In this report, we describe a 15-year-old Malaysian male patient with a de novo SCN1A mutation who experienced prolonged febrile seizures after his first seizure at 6 months of age. This boy had generalized tonic clonic seizure (GTCS) which occurred with and without fever. Sequencing analysis of voltage-gated sodium channel a1-subunit gene, SCN1A, confirmed a homozygous A to G change at nucleotide 5197 (c.5197A > G) in exon 26 resulting in amino acid substitution of asparagines to aspartate at codon 1733 of sodium channel. The mutation identified in this patient is located in the pore-forming loop of SCN1A and this case report suggests missense mutation in pore-forming loop causes generalized epilepsy with febrile seizure plus (GEFS+) with clinically more severe neurologic phenotype including intellectual disabilities (mental retardation and autism features) and neuropsychiatric disease (anxiety disorder).
Introduction: Elimination of viral hepatitis as a major public health threat by 2030 was announced by the Global Health Sector Strategy (GHSS) on viral hepatitis in 2016. Hepatitis C is one of the major causes of liver cirrhosis and liver cancer. Complications as a results of hepatitis C infection can be prevented as hepatitis C infection is now con-sidered as a curable disease with the availability of Direct Acting Agents (DAAs). However, the main barrier towards treating and curing all HCV infected patients is a high cost of DAAs. The treatment regime of hepatitis C infection in Malaysia is sofosbuvir and daclastavir, an NS5A inhibitor. Daclastavir was reported as inherently resistant to HCV GT 3. Thus, this study aimed to develop an assay to detect the resistance associated substitution (RAS) towards the NS5A inhibitor among HCV GT 3 infected patients. Methods: Samples for the study were obtained from various hospitals in Malaysia. The samples were collected from DAAs naïve HCV GT 3 infected patients. From the literature review, a specific assay was chosen with different sets of primers were selected for the study. The DNA sequences of NS5A region of HCV genome were submitted to the geno2pheno [HCV] resistance database by Max Planck Institut (MPI) Informatics to yield RAS. Results: Suitable primers were identified based on generated amplicons produced for the samples which NS5A region were successfully sequenced. Results were obtained based on the 213 codon generated from the population based Sanger sequencing. RAS/ RASs towards Daclastavir were produced and the susceptibility result towards the drug was generated. Conclusion: The assay was successfully optimised and able to generate drug resistance results towards Daclastavir which might have impacts on the duration of treatment and/or inclusion of ribavirin in managing HCV infected patients in Malaysia.
Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.
The House Crow (Corvus splendens) is a useful study system for investigating the genetic basis of adaptations underpinning successful range expansion. The species originates from the Indian subcontinent, but has successfully spread through a variety of thermal environments across Asia, Africa and Europe. Here, population mitogenomics was used to investigate the colonisation history and to test for signals of molecular selection on the mitochondrial genome. We sequenced the mitogenomes of 89 House Crows spanning four native and five invasive populations. A Bayesian dated phylogeny, based on the 13 mitochondrial protein-coding genes, supports a mid-Pleistocene (~630,000 years ago) divergence between the most distant genetic lineages. Phylogeographic patterns suggest that northern South Asia is the likely centre of origin for the species. Codon-based analyses of selection and assessments of changes in amino acid properties provide evidence of positive selection on the ND2 and ND5 genes against a background of purifying selection across the mitogenome. Protein homology modelling suggests that four amino acid substitutions inferred to be under positive selection may modulate coupling efficiency and proton translocation mediated by OXPHOS complex I. The identified substitutions are found within native House Crow lineages and ecological niche modelling predicts suitable climatic areas for the establishment of crow populations within the invasive range. Mitogenomic patterns in the invasive range of the species are more strongly associated with introduction history than climate. We speculate that invasions of the House Crow have been facilitated by standing genetic variation that accumulated due to diversifying selection within the native range.
