Pattamawadee Yanatatsaneejit

Electrochemical detection of human papillomavirus DNA type 16 using a pyrrolidinyl peptide nucleic acid probe immobilized on screen-printed carbon electrodes

An electrochemical biosensor based on an immobilized anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe was successfully developed for the selective detection of human papillomavirus (HPV) type 16 DNA. A 14-mer acpcPNA capture probe was designed to recognize a specific 14 nucleotide region of HPV type 16 L1 gene. The redox-active label anthraquinone (AQ) was covalently attached to the N-terminus of the acpcPNA probe through an amide bond. The probe was immobilized onto a chitosan-modified disposable screen-printed carbon electrode via a C-terminal lysine residue using glutaraldehyde as a cross-linking agent. Hybridization with the target DNA was studied by measuring the electrochemical signal response of the AQ label using square-wave voltammetric analysis. The calibration curve exhibited a linear range between 0.02 and 12.0 µM with a limit of detection and limit of quantitation of 4 and 14 nM, respectively. This DNA sensing platform was successfully applied to detect the HPV type 16 DNA from a PCR amplified (240 bp fragment of the L1 gene) sample derived from the HPV type 16 positive human cancer cell line (SiHa), and failed to detect the HPV-negative c33a cell line. The sensor probe exhibited very high selectivity for the complementary 14 base oligonucleotide over the non-complementary oligonucleotides with sequences derived from HPV types 18, 31 and 33. The proposed sensor provides an inexpensive tool for the early stage detection of HPV type 16, which is an important biomarker for cervical cancer.

TM4SF20 Ancestral Deletion and Susceptibility to a Pediatric Disorder of Early Language Delay and Cerebral White Matter Hyperintensities

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ~70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles.

Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation

Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV‐associated cancer. CCNA1 methylation is found in HPV‐associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV‐positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA‐transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7‐overexpressing cells. To confirm whether the binding of the E7–Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7‐, del CR3‐E7 and vector control‐overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome‐wide mechanism of E7 oncoprotein‐mediated DNA methylation.

Human papillomavirus's physical state and cyclin A1 promoter methylation in cervical cancer.

Background: Cervical cancer is the second biggest cause of death among human female cancers. Human papillomavirus (HPV) is the main factor in this cancer, especially HPV types 16 and 18, which constitute the high-risk group. There are 2 physical states of HPV in host cells: integrated and episomal forms. Our previous study explored the very high degree of cyclin A1 (CCNA1) promoter methylation in invasive cervical cancer in which all cases were infected by HPV. Objective: From previous evidence, it seemed that HPV might affect CCNA1 promoter methylation. Therefore, both the quantity and physical state of HPV were investigated in this study for their effects on CCNA1 promoter methylation. Materials and Methods: To determine the correlation of HPV quantity and CCNA1 methylation, the proportion of HPV L1/HAT (histone acetyltransferase, which is a human housekeeping gene) and the percentage intensity of CCNA1 promoter methylation were observed. CCNA1 promoter methylation was detected by methylation-specific primer polymerase chain reaction. To investigate the physical state, the HPV E2 region was amplified. The effect of the physical state on CCNA1 methylation was observed. Results: No correlation was found between the quantity of HPV and CCNA1 promoter methylation. Interestingly, the physical state of HPV had the potential to affect methylation of this gene. The integrated form of HPV had a significantly higher impact on CCNA1 methylation than HPV in episomal form (P = 0.001; 95% confidence interval, 11.96-38.44). Conclusion: We suggest that the integrated form of HPV might lead to CCNA1 promoter methylation in cervical cancer by some mechanisms.

Association of P53 codon 72 polymorphism and ameloblastoma.

OBJECTIVE To test the hypothesis that P53 codon 72 polymorphism was associated with an increased risk of developing ameloblastoma in the Thai population. MATERIALS AND METHODS Seventy-eight ameloblastomas and 94 healthy controls were genotyped for the P53 codon 72 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS The frequencies of the Arg/Arg, Arg/Pro and Pro/Pro genotypes were 28.72%, 50.00% and 21.28%, respectively, in the controls; and 44.87%, 51.28% and 3.85%, respectively, in ameloblastomas. Therefore, P53 Arg is an ameloblastoma-susceptible allele [OR (95% CI) = 2.06 (1.28-3.31), P = 0.002]. Sex-adjusted OR (95% CI) is 2.08 (1.29-3.34), P = 0.002; and adjusted OR by clinical type (95% CI) is 2.04 (1.34-3.13), P < 10(-3). Therefore, the increased risk associated with P53 Arg may not be influenced by either the sex of patients or clinical characteristics of the tumours. Moreover, when compared with homozygous P53 Pro, people who carried the Arg allele had a remarkably high risk of developing ameloblastoma [adjusted OR (95% CI) = 7.26 (2.34-23.41), P < 10(-3)]. CONCLUSION The Arg allele of P53 gene codon 72 may increase susceptibility, and P53 may be important in the aetiology of ameloblastoma.

