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Abstract Background Arachidonic acid (AA) is a long-chain omega-6 polyunsaturated fatty acid (PUFA) synthesized from the precursor dihomo-gamma-linolenic acid (DGLA) that plays a vital role in immunity and inflammation. Variants in the Fatty Acid Desaturase (FADS) family of genes on chromosome 11q have been shown to play a role in PUFA metabolism in populations of European and Asian ancestry; no work has been done in populations of African ancestry to date. Results In this study, we report that African Americans have significantly higher circulating levels of plasma AA (p = 1.35 × 10-48) and lower DGLA levels (p = 9.80 × 10-11) than European Americans. Tests for association in N = 329 individuals across 80 nucleotide polymorphisms (SNPs) in the Fatty Acid Desaturase (FADS) locus revealed significant association with AA, DGLA and the AA/DGLA ratio, a measure of enzymatic efficiency, in both racial groups (peak signal p = 2.85 × 10-16 in African Americans, 2.68 × 10-23 in European Americans). Ancestry-related differences were observed at an upstream marker previously associated with AA levels (rs174537), wherein, 79-82% of African Americans carry two copies of the G allele compared to only 42-45% of European Americans. Importantly, the allelic effect of the G allele, which is associated with enhanced conversion of DGLA to AA, on enzymatic efficiency was similar in both groups. Conclusions We conclude that the impact of FADS genetic variants on PUFA metabolism, specifically AA levels, is likely more pronounced in African Americans due to the larger proportion of individuals carrying the genotype associated with increased FADS1 enzymatic conversion of DGLA to AA.

Wilson's disease (WD) is a rare metabolic disorder caused by variations in the ATP7B gene. It usually manifests hepatic, neurologic, and psychiatric symptoms due to excessive copper accumulation. The prevalence of WD and its common variants differ across populations. This study aimed to examine these aspects of WD within the Thai population, where information has been limited. We reviewed ClinVar and the Wilson Disease Mutation Database, organizing variants classified as pathogenic or likely pathogenic in one or both databases as "relaxed" and "strict" lists. Allele frequencies were estimated from genotyping array data (Asian Screening Array: ASA; Illumina Corp, CA) of 6291 Thai subjects, which also underwent genotype imputation. The prevalence of WD in the Thai population was estimated assuming Hardy-Weinberg Equilibrium. The strict list yielded a prevalence of 1/24,128 (carrier frequency=1/78), while the relaxed list yielded a prevalence of 1/9971 (carrier frequency=1/50). The most common WD variants in Thai subjects were c.2333 G > T, c.3443 T > C, and c.813 C > A from the strict list, and c.3316 G > A and c.2605 G > A from the relaxed list. The ASA chip covered approximately 59 and 24% of WD variants from the strict and relaxed lists, respectively. Based on the estimated prevalence, a carrier screening program for WD is not currently required in Thailand. However, as genotyping services become more affordable and accessible, such a program would facilitate early identification, treatment, and prevention of WD., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)

