Since the discovery of the double-helical structure of DNA, genetic regulation of gene expression has been well elucidated. More recently, another equally, if not more, important scheme of regulation of gene expression, called epigenetics, has emerged to explain the many biological observations that traditional genetic mechanisms have failed to decipher. Epigenetics is a discipline of study on the biological consequences of cellular alterations that do not involve nucleotide changes, as opposed to genetic mutations. Epigenetic changes are reversible and may lead to loss or gain of biological functions. The three most reported mechanisms of epigenetic regulation of gene expression involve changes in: (i) chromatin remodelling, (ii) DNA methylation and (iii) microRNA (miRNA). More importantly, many of the elucidated epigenetic changes are linked to the pathogenesis of human diseases and cancers. In this mini review, core concepts and basic experimental approaches in the study of epigenetic regulation of gene expression are briefly reviewed in relation to disease, with emphasis on cancer. This mini review also intends to highlight the fact that, besides genetics, epigenetics is now a discipline physicians and clinical research scientists can no longer ignore in their pursuit to understand disease and cancer and to develop new therapeutic strategies for treatment.
The 2009 pandemic influenza A(H1N1) was first detected in Malaysia in May 2009. It quickly spread in the general population and contributed to a number of influenza-like illness. The objective of the study is to characterize genetic changes in early Malaysian isolates of mild and severe illness of the novel influenza, and to compare sequences of viruses circulating in Malaysia to those in other countries between May to September 2009. Viral isolates of 56 mild cases and 10 severe (intensive care unit or fatal) cases were sequenced for haemagglutinin (HA) and neuraminidase (NA). Genome sequencing of the viral RNA was conducted on 5 isolates (3 were from fatal cases). Highly conserved sequences with few sporadic variations were identified in HA and NA. E374K and D222N were identified in 2 viral isolates from patients with severe illness. Phylogenetic analysis showed close genetic relatedness to the vaccine strain A/California/07/09 and other isolates circulating worldwide during the same period. Sporadic variations were identified in the viral isolates, however a larger sample size is required to make associations with disease severity.
Leptospirosis is a re-emerging zoonotic infection. In developing countries large outbreaks have occurred in urban slums and following floods. Individuals from developed nations are also now more frequently exposed to the infection as a result of international travel and greater participation in certain outdoor recreational activities. Leptospirosis remains a diagnostic challenge since it often presents as a non-specific febrile event and laboratory diagnosis is still currently inadequate. Rapid tests may not be sufficiently sensitive in early disease and culture facilities are not widely available. A severe pulmonary haemorrhagic form of the infection is increasingly being encountered in many countries including Malaysia. The control of leptospirosis is largely dependent on general hygienic measures and rodent control. An effective human vaccine is still not available. There remains much that is unknown about this disease and there is scope and opportunity for good quality research.
Aim: Autoantibodies against cyclic citrullinated peptide (anti-CCP) are considered to be a sensitive and specific marker for rheumatoid arthritis (RA). This study evaluated the diagnostic and analytical performances of the automated anti-CCP assay.
Materials and Method: Sera from 80 patients with established RA, 65 from other rheumatic diseases (non-RA) and 55 from healthy controls were studied using second generation anti-CCP. Rheumatoid factor (RF) was also assayed in each sample, and the results were compared to the anti-CCP fi ndings. Serum pools were used to determine the precision and linearity.
Results: At a cut-off of 7.4 U/ml for anti-CCP, the sensitivity and specificity for RA were 65% and 98% respectively. RF had a sensitivity of 58% and a lower specifi city of 93 % than anti-CCP. Conclusion: The high specificity of the assay suggests that anti-CCP is useful in the diagnosis of rheumatoid arthritis and in our cohort of study population anti-CCP exhibits a better diagnostic value than RF. A considerable proportion (28%) of RF-negative RA patients were anti-CCP positive. Based on analytical performance of the assay, we conclude that full automation and high throughput features of AxSYM makes it an ideal platform for routine testing of anti-CCP.