Maternal uniparental disomy of chromosome 16 resulting in hemoglobin Bart's hydrops fetalis.

To the Editor: Since the first cases of maternal uniparental disomy for chromosome 16 or UPD(16)mat was described by Kalousek et al. in 1993, a handful of cases have been diagnosed in newborn infants and terminated fetuses (1–4). Most cases are associated with confined placental mosaicism, with high incidence of adverse pregnancy outcome (4, 5). The most common findings include intrauterine growth retardation, single umbilical artery, imperforate anus, and pulmonary and cardiac malformations (1, 2, 4, 6, 7). Also, UPD(16)mat individual with normal phenotypes has been reported (8). The only confirmed case with UPD(16)pat was isodisomic and identified through pre-natal detection for abnormal maternal serum screening; eventually, the child was quite normal (9). Herein, we describe a hemoglobin (Hb) Bart’s hydroptic fetus and fetal malformations caused by UPD(16)mat. The counselee is a 34-year-old G1P0 pregnant Thai woman affected with Hb H disease caused by heterozygous Southeast Asian (–SEA) and the 3.7-kb deletions of the alpha1 and 2 globin genes, respectively. Her husband is Hb E heterozygote. Ultrasound performed at a gestation of 23 weeks showed a hydroptic fetus. Fetal blood from cordocentesis showed Hb Bart’s hydrops fetalis with –SEA globin1 gene deletion but not the other types of alpha1 globin gene mutations, suggesting the following possibilities: non-paternity, another rare but as yet unidentified alphathalassemia 1 mutation inherited from the father, or UPD(16)mat. Fetal autopsy showed characteristic features of Hb Bart’s hydrops including interstitial edema of skin, cardiomegaly, and hepatosplenomegaly with extramedullary hemopoiesis. Single umbilical artery, bilobed right lung and ileal Meckel’s diverticulum were found. Retrospectively, a two-vessel cord was noted on pre-natal ultrasonogram. Peripheral blood culture for fetal karyotype analysis was unsuccessful. It is unlikely that the husband has an unidentified alpha-thalassemia 1 gene deletion, given normal hematocrit, mildly small red cell size, and percent Hb E as expected in a heterozygous Hb E individual. Normally, heterozygote for Hb E with co-inheritance of alpha-thalassemia 1 will

XRCC1 gene polymorphisms and risk of ameloblastoma

OBJECTIVE Ameloblastoma is a common benign odontogenic tumour with inherently aggressive behaviour. Genetic susceptibility of single nucleotide polymorphism (SNP) can likely predict ameloblastoma at risk patients but this data remains limited. Here, we studied XRCC1 polymorphism as a risk factor for ameloblastoma. DESIGN Eighty-two ameloblastoma samples and blood from 140 healthy controls were used to perform polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for XRCC1 at codons 194, 280 and 399, and confirmed by sequence analysis. RESULTS Compare to healthy control, a significant increase was noted in the occurrence of polymorphism at codon 194 and 399 in ameloblastoma patients. At codon 194, tryptophan encoded by T, was the susceptibility allele showed an ODD ratio of (95% CI)=1.62 (1.05-2.48), p=0.027. At codon 399, glycine encoded by A was the susceptibility allele showing ODD ratio of (95% CI)=1.83 (1.19-2.84), p=0.005. Moreover at codon 399, we found AG as the susceptibility genotype (2.06 (1.14-3.72), p=0.015). However, we did not find any significant increase in polymorphic occurrence in ameloblastoma patients at codon 280. For haplotype analysis of 3 codons, we found GGC as protective haplotype, and AGT as the risk haplotype. CONCLUSION Our data suggest that polymorphism at codons 194 and 399, likely contributes to the risk of developing ameloblastoma.

P53 polymorphism at codon 72 is associated with keratocystic odontogenic tumors in the Thai population.

