Chupong Ittiwut

Hypomethylation of Intragenic LINE-1 Represses Transcription in Cancer Cells through AGO2

In human cancers, the methylation of long interspersed nuclear element -1 (LINE-1 or L1) retrotransposons is reduced. This occurs within the context of genome wide hypomethylation, and although it is common, its role is poorly understood. L1s are widely distributed both inside and outside of genes, intragenic and intergenic, respectively. Interestingly, the insertion of active full-length L1 sequences into host gene introns disrupts gene expression. Here, we evaluated if intragenic L1 hypomethylation influences their host gene expression in cancer. First, we extracted data from L1base (http://l1base.molgen.mpg.de), a database containing putatively active L1 insertions, and compared intragenic and intergenic L1 characters. We found that intragenic L1 sequences have been conserved across evolutionary time with respect to transcriptional activity and CpG dinucleotide sites for mammalian DNA methylation. Then, we compared regulated mRNA levels of cells from two different experiments available from Gene Expression Omnibus (GEO), a database repository of high throughput gene expression data, (http://www.ncbi.nlm.nih.gov/geo) by chi-square. The odds ratio of down-regulated genes between demethylated normal bronchial epithelium and lung cancer was high (p<1E−27; OR = 3.14; 95% CI = 2.54–3.88), suggesting cancer genome wide hypomethylation down-regulating gene expression. Comprehensive analysis between L1 locations and gene expression showed that expression of genes containing L1s had a significantly higher likelihood to be repressed in cancer and hypomethylated normal cells. In contrast, many mRNAs derived from genes containing L1s are elevated in Argonaute 2 (AGO2 or EIF2C2)-depleted cells. Hypomethylated L1s increase L1 mRNA levels. Finally, we found that AGO2 targets intronic L1 pre-mRNA complexes and represses cancer genes. These findings represent one of the mechanisms of cancer genome wide hypomethylation altering gene expression. Hypomethylated intragenic L1s are a nuclear siRNA mediated cis-regulatory element that can repress genes. This epigenetic regulation of retrotransposons likely influences many aspects of genomic biology.

Maternal 677CT/1298AC genotype of the MTHFR gene as a risk factor for cleft lip

Non-syndromic cleft lip with or without cleft palate (CL/P) is one of the most common congenital anomalies world wide. It has a prevalence of approximately 1/1000 among white populations1 and 1/600 among Thai newborns.2 Environmental and genetic factors have been implicated in CL/P and several different loci and genes have been associated with them.3 Maternal folic acid supplementation during early pregnancy may reduce the risk for oral clefts,4,5 but this is controversial.6 One of the mechanisms by which low folate levels predispose some subjects to oral clefts could be the presence of polymorphisms in the genes encoding enzymes of the folate pathway, such as 5,10-methylenetetrahydrofolate reductase ( MTHFR , MIM 236250). MTHFR catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and the carbon donor for the remethylation of homocysteine to methionine. Two polymorphisms, 677C>T and 1298A>C, in the MTHFR gene have been shown to have reduced MTHFR activity.7,8 The 677C>T transition, producing an alanine to valine amino acid substitution within the catalytic domain of the MTHFR enzyme,7 has been associated with many disorders and conditions including neural tube defects,9 vascular disease,7 migraine,10 smoking behaviour,11 and oral clefts.12,13 However, the last is controversial.14 Recent studies reported an association between the maternal polymorphism and the anomalies,15,16 but this again is not a consistent finding.17,18 No studies have investigated 1298A>C, the second most common polymorphism in MTHFR resulting in a glutamate to alanine substitution, in CL/P patients and their parents. We therefore carried out a case-control study to determine whether the two MTHFR polymorphisms in Thai patients with CL/P or their parents were associated with an increased risk of the anomaly. The study sample consisted of 109 CL/P patients, …

NUDT15 c.415C>T increases risk of 6-mercaptopurine induced myelosuppression during maintenance therapy in children with acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children[1][1]. Prolongation of therapy by incorporating a maintenance phase, containing 6-mercaptopurine (6-MP) as the backbone, has improved treatment outcomes in pediatric ALL[2][2]–[4][3]. However, 6-MP can cause

Targeted therapies for rare gynaecological cancers.

Some gynaecological cancers are uncommon, such as sex cord-stromal tumours, malignant germ-cell tumours, vulvar carcinoma, melanoma of the female genital tract, clear-cell carcinoma of the ovary and endometrium, neuroendocrine tumours of the cervix, and gestational trophoblastic neoplasia. All these cancers have different clinicopathological characteristics, suggesting different molecular biological pathogeneses. Despite aggressive treatment, some cancers recur or respond poorly to therapy. Comprehensive knowledge of the molecular biology of each cancer might help with development of novel treatments that maximise efficacy and minimise toxic effects. Targeted therapy is a new treatment strategy that has been investigated in various tumours in clinical and laboratory settings. Since these cancers are rare and large clinical trials are difficult to do, molecular biological techniques might allow rapid proof-of-principle experiments in few patients. Novel targeted agents either alone or in combination with other treatments offer promising therapeutic options.

