[Serum MiRNA as Predictive and Prognosis Biomarker in Advanced Stage Non-small Cell Lung Cancer in Indonesia].

BACKGROUND: Lung cancer is the most common cause of death in men in the world and in Indonesia where non-small cell carcinoma lung cancer (NSCLC) constitutes 85% of all lung cancer cases. The high mortality rate is due to a poor prognosis and is often diagnosed as having advanced stages. If it is known at the initial stage, the prognosis of lung cancer will be better. Prognosis can be predicted with a marker of prognostic biology, one of which is micro RNA (miRNA). This study aims to prove that serum miRNA can be predictive biological marker and prognosis in NSCLC patients in Indonesia. METHODS: This study was cohort retrospective among 52 subjects in "Dharmais" Hospital National Cancer Center. Sample was obtained from patients' serum. MiR-34, miR-148, miR-155 and miR-222 serum are measured through Real-Time PCR (qPCR). Data were analyzed and interpreted with descriptive analysis, bivariate analysis (Mann Whitney-U for two type of variables or Kruskal-Wallis for more than two type of variables. Kaplan-Meier analysis was used to know association between characteristic which are sociodemographic, performance status, clinico-pathology, and survival rate in miRNA expression. RESULTS: From this study, miRNA expression: miR-34 (46.15%), miR-148 (23.08%), miR-155 (40.38%) and miR-222 (32.69%). Performance status score was statistically significant correlation with miR-148 (P=0.049) and miR-222 (P=0.018). High miR-34 is associated with multiple M1b metastatic type (P=0.020), cancer cell type (adenocarcinoma, P=0.009) and adenocarcinoma epidermal growth factor receptor (EGFR) mutation (negative, P=0.031). There was a significant correlation between the high miR-222 as a poor prognosis in advanced stage NSCLC with M1b metastasis (Median Survival/MS: 27 d, P=0.049) and positive EGFR mutations (MS: 74 d, P=0.049) and correlation of miR-155 with adenocarcinoma (MS: 69 d, P=0.034) and positive EGFR gene mutations (MS: 58 d, P=0.023). CONCLUSIONS: High miR-34 expression in advanced stage NSCLC is the predictive factor for multiple metastatic, adenocarcinoma cell type and adenocarcinoma negative EGFR mutation. High expression of miR-155 and miR-222 are poor prognoses, especially high miR-222 found in metastasis M1b and positive EGFR mutation and miR-155 found in adenocarcinoma and positive EGFR gene mutations. Further studies regarding correlation between miRNA and survival rate are needed.

What information and the extent of information research participants need in informed consent forms: a multi-country survey.

BACKGROUND: The use of lengthy, detailed, and complex informed consent forms (ICFs) is of paramount concern in biomedical research as it may not truly promote the rights and interests of research participants. The extent of information in ICFs has been the subject of debates for decades; however, no clear guidance is given. Thus, the objective of this study was to determine the perspectives of research participants about the type and extent of information they need when they are invited to participate in biomedical research. METHODS: This multi-center, cross-sectional, descriptive survey was conducted at 54 study sites in seven Asia-Pacific countries. A modified Likert-scale questionnaire was used to determine the importance of each element in the ICF among research participants of a biomedical study, with an anchored rating scale from 1 (not important) to 5 (very important). RESULTS: Of the 2484 questionnaires distributed, 2113 (85.1%) were returned. The majority of respondents considered most elements required in the ICF to be 'moderately important' to 'very important' for their decision making (mean score, ranging from 3.58 to 4.47). Major foreseeable risk, direct benefit, and common adverse effects of the intervention were considered to be of most concerned elements in the ICF (mean score = 4.47, 4.47, and 4.45, respectively). CONCLUSIONS: Research participants would like to be informed of the ICF elements required by ethical guidelines and regulations; however, the importance of each element varied, e.g., risk and benefit associated with research participants were considered to be more important than the general nature or technical details of research. Using a participant-oriented approach by providing more details of the participant-interested elements while avoiding unnecessarily lengthy details of other less important elements would enhance the quality of the ICF.

Patient-factors associated with metformin steady-state levels in type 2 diabetes mellitus with therapeutic dosage.

