Population data and forensic evaluation of six alu insertions in indigenous groups from Sabah, Malaysia

Background and aim: The present study is the first to report the genetic relatedness of indigenous populations of Sabah, Malaysia, using a set of Indel markers (HS4.32, TPA25, APO, PV92, B65 and HS3.23). The primary aim was to assess the genetic relationships among these populations and with populations from other parts of the world by examining the distribution of these markers. Subjects and methods: A total of 504 volunteers from the three largest indigenous groups, i.e. Kadazan-Dusun, Bajau and Rungus, were recruited for the study. Six Alu insertions were typed by PCR with specific primer sets. Results: All insertions were found to present at different frequencies, ranging from 0.170–0.970. The heterozygosity of most of the markers was high (.0.4), with the exception of HS3.23 and APO. A genetic differentiation study revealed that these populations are closely related to each other (GST ¼0.006). A principle component plot showed that these populations have higher affinity to Mainland South East Asia/East Asia populations, rather than Island Southeast Asia (ISEA) populations. Conclusion: In summary, these indigenous groups were closely associated in terms of their genetic composition. This finding also supports the colonization model of ISEA, which suggests that the inhabitants of this region were mostly descendants from Southern Chin

Genetic data for 15 STR loci in Kadazan-Dusun population from East Malaysia

Allele frequencies of 15 short tandem repeat (STR) loci, namely D5S818, D7S820, D13S317, D16S539, TH01, TPOX, Penta D, Penta E, D3S1358, D8S1179, D18S51, D21S11, CSF1PO, vWA, and FGA, were determined for 154 individuals from the Kadazan-Dusun tribe, an indigenous population of East Malaysia. All loci were amplified by polymerase chain reaction, using the Powerplex 16 system. Alleles were typed using a gene analyzer and the Genemapper ID software. Various statistical parameters were calculated and the combined power of discrimination for the 15 loci in the population was calculated as 0.999999999999999. These loci are thus, informative and can be used effectively in forensic and genetic studies of this indigenous population

Implementing HLA-B*58:01 testing prior to allopurinol initiation in Malaysian primary care setting: A qualitative study from doctors’ and patients’ perspective

IntroductionAllopurinol, the first-line treatment for chronic gout, is a common causative drug for severe cutaneous adverse reactions (SCAR). HLA-B*58:01 allele was strongly associated with allopurinol-induced SCAR in Asian countries such as Taiwan, Japan, Thailand and Malaysia. HLA-B*58:01 screening before allopurinol initiation is conditionally recommended in the Southeast-Asian population, but the uptake of this screening is slow in primary care settings, including Malaysia. This study aimed to explore the views and experiences of primary care doctors and patients with gout on implementing HLA-B*58:01 testing in Malaysia as part of a more extensive study exploring the feasibility of implementing it routinely.MethodsThis qualitative study used in-depth interviews and focus group discussions to obtain information from patients with gout under follow-up in primary care and doctors who cared for them. Patients and doctors shared their gout management experiences and views on implementing HLA-B*58:01 screening in primary care. Data were coded and analysed using thematic analysis.Results18 patients and 18 doctors from three different healthcare settings (university hospital, public health clinics, private general practitioner clinics) participated. The acceptability to HLA-B*58:01 screening was good among the doctors and patients. We discovered inadequate disclosure of severe side effects of allopurinol by doctors due to concerns about medication refusal by patients, which could potentially be improved by introducing HLA-B*58:01 testing. Barriers to implementation included out-of-pocket costs for patients, the cost-effectiveness of this implementation, lack of established alternative treatment pathway besides allopurinol, counselling burden and concern about genetic data security. Our participants preferred targeted screening for high-risk populations instead of universal screening.ConclusionImplementing HLA-B*58:01 testing in primary care is potentially feasible if a cost-effective, targeted screening policy on high-risk groups can be developed. A clear treatment pathway for patients who test positive should be made available

Assessment and analysis of genomic diversity and biomarkers in sabahan indigenous populations / Kee Boon Pin

Ever since the proposal of the recent African origin of modern humans and its various opposing concepts, evolutionary studies have been focusing on the discovery of timelines for such events based on traceable records in archaic remains and contemporary human populations. Although the migration of anatomically modern humans between the 7 major continents in the near 120,000 years has been explained and fit well in the “out of Africa” hypothesis, human movement within these continents remains elusive. Human demic events in Southeast Asia region have also been the heat of debate among researchers. It has been proposed that this region was populated by a recent migration wave from Taiwan about 5,000 years ago, accompanying by the expansion of Austronesian languages into various parts of this region and further into Oceania – known as the “out of Taiwan” model. However, genetic studies in the Southeast Asia have been centred on populations lying along the proposed migratory paths, i.e., southern China, Taiwan, Philippines, Eastern Indonesia, and Near Oceania. The study on population in other parts of this region could shed important information, to complement the proposed model, on the understanding of the historical migration within Southeast Asia and also between other neighboring regions. Sabah, at the northern tip of Borneo Island, is strategically located in the centre of Southeast Asia. In the present study, the aim was to characterize the genetic structure of the 3 major indigenous populations in Sabah, i.e., Kadazan-Dusun, Bajau, and Rungus, and correlate them to the migration patterns in this region. A total of 639 indigenous individuals were recruited and genomic DNA was extracted from the blood/buccal samples. Polymorphisms on the nuclear DNA (VNTRs and InDels) were accessed by direct PCR method. In addition, typing of 15 STR markers on each sample was completed via fragment analysis study. Furthermore, the mitochondrial DNA was examined by the screening of the 9-bp deletion in the region V and the nucleotide ABSTRACT │ IV sequence of the 3 hypervariable regions in the D-loop was determined via sequencing reactions. The genetic data generated was subsequently subjected to statistical and comparative analysis. In an overview, these indigenous populations were shown to have high genetic similarity (AMOVA < 5 %). The Kadazan-Dusun and Rungus populations exhibited a closer relationship compared to the Bajaus. Based on the mitochondrial lineages, different waves/directions of dispersal into the Borneo Island that perhaps shaped the genetic discrepancies of the Bajau with the Kadazan-Dusun and Rungus groups were proposed. The Sabahan Bajau population could have persisted and originated from South Philippines since the earliest entry about 50,000 years ago. There was more interaction found in the Bajau with the surrounding lineages, such as East Asia, Mainland SEA, South Asia, and Oceania, which contributed to their high diversity. The Kadazan-Dusun and Rungus on the other hand, may have arrived in nearer timeframes, possibly following a western route through the Palawan Islands after their exodus from Taiwan some 5,000 to 10,000 years ago

