Nonsense mutations contribute to approximately 10-30% of the total human inherited diseases via disruption of protein translation. If any of the three termination codons (UGA, UAG and UAA) emerges prematurely [known as premature termination codon (PTC)] before the natural canonical stop codon, truncated nonfunctional proteins or proteins with deleterious loss or gain-of-function activities are synthesized, followed by the development of nonsense mutation-mediated diseases. In the past decade, PTC-associated diseases captured much attention in biomedical research, especially as molecular therapeutic targets via nonsense suppression (i.e. translational readthrough) regimens. In this review, we highlighted different treatment strategies of PTC targeting readthrough therapeutics including the use of aminoglycosides, ataluren (formerly known as PTC124), suppressor tRNAs, nonsense-mediated mRNA decay, pseudouridylation and CRISPR/Cas9 system to treat PTC-mediated diseases. In addition, as thrombotic disorders are a group of disease with major burdens worldwide, 19 potential genes containing a total of 705 PTCs that cause 21 thrombotic disorders have been listed based on the data reanalysis from the 'GeneCards® - Human Gene Database' and 'Human Gene Mutation Database' (HGMD®). These PTC-containing genes can be potential targets amenable for different readthrough therapeutic strategies in the future.
We report a case of Griscelli Syndrome (GS). Our patient initially presented with a diagnosis of haemophagocytic lymphistiocytosis (HLH). Subsequent microscopic analysis of the patient's hair follicle revealed abnormal distribution of melanosomes in the shaft, which is a hallmark for GS. Analysis of RAB27A gene in this patient revealed a homozygous mutation in exon 6, c.550C>T, p.R184X . This nonsense mutation causes premature truncation of the protein resulting in a dysfunctional RAB27A. Recognition of GS allows appropriate institution of therapy namely chemotherapy for HLH and curative haemotopoeitic stem cell transplantation.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common type of inherited cystic kidney disease. The feasibility of whole‑exome sequencing (WES) to obtain molecular diagnosis of ADPKD is still in question as previous studies showed conflicting results. Utilizing WES on a patient with ADPKD, standard bioinformatics pipeline demonstrated no pathogenic variant in the genes of interest. By visualizing read alignments using the Integrative Genomics Viewer, a region with atypical alignment of numerous soft‑clipped reads at exon 45 of polycystin 1, transient receptor potential channel interacting (PKD1) gene was demonstrated. A total of four visual inspection steps were outlined to assess the origin of these soft‑clipped reads as strand bias during capture, poor mapping, sequencing error or DNA template contamination. Following assessment, the atypical alignment at PKD1 was hypothesized to be caused by an insertion/deletion mutation. Sanger sequencing confirmed the presence of a novel 20‑bp insertion in PKD1 (NM_001009944.3; c.12143_12144insTCCCCGCAGTCTTCCCCGCA; p.Val4048LeufsTer157), which introduced a premature stop codon and was predicted to be pathogenic. The present study demonstrated that WES could be utilized as a molecular diagnostic tool for ADPKD. Furthermore, visual inspection of read alignments was key in identifying the pathogenic variant. The proposed visual inspection steps may be incorporated into a typical WES data analysis workflow to improve the diagnostic yield.
In Duchenne muscular dystrophy (DMD), identification of one nonsense mutation in the DMD gene has been considered an endpoint of genetic diagnosis. Here, we identified two closely spaced nonsense mutations in the DMD gene. In a Malaysian DMD patient two nonsense mutations (p.234S>X and p.249Q>X, respectively) were identified within exon 8. The proband's mother carried both mutations on one allele. Multiple mutations may explain the occasional discrepancies between genotype and phenotype in dystrophinopathy.
The present study was carried out to characterize the causative genetic mutation in a medium-sized Malaysian Chinese pedigree of three generations affected with familial adenomatous polyposis (FAP). Clinical data and genetic studies revealed considerable phenotypic variability in affected individuals in this family. Blood was obtained from members of the FAP-01 family and genomic DNA was extracted. Mutation screening of the adenomatous polyposis coli (APC) gene was carried out using the single strand conformation polymorphism (SSCP) technique. The possibility of exon skipping was predicted by splicing motif recognition software (ESEfinder release2.0). SSCP results showed mobility shifts in exon 8 of the APC gene which segregated with affected members of the family. Sequence analysis revealed that the affected individuals are heterozygous for a C847T transition, whilst all the unaffected family members and control individuals are homozygous C at the same position. This nucleotide substitution generates a stop codon at amino acid position 283, in place of the usual arginine (Arg283Ter). We conclude that an Arg283Ter mutation in the APC gene is causative of the FAP phenotype in this family, although there is considerable variation in the presentation of this disease among affected individuals. Computational analysis predicts that this mutation occurs within sequences that may function as splicing signals, so that the sequence change may affect normal splicing.
Glutaric aciduria type 1 (GA1) is an autosomal recessive metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase enzyme encoded by the GCDH gene. In this study, we presented the clinical and molecular findings of seven GA1 patients in Malaysia. All the patients were symptomatic from infancy and diagnosed clinically from large excretion of glutaric and 3-hydroxyglutaric acids. Bidirectional sequencing of the GCDH gene revealed ten mutations, three of which were novel (Gln76Pro, Glu131Val, and Gly390Trp). The spectrum of mutations included eight missense mutations, a nonsense mutation, and a splice site mutation. Two mutations (Gln76Pro and Arg386Gln) were homozygous in two patients with parental consanguinity. All mutations were predicted to be disease causing by MutationTaster2. In conclusion, this is the first report of both clinical and molecular aspects of GA1 in Malaysian patients. Despite the lack of genotype and phenotype correlation, early diagnosis and timely treatment remained the most important determinant of patient outcome.
The Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency condition characterized by microthrombocytopenia, eczema and recurrent infections. It is caused by mutations in the Wiskott-Aldrich Syndrome protein (WASP) gene. We investigated two Malay boys who presented with congenital thrombocytopenia, eczema and recurrent infections. Here we report two cases of WASP mutation in Malaysia from two unrelated families. One had a novel missense mutation in exon 1 while the other had a nonsense mutation in exon 2. Both patients succumbed to diseaserelated complications. A differential diagnosis of WAS should be considered in any male child who present with early onset thrombocytopenia, especially when this is associated with eczema and recurrent infections.
The availability of molecular genetic testing for retinoblastoma (RB) in Malaysia has enabled patients with a heritable predisposition to the disease to be identified, which thus improves the clinical management of these patients and their families. In this paper, we presented our strategy for performing molecular genetic testing of the RB1 gene and the findings from our first 2 years of starting this service.
Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.