AIMS: This prospective study aimed to analyze metformin steady-state concentration in repeated constant dosage and the influencing patient-factors as well as to correlate them with glycemic control. METHODS: The validated HPLC-UV method was used to examine metformin steady-state concentration, while FBG and glycated albumin were used as the parameters of glycemic control during metformin administration. RESULTS: A total of 82 type-2 diabetes patients were involved with 32.1% of them having metformin Cssmin and 84.1% having Cssmax of metformin within the recommended therapeutic range. One patient had metformin Css that exceeded minimum toxic concentration despite his normal renal function and administered therapeutic dosage of metformin. Higher Cssmax was found in patients with metformin monotherapy, while patients with longer duration of metformin use had significantly higher Cssmin. CONCLUSIONS: Along with initial hyperglycemia and eGFR, metformin Cssmin became the only parameter that influenced FBG level (P < 0.05). Duration of previous metformin use should be considered in the strategy of optimizing metformin dosage. The type-2 diabetes patients with obesity are more suggested to take shorter interval of metformin administration (or possibly with sustained-release formulation) to keep Cssmin within the therapeutic range.

The role of vascular endothelial growth factor -634 G/C and its soluble receptor on chronic liver disease and hepatocellular carcinoma.

BACKGROUND AND STUDY AIMS: The single nucleotide polymorphism (SNP) of the vascular endothelial growth factor (VEGF) gene -634 G/C (rs2010963) influences the progression of hepatocellular carcinoma (HCC). There have been no studies on the role of VEGF SNP -634 G/C in chronic liver disease (CLD). The aim of the present study was to analyse the correlation between VEGF SNP -634 and the clinical severity of CLD and HCC. PATIENTS AND METHODS: A cross sectional study was conducted on 182 subjects (46 HCC, 39 liver cirrhotic/LC, 38 chronic hepatitis/CH; and 57 healthy subjects). The study was conducted from 2010 to 2014 at the Dr. Sardjito Hospital Yogyakarta, Indonesia. All subjects submitted blood serum for DNA sequencing examination using primer. The clinical data of CLD and HCC were assessed, and sVEGFR-2 was examined in 149 subjects. All data were analysed using STATA programme 11.0. RESULTS: Significant differences were observed in genotypic frequency (GG/GC/CC) between HCC, LC, CH and healthy subjects (p=0.004), but though no significant differences were observed between the G>G and C>G genotypic frequencies (p=0.337). The frequency of genotype GG was significantly higher than genotype GC or CC in HCC and was associated with declining of clinical conditions (p<0.05). No significant difference in the distribution genotypes was observed with respect to the level of sVEGFR-2 in the serum. However, we observed a significant correlation between sVEGFR-2 and clinical characteristics in LC and CH (p<0.05). CONCLUSION: Genotype GG of the VEGF SNP -634 is the dominant genotype in severe CLD and HCC. sVEGFR-2 correlates with the disease severity but is not directly associated with the SNP -634 genotype.

Dyslipidemia, insulin resistance and dietary fat intake in obese and normal weight adolescents: the role of uncoupling protein 2 -866G/A gene polymorphism.

Obesity in adolescents has been associated with increased cardiovascular risk factors such as dyslipidemia and insulin resistance. Several factors have been proposed to be associated with cardiovascular risk factors in adolescents including dietary habit, physical activity and genetic. This study was aimed to evaluate the interaction between genetic variation and dietary intake on cardiovascular metabolic risk factors in obese and normal weight adolescents. The UCP2 gene was chosen because it was previously correlated with dietary intake and cardiovascular risk factors. This study is a case control study done in 10 senior high school in Yogyakarta. Subjects were obese and normal weight adolescents taken from an obesity screening with age ranged between 16 and 18 years old. Dyslipidemia was observed by measuring total cholesterol, triglyceride, LDL dan HDL level while insulin resistance was determined by calculating fasting glucose and insulin level. Lipid profile, glucose and insulin level were measured after 8 hours of fasting. UCP2 -866G/A gene polymorphism were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results show that obese adolescents had significantly higher blood pressure, total cholesterol, LDL, triglyceride, insulin level and lower HDL level than their normal weight counterparts (all p<0.001). In obese adolescents, UCP2 -866G/A was associated with blood pressure (p=0.025), total cholesterol level (p=0.025), LDL (p=0.024) level and HOMA IR (p<0.001) but not with dietary fat intake (p=0.386). Additionally, subjects with UCP2 -866G/A gene polymorphism and high dietary fat intake had lower risk on obesity compared to those without UCP2 -866G/A gene polymorphism and low dietary fat intake. We conclude that the UCP2 -866G/A was associated with dyslipidemia, insulin resistance in obese adolescents. Additionally, we also observed the interaction between UCP2 -866G/A gene polymorphism and dietary intake on the risk of obesity.