Study site: Rheumatology clinic, University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia
A number of genetic risk factors have been implicated in the development of neonatal severe hyperbilirubinaemia. This includes mutations in the uridine glucoronosyl transferase 1A1 (UGT1A1) gene which is responsible for unconjugated hyperbilirubinemia in Gilbert's Syndrome. We studied the prevalence of UGT1A1 gene mutations in a group of Malay neonates to determine whether they are risk factors to severe neonatal jaundice. One hundred and twenty-five Malay neonates with severe hyperbilirubinemia were studied. Ninety-eight infants without severe hyperbilirubinaemia were randomly selected from healthy Malay term infants (controls). DNA from EDTA cord blood samples were examined for UGT1A1 mutations nt211G > A and nt247T > C using established Taqman SNP genotyping assays and the UGT1A1*28 variant was detected by the Agilent 2100 bioanalyzer. All samples were also screened for common Malay G6PD variants using established techniques. The frequency of UGT1A1 211G > A mutation is significantly higher in the severely hyperbilirubinemic group (13%) than the control group (4%; p = 0.015) and all the positive cases were heterozygous for the mutation. There was no significant difference in the frequency of UGT1A1*28 mutation between the severely hyperbilirubinemic (3.5%) and the control group (0.01%; p = 0.09). None of the neonates in both groups carried the nt247 T > C mutation. The prevalence of G6PD mutation was significantly higher in the severely jaundiced group than control (9% vs 4%; p = 0.04). In conclusion, nt 211 G > A alleles constitute at least 12% of UGT1A1 mutations underlying unconjugated hyperbilirubinemia and appears to be a significant independent risk factor associated with severe neonatal hyperbilirubinemia in the Malay newborns.
Sickle cell disease (SCD) is an inherited red cell disorder, characterized by the tendency of haemoglobin S or sickle haemoglobin to polymerize and assume a characteristic sickle shape. Molecular analysis has been the mainstay of detection method when confirmation is required. Previously a polymerase chain reaction (PCR)-based restriction enzyme analysis was used for this purpose. A simple bidirectional allele-specific amplification, recently described by Waterfall in 2001 was used to detect the GAG --> GTG mutation on codon 6 of the beta globin gene. Two sets of primers for the mutant and the wild type alleles were used in a single PCR reaction to amplify the regions of interest. The resultant PCR products will produce two fragments at 517 and 267 base pair (bp) respectively. This report highlights the investigations for SCD in the family of a 16-year old girl with recurrent painful crisis affecting the lower limbs whereby the family members are asymptomatic for the disease. Her haemoglobin electrophoresis at an alkaline pH showed dense bands at the HbS and HbF regions, while her father and two sisters had bands at HbS, HbF and HbA. The PCR analysis showed that she was homozygous for the mutation by the presence of only one band at 267 bp fragment, while the father and her sisters were heterozygotes, with the presence of two bands at 267 as well as 517 bp fragments. DNA sequencing of the sample confirmed the mutation. In conclusion, this case report highlighted the simple and cheap yet practical method for molecular confirmation of the presence of HbS gene in subjects with homozygous or heterozygous state of the condition.
Argininosuccinic aciduria is an inborn error of the urea cycle caused by deficiency of argininosuccinate lyase (ASL). ASL-deficient patients present with progressive intoxication due to accumulation of ammonia in the body. Early diagnosis and treatment of hyperammonemia are necessary to improve survival and prevent long-term handicap. Two clinical phenotypes have been recognized--neonatal acute and milder late-onset form. We investigated patients with hyperammonemia by a stepwise approach in which quantitative amino acids analysis was the core diagnostic procedure. Here, we describe the clinical phenotypes and biochemical characteristics in diagnosing this group of patients. We have identified 13 patients with argininosuccinic aciduria from 2003 till 2009. Ten patients who presented with acute neonatal hyperammonemic encephalopathy had markedly elevated blood ammonia (> 430 micromol/L) within the first few days of life. Three patients with late-onset disease had more subtle clinical presentations and they developed hyperammonemia only during the acute catabolic state at two to twelve months of age. Their blood ammonia was mild to moderately elevated (> 75-265 micromol/L). The diagnosis was confirmed by detection of excessive levels of argininosuccinate in the urine and/or plasma. They also have moderately increased levels of citrulline and, low levels of arginine and ornithine in their plasma. Two patients succumbed to the disease. To date, eleven patients remained well on a dietary protein restriction, oral ammonia scavenging drugs and arginine supplementation. The majority of them have a reasonable good neurological outcome.
