rare homozygous missense mutation in ATP7B exon 19 in a case of Wilson disease

Introduction Wilson disease (WD), an autosomal recessive disorder of copper transport, is characterized by impaired biliary excretion and deficient incorporation of copper into ceruloplasmin [1]. This leads to toxic accumulation of copper in the liver and subsequent overflow and accumulation in the brain, kidney and cornea. Thus, excess accumulation of copper can cause tissue damage leading to hepatic, neurological and psychiatric disturbances or a combination of these [1, 2]. Patients with liver disease generally present in childhood or adolescence. Neurological and psychiatric symptoms begin at the age of 12 years or later [2]. WD occurs in populations of every geographic and ethnic origin. The worldwide prevalence of WD is estimated to be 1 in 30,000, with a corresponding gene frequency of 0.56% and a carrier frequency of about 1/90 [1, 2]. ATP7B, the gene mutated in WD, has 21 exons and encodes a protein of 1,465 amino acids [1]. The protein is a copper-transporting P-type ATPase. To date more than 100 mutations have been detected in the ATP7B gene, few of which are common to several populations, the majority being population specific [3–5]. In this report, we present a patient with WD caused by a novel homozygous missense mutation associated with severe neurological and psychiatric disturbances leading to his death.

A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening

Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/μL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/μL. The ddPCR lower limit of quantitation was 14 TREC copies/μL, and the limit of detection was 4 TREC copies/μL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/μL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/μL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS.

Allelic spectrum of formiminotransferase-cyclodeaminase gene variants in individuals with formiminoglutamic aciduria

Elevated plasma and urine formiminoglutamic acid (FIGLU) levels are commonly indicative of formiminoglutamic aciduria (OMIM #229100), a poorly understood autosomal recessive disorder of histidine and folate metabolism, resulting from formiminotransferase‐cyclodeaminase (FTCD) deficiency, a bifunctional enzyme encoded by FTCD.