RET and EDNRB mutation screening in patients with Hirschsprung disease: Functional studies and its implications for genetic counseling.

Hirschsprung disease (HSCR) is a major cause of chronic constipation in children. HSCR can be caused by germline mutations in RET and EDNRB. Defining causality of the mutations identified is difficult and almost exclusively based on in silico predictions. Therefore, the reported frequency of pathogenic mutations might be overestimated. We combined mutation analysis with functional assays to determine the frequencies of proven pathogenic RET and EDNRB mutations in HSCR. We sequenced RET and EDNRB in 57 HSCR patients. The identified RET-coding variants were introduced into RET constructs and these were transfected into HEK293 cells to determine RET phosphorylation and activation via ERK. An exon trap experiment was performed to check a possible splice-site mutation. We identified eight rare RET-coding variants, one possible splice-site variant, but no rare EDNRB variants. Western blotting showed that three coding variants p.(Pr270Leu), p.(Ala756Val) and p.(Tyr1062Cys) resulted in lower activation of RET. Moreover, only two RET variants (p.(Ala756Val) and p.(Tyr1062Cys)) resulted in reduced ERK activation. Splice-site assays on c.1880-11A>G could not confirm its pathogenicity. Our data suggest that indeed almost half of the identified rare variants are proven pathogenic and that, hence, functional studies are essential for proper genetic counseling.

Relationship of angiotensin converting enzyme gene polymorphism and hypertension in Yogyakarta, Indonesia.

AIM: To evaluate the association between ACE gene polymorphism I/D and hypertension in Yogyakarta population. METHODS: This study is a cross-sectional. Sample was taken by random sampling method from hypertensive, prehypertensve and normotensive subjects; from that were obtained 125 subjects, 97 subjects and 108 subjects, consecutively. ACE gene polymorphism I/D was examined by PCR. Genotype was classified as II, ID, or DD based on positive or negative insertion/delation allele. RESULTS: This study shows significant differences of three groups (ages, body mass index (BMI), and family history of hypertension) and total cholesterol level in blood, which tends to have greater value in the hypertension group. Frequency of genotype II, ID, DD are 85 (68%), 39 (31.2%), 1 (0.8%) in hypertension, 66 (61.1%), 38 (35.2%), 4 (3.7%) in normo-tension and 56 (57.7%), 37 (38.1%), 4 (4.1%) in pre-hypertension subject, consecutively. Chi-square analysis shows statistically significant association between ID+DD vs. II genotype and hypertension. Multiple logistic regression analysis shows four variables that significantly influence to hypertension, namely ages, family history of hypertension, BMI, and ACE gene polymorphism. CONCLUSION: ACE ID+DD genotype has significant relationship with hypertension in Melati population, Sleman, Yogyakarta, Indonesia.

Germ-line mutation of KCNQ2, p.R213W, in a Japanese family with benign familial neonatal convulsion.