OBJECTIVE To clarify the association between the p53 polymorphism at codon 72 and susceptibility to the sporadic keratocystic odontogenic tumor (KCOT). DESIGN One hundred KCOTs and 160 match-group healthy controls were genotyped to ascertain the frequency of the p53 codon 72 polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), confirmed by direct sequencing. RESULTS The frequencies of the Pro/Pro, Arg/Pro, and Arg/Arg genotypes were 23.8%, 49.4%, and 26.9%, respectively, in the controls, while the KCOT cohort demonstrated 43.0%, 39.0%, and 18.0%, respectively. Further analysis suggested that p53 Pro could be a KCOT-susceptible allele (OR (95%CI)=1.77 (1.22 to 2.59), p=0.0024), with a sex-adjusted OR (95%CI) of 1.71 (1.17-2.50), p=0.0046. Moreover, the results indicated that p53 codon 72 Pro homozygous was associated with a two-fold risk of developing KCOT (adjusted OR (95%CI) =2.17(1.23-3.84), p=0.0062). CONCLUSIONS The C/C genotype of P53 gene codon 72 increases the risk of developing sporadic KCOT and may be a useful tool for screening and diagnostic purposes.

Combined Bisulfite Restriction Analysis for Brain Tissue Identification

According to the tissue-specific methylation database (doi: 10.1016/j.gene.2014.09.060), methylation at CpG locus cg03096975 in EML2 has been preliminarily proven to be specific to brain tissue. In this study, we enlarged sample size and developed a technique for identifying brain tissue in aged samples. Combined Bisulfite Restriction Analysis-for EML2 (COBRA-EML2) technique was established and validated in various organ samples obtained from 108 autopsies. In addition, this technique was also tested for its reliability, minimal DNA concentration detected, and use in aged samples and in samples obtained from specific brain compartments and spinal cord. COBRA-EML2 displayed 100% sensitivity and specificity for distinguishing brain tissue from other tissues, showed high reliability, was capable of detecting minimal DNA concentration (0.015ng/μl), could be used for identifying brain tissue in aged samples. In summary, COBRA-EML2 is a technique to identify brain tissue. This analysis is useful in criminal cases since it can identify the vital organ tissues from small samples acquired from criminal scenes. The results from this analysis can be counted as a medical and forensic marker supporting criminal investigations, and as one of the evidences in court rulings.

VDR and TNFSF11 polymorphisms are associated with osteoporosis in Thai patients

Determining molecular markers for osteoporosis may be valuable for improving the quality of life of affected elderly patients by aiding in early detection and disease management. In the present study, the association between single nucleotide polymorphisms (SNPs) of the vitamin D receptor (VDR) and tumour necrosis factor superfamily number 11 (TNFSF11) genes and the susceptibility of developing osteoporosis was investigated in a Thai female cohort. The study group consisted of 105 Thai postmenopausal patients diagnosed with osteoporosis and 132 healthy Thai postmenopausal female volunteers. DNA extracted from blood samples was used to genotype the VDR and TNFSF11 genes using polymerase chain reaction-restriction fragment length polymorphism and sequencing analysis. For VDR, the frequencies of the genotypes TT, CT and CC for the TaqI SNP (rs731236) were 87.88, 11.36 and 0.76%, respectively, in the control group, while in the osteoporosis cohort were 92.38, 5.71 and 1.91%, respectively. For the FokI SNP (rs2228570), the frequencies of the genotypes CC, CT and TT were 31.06, 55.30 and 13.64%, respectively, in the control group, and in the osteoporosis group were 29.52, 43.81 and 26.67%, respectively. For BsmI SNP (rs1544410), the frequencies of the genotypes GG, GA and AA were 78.03, 18.94 and 3.03%, respectively, in control group, and in the osteoporosis group were 80.95, 18.10 and 0.95%, respectively. The significant risk of osteoporosis associated with the FokI SNP was determined. The odds ratio (95% confidence interval) was 2.30 (1.14-4.69; P=0.01) among patients with osteoporosis with TT as the susceptibility genotype. For TNFSF11, the frequencies of the genotypes TT, CT and CC for the -290C>T SNP (rs9525641) in the control group were 36.36, 50.76 and 12.88%, respectively, while in the osteoporosis group were 31.43, 56.19 and 12.38%, respectively. For the -643C>T SNP (rs9533156), the frequencies of the genotypes TT, CT and CC in the control group were 35.61, 48.48 and 15.91%, respectively, while in the osteoporosis group were 32.38, 55.24 and 12.38%, respectively. For the -693G>C SNP (rs9533155), the frequencies of the genotypes CC, CG, and GG in the control group were 39.39, 46.97 and 13.64%, respectively, and in the osteoporosis group were 36.19, 53.33 and 10.48%, respectively. No significant associations of the TNFSF11 SNPs with osteoporosis were determined; however, it was notable that the GCT haplotype of TNFSF11 may be a protective haplotype for osteoporosis. Therefore, it was concluded that the SNP FokI of VDR may be a potential molecular biomarker for the development of osteoporosis in Thai females.