Association of NOD1, CXCL16, STAT6 and TLR4 gene polymorphisms with Malaysian patients with Crohn’s disease

Crohn’s disease (CD) is a prominent type of inflammatory bowel disease (IBD) that can affect any part of the gastrointestinal tract. CD is known to have higher prevalence in the Western countries, but the number of cases has been increasing in the past decades in Asia, including Malaysia. Therefore, there is a need to investigate the underlining causes of CD that may shed light on its prevention and treatment. In this study, genetic polymorphisms in NOD1 (rs2075820), CXCL16 (rs2277680), STAT6 (rs324015) and TLR4 (rs4986791) genes were examined in a total of 335 individuals (85 CD patients and 250 healthy controls) with PCR-RFLP approach. There was no significant association observed between NOD1 rs2075820 and STAT6 rs324015 with the onset of CD in the studied cohort. However, the G allele of CXCL16 rs2277680 was found to have a weak association with CD patients (P = 0.0482; OR = 1.4310). The TLR4 rs4986791 was also significantly associated to CD. Both the homozygous C genotype (P = 0.0029; OR = 0.3611) and C allele (P = 0.0069; OR = 0.4369) were observed to confer protection against CD. On the other hand, the heterozygous C/T genotype was a risk genotype (P = 0.0015; OR = 3.1392). Further ethnic-stratified analysis showed that the significant associations in CXCL16 rs2277680 and TLR4 rs4986791 were accounted by the Malay cohort. In conclusion, the present study reported two CD-predisposing loci in the Malay CD patients. However, these loci were not associated to the onset of CD in Chinese and Indian patients

Whole mitochondrial genome sequencing of Malaysian patients with cardiomyopathy

Cardiomyopathy (CMP) constitutes a diverse group of myocardium diseases affecting the pumping ability of the heart. Genetic predisposition is among the major factors affecting the development of CMP. Globally, there are over 100 genes in autosomal and mitochondrial DNA (mtDNA) that have been reported to be associated with the pathogenesis of CMP. However, most of the genetic studies have been conducted in Western countries, with limited data being available for the Asian population. Therefore, this study aims to investigate the mutation spectrum in the mitochondrial genome of 145 CMP patients in Malaysia. Long-range PCR was employed to amplify the entire mtDNA, and whole mitochondrial genome sequencing was conducted on the MiSeq platform. Raw data was quality checked, mapped, and aligned to the revised Cambridge Reference Sequence (rCRS). Variants were named, annotated, and filtered. The sequencing revealed 1,077 variants, including 18 novel and 17 CMP and/or mitochondrial disease-associated variants after filtering. In-silico predictions suggested that three of the novel variants (m.8573G>C, m.11916T>A and m.11918T>G) in this study are potentially pathogenic. Two confirmed pathogenic variants (m.1555A>G and m.11778G>A) were also found in the CMP patients. The findings of this study shed light on the distribution of mitochondrial mutations in Malaysian CMP patients. Further functional studies are required to elucidate the role of these variants in the development of CMP

Development of a species-specific PCR-RFLP targeting rpoD gene fragment for discrimination of Aeromonas species

Purpose. The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis. Methodology. A pair of degenerate primers (Aero F: 5¢-YGARATCGAYATCGCCAARCGB-3¢ and Aero R: 5¢-GRCCDATGCTCATRCGRCGGTT-3¢) was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T ) were randomly selected from the 27 Aeromonas species for experimental validation. Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement. Conclusion. This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens

Plant-derived chimeric antibodies inhibit the invasion of human fibroblasts by Toxoplasma gondii

The parasite Toxoplasma gondii causes an opportunistic infection, that is, particularly severe in immunocompromised patients, infants, and neonates. Current antiparasitic drugs are teratogenic and cause hypersensitivity-based toxic side effects especially during prolonged treatment. Furthermore, the recent emergence of drug-resistant toxoplasmosis has reduced the therapeutic impact of such drugs. In an effort to develop recombinant antibodies as a therapeutic alternative, a panel of affinity-matured, T. gondii tachyzoite-specific single-chain variable fragment (scFv) antibodies was selected by phage display and bioinformatic analysis. Further affinity optimization was attempted by introducing point mutations at hotspots within light chain complementarity-determining region 2. This strategy yielded four mutated scFv sequences and a parental scFv that were used to produce five mouse–human chimeric IgGs in Nicotiana benthamiana plants, with yields of 33–72 mg/kg of plant tissue. Immunological analysis confirmed the specific binding of these plant-derived antibodies to T. gondii tachyzoites, and in vitro efficacy was demonstrated by their ability to inhibit the invasion of human fibroblasts and impair parasite infectivity. These novel recombinant antibodies could therefore be suitable for the development of plant-derived immunotherapeutic interventions against toxoplasmosis