BACKGROUND: Benign familial neonatal convulsion (BFNC) is an autosomal-dominantly inherited epilepsy of neonates. The KCNQ2 and KCNQ3 genes have been cloned as the responsible genes for BFNC. Detection of mutations in these genes is helpful for confirmation of BFNC or differential diagnosis of convulsive disorders in the neonatal period. METHODS: A Japanese family with BFNC was investigated. Two siblings were clinically diagnosed as having BFNC. KCNQ2 and KCNQ3 were screened for mutations using a combination of polymerase chain reaction and denaturing high-performance liquid chromatography. Nucleotide substitutions were confirmed by direct sequencing. RESULTS: In the affected siblings a C-to-T heterozygous substitution was detected at nucleotide 683 (c.683C>T) in KCNQ2, leading to substitution of arginine with tryptophan at amino acid position 213 (p.R213W) in the S4 voltage-sensing domain of the KCNQ2 protein. The detected mutation may disrupt this highly conserved region among potassium channel proteins. The c.683C>T substitution in KCNQ2 was not present in the parents. KCNQ3 was also analyzed and a single nucleotide polymorphism, c.1241A>G (National Center for Biotechnology Information (NCBI), SNP ID: rs2303995), was detected in the index family. CONCLUSIONS: Two siblings with BFNC had a novel heterozygous missense mutation, p.R213W, in KCNQ2. This mutation may affect potassium gating, leading to neuronal excitability or convulsions in the patients. Furthermore, neither of the parents had the p.R213W mutation, indicating that it was a germ-line mutation. The possibility of recurrence of such a germ-line mutation in the next siblings should be explained during genetic counseling.

Somatic and germline mosaicism for a mutation of the PHEX gene can lead to genetic transmission of X-linked hypophosphatemic rickets that mimics an autosomal dominant trait.

CONTEXT: Familial hypophosphatemic rickets is usually transmitted as an X-linked dominant disorder (XLH), although autosomal dominant forms have also been observed. Genetic studies of these disorders have identified mutations in PHEX and FGF23 as the causes of X-linked dominant disorder and autosomal dominant forms, respectively. OBJECTIVE: The objective of the study was to describe the molecular genetic findings in a family affected by hypophosphatemic rickets with presumed autosomal dominant inheritance. PATIENTS: We studied a family in which the father and the elder of his two daughters, but not the second daughter, were affected by hypophosphatemic rickets. The pedigree interpretation of the family suggested that genetic transmission of the disorder occurred as an autosomal dominant trait. METHODS AND RESULTS: Direct nucleotide sequencing of FGF23 and PHEX revealed that the elder daughter was heterozygous for an R567X mutation in PHEX, rather than FGF23, suggesting that the genetic transmission occurred as an X-linked dominant trait. Unexpectedly, the father was heterozygous for this mutation. Single-nucleotide primer extension and denaturing HPLC analysis of the father using DNA from single hair roots revealed that he was a somatic mosaic for the mutation. Haplotype analysis confirmed that the father transmitted the genotypes for 18 markers on the X chromosome equally to his two daughters. The fact that the father transmitted the mutation to only one of his two daughters indicated that he was a germline mosaic for the mutation. CONCLUSIONS: Somatic and germline mosaicism for an X-linked dominant mutation in PHEX may mimic autosomal dominant inheritance.

Screening for G71R mutation of the UGT1A1 gene in the Javanese-Indonesian and Malay-Malaysian populations.

BACKGROUND: 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. METHODS: One hundred and thirty-six subjects were enrolled in this study: 68 Javanese-Indonesian adults and 68 Malay-Malaysian newborns (32 with jaundice and 36 without jaundice). Denaturing high-performance liquid chromatography (DHPLC) was used to screen for the G71R mutation, and the results were confirmed by nucleotide sequencing analysis. RESULTS: With DHPLC, the authors easily and clearly detected seven subjects carrying the G71R mutation: two Javanese-Indonesian adults and five Malay-Malaysian newborns. In the 68 Javanese-Indonesian adults, the genotype distribution for G71R mutation was 66 G/G, two G/R and no R/R genotypes, and the mutated allele frequency was 0.015. In the 68 Malay-Malaysian newborns, genotype distribution for the mutation was 63 G/G, five G/R and no R/R genotypes, and the mutated allele frequency was 0.037. The genotype distributions did not differ significantly between the newborns with jaundice and those without jaundice. CONCLUSION: The G71R mutation is present, but very rare, in Javanese-Indonesians and Malay-Malaysians. Thus, G71R mutation may not contribute to the high incidence of the neonatal jaundice in South-east Asian populations. DHPLC analysis is a very useful method for detecting the G71R mutation.