This article describes the homicide pattern in Penang Island, Malaysia over a three-year period (2007-2009). 65 homicide autopsies were performed at the Department of Forensic Medicine, Penang Hospital over the study period. The homicide rates ranged from 0.01 to 0.09/1000 population, the highest being in the Indian ethnic group. The majority (37%) of victims were in the 20-39 years age group. The male: female ratio was 3:1. The majority of deaths were caused by blunt instruments (46%), followed by stab/slash wounds (25%) and asphyxiation (12%). 63% of homicides occurred in areas served by the police stations at Jalan Patani (23.1%), Sg. Nibong (16.9%), Central (12.3%) and Bayan Lepas (10.9%). 56 (86%) victims were brought in dead to the hospital, while 9 (14%) died after admission. Most (39%) incidences occurred in the morning. The methods of homicide were different from Kuala Lumpur, another highly urbanised area of Malaysia.
The GATA3 gene is a potential tumour marker and putative tumour suppressor gene in breast cancer. Its expression is associated with better prognosis and disease free survival in breast cancer patients. We aimed to evaluate GATA3 transcriptome expression and mutation in breast carcinomas and correlate its expression with oestrogen receptor (ER), progesterone receptor (PR), lymph node (LN) status, tumour grade and c-erbB-2 expression. Twenty-two breast infiltrating ductal carcinomas and paired normal tissues were used in Branch DNA assay to detect GATA3 mRNA expression. Normalized data for GATA3 mRNA expression were grouped according to the ER, PR and LN status, tumour grade and c-erbB-2 expression of the tumours. Statistical significance was tested using t-test and ANOVA at 95% confidence interval level. Mutational analysis of GATA3 was performed by direct sequencing of the coding regions of GATA3 mRNA. Our findings showed that GATA3 gene were over-expressed and under-expressed by > 2 fold change in 12 and 4 tested samples, respectively. Eighty per cent of ER positive breast carcinomas were GATA3 positive. There was a statistically significant correlation between GATA3 expression and ER at 95% confidence interval level between the study groups. On the contrary, GATA3 expression was not statistically significant with PR, LN, tumour grade and c-erbB-2 expression in our study. In addition, we observed that there was no mutation in mRNA coding region in 16 breast carcinomas that showed GATA3 differential gene expression. Our preliminary results suggested that GATA3 is linked to the ER. This scenario suggests that GATA3 may play a crucial role in oestrogen receptor positive breast cancer patients. Whether GATA3 expression is involved in regulating tumour cell growth in oestrogen responsive breast cancer is a key question that remains to be answered.
Colorectal carcinogenesis is a complex multistep process that includes changes in histomorphological appearance of the colonic mucosa and changes at molecular level. Aberrant crypt foci (ACF) was first described by Bird in 1987 on examination of methylene-blue-stained colonic mucosa of azoxymethane-treated mice under light microscopy. Since then ACF was considered as the earliest preneoplastic change that can be seen in the colonic mucosa. The aim of this study was to look at the histomorphology and distribution of ACF in colorectal carcinoma. 50 formalin-fixed archival colectomy specimens for colorectal carcinoma were examined under light microscopy after staining with 0.2% methylene blue. ACF was identified by larger and darker crypts with thickened epithelium, and often elevated from adjacent normal mucosa. ACF was found in 41 of 50 colectomy specimens examined. There were 328 ACF consisting of 36 (11.0%) ACF without hyperplasia or dysplasia, 263 (80.2%) ACF with hyperplasia and 29 (8.8%) ACF with dysplasia. Of these 29 ACF with dysplasia, 25 showed low grade dysplasia and four high grade dysplasia. The density of ACF was higher in the left colon, those older than 65 years of age and among males but these findings were statistically not significant. The crypt multiplicity of hyperplastic ACF (30.149, SD 28.395) was larger than dysplastic ACF (20.613, SD 40.128). The spectrum of histological changes observed probably represent the evolution of ACF in colorectal carcinogenesis.