Chloroquine resistance transporter of Plasmodium falciparum (PfCRT) is a food vacuolar transmembrane protein that mediates susceptibility of the parasite to chloroquine. A mutation at K76T of the Pfcrt gene is a key determinant for chloroquine resistance phenotype. In the absence of drug pressure, in vitro growth rate of chloroquine-resistance parasites was outcompeted by wild-type parasites unless intragenic compensatory mutations occurred. Chloroquine-resistant P. falciparum bearing the Cam734 haplotype known to circulate in endemic areas of Cambodia bordering Thailand contains 9 mutations in Pfcrt and exhibits both chloroquine resistance and comparable growth rate to the chloroquine-sensitive 3D7 strain. To analyze the evolution of the Cam734 haplotype, codon-based analysis was performed by using the mixed effects model of evolution (MEME), branch-site random effects likelihood (BR-REL) and other related methods. Results revealed that the Cam734 haplotype has evolved distinctively from other known mutant haplotypes including the most common Dd2 haplotype in Southeast Asia. Evidence of episodic positive selection was detected at codon 144, characterized by c.[430G>T; 431C>T] (p.A144F), known to be indispensable for both chloroquine resistance and restoration of growth rate of the parasites. To survey the prevalence of mutations at codons 76 and 144 in Pfcrt among Thai isolates, restriction fragment analysis of 548 P. falciparum isolates collected from six endemic provinces of Thailand during 1991 and 2016 was performed. The 144F Pfcrt mutant was detected in 7 (1.28%) isolates. All Thai isolates analyzed herein harbored a mutation at codon 76 whilst the wild-type parasite was not found. The low prevalence of isolates bearing the mutation 144F in PfCRT could imply little or lack of survival advantage of this mutant in endemic areas of Thailand where the wild-type parasites seem to be absent or extremely rare.
Gymnothorax minor is a moray eel of the family Muraenidae found in the Western Pacific Ocean. We report here
its complete mitogenome as determined by Illumina next-generation sequencing and the phylogenetic relationship
with its congeners and other taxa of the family Muraenidae. The whole mitogenome of G. minor had a total length
of 16,574 bp, comprising 37 genes - 13 protein-coding genes (PCGs), two ribosomal ribonucleic acid (rRNA) and 22
transfer ribonucleic acid (tRNA) genes - and a control region. Excepting cox1 with GTG, the other 12 PCGs had ATG
start codon. Seven of its PCGs had incomplete stop codon - five (nad2; cox1; cox2; nad3 and nad4) with T and two
(atp6 and cox3) with TA. Molecular phylogeny based on 13 PCGs was concordant with 15 mitochondrial genes (13 PCGs
and 2 rRNA genes). The subfamily Muraeninae as well as the subfamily Uropterygiinae were monophyletic. However,
the genus Gymnothorax was paraphyletic, with G. minor forming a sister group with Rhinomuraena quaesita in the
lineage containing also G. kidako and G. formosus forming a sister group with Enchelynassa canina. The phylogenetic
relationship of the genus Gymnothorax and related taxa of the family Muraenidae, based on the mitochondrial cob
gene, was in general similar to that based on 15 mt-genes. The mitogenome is useful for future studies on phylogenetics
and systematics of eels of the family Muraenidae and other taxa of the order Anguilliformes.
Diversifying selection between populations that inhabit different environments can promote lineage divergence within species and ultimately drive speciation. The mitochondrial genome (mitogenome) encodes essential proteins of the oxidative phosphorylation (OXPHOS) system and can be a strong target for climate-driven selection (i.e., associated with inhabiting different climates). We investigated whether Pleistocene climate changes drove mitochondrial selection and evolution within Australian birds. First, using phylogeographic analyses of the mitochondrial ND2 gene for 17 songbird species, we identified mitochondrial clades (mitolineages). Second, using distance-based redundancy analyses, we tested whether climate predicts variation in intraspecific genetic divergence beyond that explained by geographic distances and geographic position. Third, we analysed 41 complete mitogenome sequences representing each mitolineage of 17 species using codon models in a phylogenetic framework and a biochemical approach to identify signals of selection on OXPHOS protein-coding genes and test for parallel selection in mitolineages of different species existing in similar climates. Of 17 species examined, 13 had multiple mitolineages (range: 2-6). Climate was a significant predictor of mitochondrial variation in eight species. At least two amino acid replacements in OXPHOS complex I could have evolved under positive selection in specific mitolineages of two species. Protein homology modelling showed one of these to be in the loop region of the ND6 protein channel and the other in the functionally critical helix HL region of ND5. These findings call for direct tests of the functional and evolutionary significance of mitochondrial protein candidates for climate-associated selection.
A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.
The cavity-nesting honeybee Apis nuluensis inhabits only the highlands of Mount Kinabalu of Sabah, Borneo Island. The mitochondrial genome is a circular molecule of approximately 1.6 kb that includes 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one AT-rich control region. The average AT content was 84.5%. The start codons ATC, ATG, and ATT were found in one, three, and nine genes, respectively, whereas the stop codon TAA was observed in all genes. The phylogenetic relationship, inferred using 13 PCGs, was consistent with that reported in previous studies that predicted a sister taxon relationship between A. nuluensis and A. cerana.