Massive parallel sequencing as a new diagnostic approach for phenylketonuria and tetrahydrobiopterin-deficiency in Thailand

BackgroundHyperphenylalaninemia (HPA) can be classified into phenylketonuria (PKU) which is caused by mutations in the phenylalanine hydroxylase (PAH) gene, and BH4 deficiency caused by alterations in genes involved in tetrahydrobiopterin (BH4) biosynthesis pathway. Dietary restriction of phenylalanine is considered to be the main treatment of PKU to prevent irreversible intellectual disability. However, the same dietary intervention in BH4 deficiency patients is not as effective, as BH4 is also a cofactor in many neurotransmitter syntheses.MethodWe utilized next generation sequencing (NGS) technique to investigate four unrelated Thai patients with hyperphenylalaninemia.ResultWe successfully identified all eight mutant alleles in PKU or BH4-deficiency associated genes including three novel mutations, one in PAH and two in PTS, thus giving a definite diagnosis to these patients. Appropriate management can then be provided.ConclusionThis study identified three novel mutations in either the PAH or PTS gene and supported the use of NGS as an alternative molecular genetic approach for definite diagnosis of hyperphenylalaninemia, thus leading to proper management of these patients in Thailand.

In vitro Correction of a Novel Splicing Alteration in the BTK Gene by Using Antisense Morpholino Oligonucleotides

A novel sequence variant, c.240+109C>A, in the Bruton’s tyrosine kinase (BTK) gene was identified in a patient with X-linked agammaglobulinemia. This alteration resulted in an incorporation of 106 nucleotides of BTK intron 3 into its mRNA. Administration of the 25-mer antisense morpholino oligonucleotide analog in the patient’s cultured peripheral blood mononuclear cells was able to restore correctly spliced BTK mRNA, a potential treatment for X-linked agammaglobulinemia.

A novel PITX2 mutation in non-syndromic oro-dental anomalies

OBJECTIVE To identify orodental characteristics and genetic aetiology of a family affected with non-syndromic orodental anomalies. SUBJECTS AND METHODS Physical and oral features were characterised. DNA was collected from an affected Thai family. Whole-exome sequencing was employed to identify the pathogenic variants associated with inherited orodental anomalies. The presence of the identified mutation was confirmed by Sanger sequencing. RESULTS We observed unique orodental manifestations including oligodontia, retained primary teeth, taurodont molars, peg-shaped maxillary central incisors, high attached frenum with nodule and midline diastema in the proband and her mother. Mutation analyses revealed a novel heterozygous frameshift deletion, c.573_574delCA, p.L193QfsX5, in exon 5 of PITX2A in affected family members. The amino acid alterations, localised in the transcriptional activation domain 2 in the C-terminus of PITX2, were evolutionarily conserved. Mutations in PITX2 have been associated with autosomal-dominant Axenfeld-Rieger syndrome and non-syndromic eye abnormalities, but never been found to cause isolated oral anomalies. CONCLUSIONS This study for the first time demonstrates that the PITX2 mutation could lead to non-syndromic orodental anomalies in humans. We propose that the specific location in the C-terminal domain of PITX2 is exclusively necessary for tooth development.

The phenotypic and mutational spectrum of Thai female patients with ornithine transcarbamylase deficiency

Ornithine transcarbamylase deficiency (OTCD) is an X-linked urea cycle disorder affecting both males and females. Hemizygous males commonly present with severe hyperammonemic encephalopathy during the neonatal period. Heterozygous females have great phenotypic variability. The majority of female patients can manifest later in life or have unrecognized symptoms, making the diagnosis of OTCD in females very challenging. Here we report on three unrelated Thai female cases with OTCD presenting with different manifestations including aggressive behavior, acute liver failure and severe encephalopathy. Whole exome sequencing successfully identified disease-causing mutations in all three cases including two novel ones: the c.209_210delAA (p.Lys70Argfs*17) and the c.850T>A (p.Tyr284Asn). This study affirms variable symptoms in female patients with OTCD and emphasizes the importance of early recognition and prompt management for favorable outcomes. In addition, identification of two novel causative variants expands the genotypic spectrum of OTC.

Novel mutations in SPTA1 and SPTB identified by whole exome sequencing in eight Thai families with hereditary pyropoikilocytosis presenting with severe fetal and neonatal anaemia

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Novel mutations of the SRD5A2 and AR genes in Thai patients with 46, XY disorders of sex development.

Abstract Background: Abnormalities of dihydrotestosterone conversion [5α-reductase deficiency: online Mendelian inheritance in man (OMIM) 607306] or actions of androgens [partial androgen insensitivity syndrome (PAIS): OMIM 312300] during the 8th–12th weeks of gestation cause varying degrees of undervirilized external genitalia in 46, XY disorders of sex development (DSD) with increased testosterone production. The objective of the study was to determine clinical and genetic characteristics of Thai patients with 46, XY DSD. Methods: A cross-sectional study was conducted in 46, XY DSD with increased testosterone production (n=43) evaluated by a human chorionic gonadotropin (hCG) stimulation test or clinical features consistent with 5α-reductase deficiency or PAIS. PCR sequencing of the entire coding regions of the SRD5A2 and AR genes was performed. Molecular modeling analysis of the androgen receptor-ligand-binding domain (AR-LBD) of a novel mutation was constructed. Results: Mutations were found in seven patients (16.3%): five (11.6%) and two (4.7%) patients had mutations in SRD5A2 and AR, respectively. Two novel mutations, SRD5A2 c.383A>G (p.Y128C) and AR c.2176C>T (p.R726C), were identified. Dimensional structural analysis of the novel mutated AR (p.R726C) revealed that it affected the co-activator binding [binding function-3 (BF-3)], not the testosterone binding site. Short phallus length was associated with 5α-reductase deficiency. Conclusions: Around 16.3% of our patients with 46, XY DSD had 5α-reductase deficiency or PAIS. Two novel mutations of SRD5A2 and AR were identified. The novel mutated AR (p.R726C) might affect the co-activator binding (BF-3), not the testosterone binding site.