Mitochondrial Subunit ND1 (mtND1) gene is involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). Alteration of the electron transport components by mutations in mtDNA may compromise the normal electron flow. This could lead to an increase of bifurcation and generation of superoxidase radicals and increase oxidative stress in various types of cancer cells. Genomic DNA was extracted from thirty matched primary colorectal tumour tissues and matching non-tumour tissues. Blood samples were obtained from twenty-five normal people. The mtNDI coding region was amplified by step-down PCR. The purified products were then subjected to direct sequencing and subsequently, the DNA sequences obtained were compared with the revised Cambridge Reference Sequence (rCRS) and MITOMAP. From the analysis, the mtND1 gene showed 11 (45.8%) different mutations and also 13 (54.2%) polymorphisms. The heteroplasmic mutation A4123A/G (I273I/V) might have a pathogenic significance as it fulfills various pathogenic criteria. Three mutations, T3394C (Y30H), A3434G (Y43C) and C3497T (A64V) which occur in a highly conserved region were likely to alter the structure and function of the ND1 protein. We suggest that these mutations, and in combination with the polymorphic variance in mtDNA, may cause slight changes that generate subtly higher levels of toxic reactive oxygen species (ROS).
Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs.
The placenta constitutes a physical and immunological barrier against infectious agents. Toll-like receptors (TLRs) are essential components for the induction of innate immunity responses in different human tissues including the placenta. We investigated the expressions of TLR2 and TLR4 in the decidua and amniotic cells in non-inflamed placenta and placenta with infection.
Imatinib, a selective tyrosine kinase inhibitor, is the first line treatment against chronic myelogenous leukaemia (CML) and gastrointestinal stromal tumors (GIST). Several fatal cases have been associated with imatinib hepatotoxicity. Acetaminophen, an over-the-counter analgesic, anti-pyretic drug, which can cause hepatotoxicity, is commonly used in cancer pain management. We assessed renal and hepatic toxicity after imatinib and acetaminophen co-administration in a preclinical model. Four groups of male ICR mice (30-35 g) were fasted overnight and administered either saline solution orally (baseline control), imatinib 100 mg/kg orally (control), acetaminophen 700 mg/kg intraperitoneally (positive control) or co-administered imatinib 100 mg/kg orally and acetaminophen 700 mg/kg intraperitoneally (study group), and sacrificed at 15 min, 30 min, 1 h, 2 h, 4 h and 6 h post-administration (n = 4 per time point). The liver and kidneys were harvested for histopathology assessment. The liver showed reversible cell damage like feathery degeneration, microvesicular fatty change, sinusoidal congestion and pyknosis, when imatinib or acetaminophen were administered separately. The damage increased gradually with time, peaked at 2 h but resolved by 4 h. When both drugs were administered concurrently, the liver showed irreversible damage (cytolysis, karyolysis and karyorrhexis) which did not resolve by 6 h. Very minor renal changes were observed. Acetaminophen and imatinib co-administration increased hepatoxicity which become irreversible, probably due to shared P450 biotransformation pathways and transporters in the liver.