Polymorphisms within the beta2-adrenergic receptor (ADRB2) gene have been repeatedly linked to hypertension. Among the ADRB2 polymorphisms detected, Arg16Gly and Gln27Glu codons are considered the two most important variations. The amino acid substitution at these codons may lead to abnormal regulation of ADRB2 activity. The aim of the present study was to assess the association between ADRB2 polymorphisms and hypertension. This case-control study consisted of 100 unrelated subjects (50 hypertensive and 50 matched normal controls). Arg16Gly and the Gln27Glu polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism assay. There were no significant evidence of association in allelic and genotypes distribution of Arg16Gly and Glu27Gln with blood pressure and hypertension. These findings suggest that the variation within codon 16 and 27 of ADRB2 gene were unlikely to confer genetic susceptibility for hypertension in our population samples.
OBJECTIVE: Inherited anti-thrombin deficiency is an autosomal dominant disorder which is associated with increased risk for venous thromboembolism (VTE). This condition is very rare in Malaysia and there has been no documented report. Thus, the aim of the present study is to investigate the type of an inherited anti-thrombin deficiency mutation in a 25-year-old Malay woman who presented with deep vein thrombosis in her first pregnancy.
METHODS: DNA was extracted from the patient's blood sample and buccal mucosal swabs from family members. Polymerase chain reaction(PCR) assays were designed to cover all seven exons of the serpin peptidase inhibitor, clade C (antithrombin), member 1 (SERPINC1) gene; and the products were subjected to DNA sequencing. Sequences were referred to NCBI Reference Sequence: NG_012462.1.
RESULTS: A heterozygous substitution mutation at nucleotide position 13267 (CCT->ACT) was identified in the patient and two other family members, giving a possible change of codon 439 (Pro→Thr) also known as anti-thrombin Budapest 5. The genotype was absent in 90 healthy controls.
CONCLUSION: The study revealed a heterozygous antithrombin Budapest 5 mutation in SERPINC 1 giving rise to a possible anti-thrombin deficiency in a Malay-Malaysian family.
Xeroderma pigmentosum-D (XPD) is one of the genes that play a role in the Nucleotide-Excision Repair (NER). Polymorphisms in XPD gene have been identified and reported to be associated with many types of cancer with two common single nucleotide polymorphisms (SNPs), namely, XPD312 and XPD751. The XPD312 polymorphism is at exon 10 codon 312 Asp to Asn (A→G) and the association of this polymorphism with oral cancer is very little known, especially, in Malaysia. The aim of this study was to screen for XPD312 gene polymorphisms in human oral cancer patients attending Hospital Universiti Sains Malaysia (HUSM), Malaysia. Blood samples were collected from 10 oral cancer and 10 normal healthy subjects with their consent. DNA was extracted using commercial DNA extraction kit and Polymerase Chain Reaction (PCR) was performed to amplify the XPD312 gene. The PCR products were digested using restriction enzyme, Sty I and analyzed on a 3% agarose gel for the detection of polymorphisms. This was followed by DNA sequencing to confirm the findings. In the current study, only homozygous wild type polymorphisms in the XPD312 gene was noticed in the oral cancer tissues as revealed by the restriction enzyme and DNA sequencing analyses.
There are significant differences in the prevalence and severity of neonatal jaundice among various populations. Recently, it has been reported that a mutation of the UGT1A1 gene, glycine to arginine at codon 71 (G71R), is related to the development of neonatal jaundice in East Asian populations. However, whether the G71R mutation contributes to the high incidence of neonatal jaundice in different Asian populations remains unknown. The authors screened for this mutation in the Javanese-Indonesian and Malay-Malaysian populations.
To assess the prevalence of mutations associated with drug resistance in antiretroviral-naive patients in Kuala Lumpur, Malaysia, genotypic resistance testing was conducted among drug-naive HIV-1 patients attending the University Malaya Medical Center (UMMC) between July 2003 and June 2004. Reverse transcriptase (RT) and protease genes of plasma virions were sequenced from 100 individuals. The majority of the patients were recently diagnosed. Codons 20-255 of the RT and 1-96 of the protease gene were examined for major and minor mutations associated with antiretroviral resistance reported by the International AIDS Society- USA (IAS-USA) Drug Resistance Mutations Group. The prevalence of patients with at least one major mutation conferring drug resistance was 1%, with only one patient having a Y181C amino acid substitution in the RT gene that confers high-level resistance to nevirapine and delavirdine. Minor mutations were detected in high prevalence in the protease gene. Amino acid substitutions I13V, E35D, and M36I were associated with CRF01_AE while L63P, V77I, and I93L were associated with subtype B. Baseline prevalence of major mutations associated with resistance to antiretroviral drugs was low among antiretroviral-naive HIV-1 patients, suggesting that routine drug resistance testing may be unnecessary for all individuals newly diagnosed with HIV or all patients beginning antiretroviral therapy.
In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
Neoplastic transformation appears to be a multi-step process in which the normal controls of cell proliferation and cell-cell interaction are lost, thus transforming normal cells into cancer. The tumorigenic process involves the interplay between oncogenes and tumour suppressor genes. In this study, we have selected the ras family, c-myc and epidermal growth factor receptor (EGFR) genes to detect whether their abnormalities are associated with the expression and progression of glioma cases in Malay patients. We have used the polymerase chain reaction-single stranded conformation polymorphism followed by direct sequencing for the study. For the ras gene family, we screened the point mutations in codons 12 and 61 of the H-, K-, and N-ras gene; for EGFR and c-myc, we analyzed only the exon 1 in glioma samples. In mutational screening analyses of the ras family, c-myc and EGFR gene, there was no mobility shift observed in any tumour analyzed. All patterns of single stranded conformation polymorphism (SSCP) band observed in tumour samples were normal compared to those in normal samples. The DNA sequencing results in all high-grade tumours showed that all base sequences were normal. All 48 patients survived after five years of treatment. In simple logistic regression analysis, variables which were found to be significant were hemiplegia (p=0.047) and response radiotherapy (p=0.003). Hemiplegics were 25 times more likely to have high pathological grade compared to those without. Patients with vascular involvement were 5.5 times more likely to have higher pathological grade. However, these findings were not significant in multivariate analysis. Patients who had radiotherapy were nearly 14 times more likely to have higher pathological grade. Multivariate analysis revealed that patients with hemiplegia were more likely to have higher pathological grade (p= 0.008). Those with higher pathological grading were 80 times more likely to have radiotherapy (p=0.004).
Preimplantation genetic diagnosis (PGD) of monogenic autosomal hereditary disorders following assisted conception usually involves the removal of one or two blastomeres from preimplantation embryos. However, the amount of DNA from a single blastomere is insufficient to amplify the region of interest. Hence, the whole genome amplification (WGA) method is performed prior to amplifying the genes of interest before analysis of DNA material through polymerase chain reaction (PCR).
This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were cloned into P. pastoris expression vector (pPICZαA) downstream of alcohol oxidase gene (AOX1) for efficient induction of extracellular rFGH by adding 1% of absolute methanol. The results showed that recombinant P. pastoris was able to produce 2.80 ± 0.27 mg of oFGH compared to 1.75 ± 0.25 of nFGH in one litre of culture supernatant. The total body weight of tiger grouper fingerlings fed with oFGH increased significantly at third (P < 0.05) and fourth weeks (P < 0.01) of four-week experiment period compared to those fed with nFGH. Both oFGH and nFGH significantly enhanced the final biomass and fish survival percentage. In conclusion, codon optimization of FGH fragment was useful to increase rFGH quantity in the culture supernatant of P. pastoris that can be directly used as fish feed supplements. Further studies are still required for large scale production of rFGH and practical application in aquaculture production.
Synonymous codon usage bias is an inevitable phenomenon in organismic taxa across the three domains of life. Though the frequency of codon usage is not equal across species and within genome in the same species, the phenomenon is non random and is tissue-specific. Several factors such as GC content, nucleotide distribution, protein hydropathy, protein secondary structure, and translational selection are reported to contribute to codon usage preference. The synonymous codon usage patterns can be helpful in revealing the expression pattern of genes as well as the evolutionary relationship between the sequences. In this study, synonymous codon usage bias patterns were determined for the evolutionarily close proteins of albumin superfamily, namely, albumin, α-fetoprotein, afamin, and vitamin D-binding protein. Our study demonstrated that the genes of the four albumin superfamily members have low GC content and high values of effective number of codons (ENC) suggesting high expressivity of these genes and less bias in codon usage preferences. This study also provided evidence that the albumin superfamily members are not subjected to mutational selection pressure.