Mucopolysaccharidoses (MPS) are a group of inherited disorders caused by the deficiency of specific lysosomal enzymes involved in glycosaminoglycans (GAGs) degradation. Currently, there are 11 enzyme deficiencies resulting in seven distinct MPS clinical syndromes and their subtypes. Different MPS syndromes cannot be clearly distinguished clinically due to overlapping signs and symptoms. Measurement of GAGs content in urine and separation of GAGs using high-resolution electrophoresis (HRE) are very useful initial screening tests for isotyping of MPS before specific enzyme diagnostics. In this study, we measured total urinary GAGs by a method using dimethylmethylene blue (DMB), and followed by isolation and separation of GAGs using high resolution electrophoresis (HRE) technique. Of 760 urine samples analyzed, 40 have abnormal GAGs HRE patterns. Thirty-five of these 40 cases have elevated urinary GAGs levels as well. These abnormal HRE patterns could be classified into 4 patterns: Pattern A (elevated DS and HS; suggestive of MPS I, II or VII; 16 cases), Pattern B (elevated HS and CS; suggestive of MPS III; 17 cases), and Pattern C (elevated KS and CS; suggestive of MPS IV, 5 cases), and Pattern D (elevated DS; suggestive of MPS VI; 2 cases). Based on the GAGs HRE pattern and a few discriminating clinical signs, we performed selective enzymatic investigation in 16 cases. In all except one case with MPS VII, the enzymatic diagnosis correlated well with the provisional MPS type as suggested by the abnormal HRE pattern. Our results showed that GAGs HRE is a useful, inexpensive and practical first-line screening test when MPS is suspected clinically, and it provides an important guide to further enzymatic studies on a selective basis.
Citrin deficiency is an autosomal recessive disorder caused by mutation in the SLC25AJ3 gene. It has two major phenotypes: adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestatic caused by citrin deficiency (NICCD). NICCD is characterized by neonatal/infantile-onset cholestatic hepatitis syndrome associated with multiple amino acidemia and hypergalactosemia. NICCD is self-limiting in most patients. However, some patients may develop CTLN2 years later, which manifests as fatal hyperammonemia coma. We report three unrelated Malay children with genetically confirmed NICCD characterised by an insertion mutation IVS16ins3kb in SLC25A13 gene. All 3 patients presented with prolonged neonatal jaundice which resolved without specific treatment between 5 to 10 months. Of note was the manifestation of a peculiar dislike of sweet foods and drinks. Elevated plasma citrulline was an important biochemical marker. NICCD should be considered in the differential diagnosis of cholestatic jaundice in Malaysian infants regardless of ethnic origin.
In the past decade, enterovirus 71 (EV71) and chikungunya (CHIK) virus have re-emerged periodically causing serious public health problems in Malaysia, since their first emergence in 1997 and 1998 respectively. This study demonstrates that CHIK virus causes similar patterns of cytopathic effect in cultured Vero cells as some enteroviruses. They also show positive cross-reaction on direct immunofluorescence staining using monoclonal antibodies meant for typing enteroviruses. Without adequate clinical and epidemiological information for correlation, CHIK virus isolated from patients with acute febrile rash can be wrongly reported as untypeable enterovirus due to its cross-reactivity with commercial pan-enterovirus monoclonal antibodies. This is due to the diagnostic laboratory being unaware of such cross-reactions as it has not been reported previously. Final identification of the virus could be determined with specific antibodies or molecular typing using specific oligonucleotide primers for the CHIK virus.
Type IV collagen is the principal component of glomerular basement membrane and messangial matrix. Studies have shown increased levels of urinary type IV collagen (uIV) in diabetic patients compared to healthy controls. The concentration of uIV increases gradually as diabetic nephropathy progresses.
AIM AND METHOD: This study was carried out to determine whether urinary type IV collagen (uIV) can serve as an indicator of diabetic nephropathy. Using a sandwich enzyme immunoassay technique, uIV excretion was determined in 30 type 2 diabetic patients with normoalbuminuria and 20 patients with microalbuminuria.
RESULTS: uIV excretion was significantly increased in type 2 diabetics, in both normoalbuminuric and microalbuminuric patients, compared with healthy controls. The increase in urinary type IV collagen was well correlated with the amount of urinary albumin but not with HbA1C.
CONCLUSION: Our findings that uIV is higher in those with microalbuminuria and correlates with albuminuria, support uIV as an indicator of diabetic nephropathy. Whether the increased uIV excretion would predict the impending renal failure needs further confirmation.