Intrahepatic cholangiocarcinoma (ICC) is a lethal cancer with poor survival especially when it spreads. The histopathology of its rare intraductal papillary neoplasm of the bile duct type (IPNB) characteristically shows cancer cells originating within the confined bile duct space. These cells eventually invade and infiltrate the nearby liver tissues, making it a good model to study the mechanism of local invasion, which is the earliest step of metastasis. To discover potential suppressor genes of local invasion in ICC, we analyzed the somatic mutation profiles and performed clonal evolution analyses of the 11 pairs of macrodissected locally invasive IPNB tissues (LI-IPNB) and IPNB tissues without local invasion from the same patients. We identified a protein-truncating variant in an E3 ubiquitin ligase, RNF213 (c.6967C>T; p.Gln2323X; chr17: 78,319,102 [hg19], exon 29), as the most common protein-truncating variant event in LI-IPNB samples (4/11 patients). Knockdown of RNF213 in HuCCT1 and YSCCC cells showed increased migration and invasion, and reduced vasculogenic mimicry but maintained normal proliferation. Transcriptomic analysis of the RNF213-knockdown vs control cells was then performed in the HuCCT1, YSCCC, and KKU-100 cells. Gene ontology enrichment analysis of the common differentially expressed genes revealed significantly altered cytokine and oxidoreductase-oxidizing metal ion activities, as confirmed by Western blotting. Gene Set Enrichment Analysis identified the most enriched pathways being oxidative phosphorylation, fatty acid metabolism, reactive oxygen species, adipogenesis, and angiogenesis. In sum, loss-of-function mutation of RNF213 is a common genetic alteration in LI-IPNB tissues. RNF213 knockdown leads to increased migration and invasion of ICC cells, potentially through malfunctions of the pathways related to inflammation and energy metabolisms., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The study of non-communicable diseases (NCDs) in a developing country like Thailand has rarely been conducted in long-term cohorts, especially among the working-age population. We aim to assess the prevalence and incidence of risk factors and their associations underlying NCDs, especially type-2 diabetes mellitus (T2DM) among healthcare workers enrolled in the Siriraj Health (SIH) study cohort., Methods: The SIH study was designed as a longitudinal cohort and conducted at Siriraj hospital, Thailand. A total of 5,011 participants (77% women) were recruited and follow-up. Physical examinations, blood biochemical analyses, family history assessments, behavior evaluations, and genetics factors were assessed., Results: The average age was 35.44±8.24 years and 51% of participants were overweight and obese. We observed that men were more likely to have a prevalence of T2DM and dyslipidemia (DLP) compared to women. Aging was significantly associated with pre-diabetes and T2DM (P<0.001). Additionally, aging, metabolic syndrome, and elevated triglycerides were associated with the development of pre-diabetes and T2DM. The minor T allele of the rs7903146(C/T) and rs4506565 (A/T) were associated with a high risk of developing pre-diabetes with odds ratios of 2.74 (95% confidence interval [CI]: 0.32-23.3) and 2.71 (95% CI: 0.32-23.07), respectively; however, these associations were statistically insignificant (P>0.05)., Conclusion: The findings of the SIH study provide a comprehensive understanding of the health status, risk factors, and genetic factors related to T2DM in a specific working population and highlight areas for further research and intervention to address the growing burden of T2DM and NCDs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Mongkolsucharitkul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Summary: The revised WHO guidelines for classifying and grading brain tumors include several copy number variation (CNV) markers. The turnaround time for detecting CNVs and alterations throughout the entire genome is drastically reduced with the customized read incremental approach on the nanopore platform. However, this approach is challenging for non-bioinformaticians due to the need to use multiple software tools, extract CNV markers and interpret results, which creates barriers due to the time and specialized resources that are necessary. To address this problem and help clinicians classify and grade brain tumors, we developed GLIMMERS: glioma molecular markers exploration using long-read sequencing, an open-access tool that automatically analyzes nanopore-based CNV data and generates simplified reports., Availability and Implementation: GLIMMERS is available at https://gitlab.com/silol&#95;public/glimmers under the terms of the MIT license., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH-mutant gliomas. Here, we demonstrated the use of a nanopore-based copy-number variation sequencing (nCNV-seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV-seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH-mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole-arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV-seq, FISH, DNA methylation profiling, and whole-genome sequencing. For the CDKN2A/B deletion, nCNV-seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole-genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10 -16 to r = 0.99, P < 2.2 × 10 -16 ) and methylome profiling. Furthermore, nCNV-seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV-seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH-mutant gliomas without capital expenditure for a sequencer., (© 2023 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Objectives: Clinical manifestations of patients with Hemoglobin E/beta-thalassemia vary from mild to severe phenotypes despite exhibiting the same genotype. Studies have partially identified genetic modifiers. We aimed to study the association between rare variants in protein-coding regions and clinical severity in Thai patients., Methods: From April to November 2018, a case-control study was conducted based on clinical information and DNA samples collected from Thai patients with hemoglobin E/beta-thalassemia over the age of four years. Cases were patients with severe symptoms, while patients with mild symptoms acted as controls. Whole exome sequencing and rare variant association study were used to analyze the data., Results: All 338 unrelated patients were classified into 165 severe and 173 mild cases. Genotypes comprised 81.4% of hemoglobin E/beta-thalassemia, 2.7% of homozygous or compound heterozygous beta-thalassemia, and 0.3% of (δβ) 0 thalassemia Hb E while 15.7% of samples were not classified as beta-thalassemia. A novel cis heterozygotes of IVS I-7 (A > T) and codon 26 (G > A) was identified. Six genes ( COL4A3 , DLK1 , FAM186A , PZP , THPO , and TRIM51 ) showed the strongest associations with severity (observed p -values of <0.05; significance lost after correction for multiplicity). Among known modifiers, KLF1 variants were found in four mild patients and one severe patient., Conclusion: No rare variants were identified as contributors to the clinical heterogeneity of hemoglobin E/beta-thalassemia. KLF1 mutations are potential genetic modifiers. Studies to identify genetic factors are still important and helpful for predicting severity and developing targeted therapy.

Protein-energy malnutrition still impacts children's growth and development. We investigated the prolonged effects of egg supplementation on growth and microbiota in primary school children. For this study, 8-14-year-old students (51.5% F) in six rural schools in Thailand were randomly assigned into three groups: (1) whole egg (WE), consuming 10 additional eggs/week ( n = 238) ( n = 238); (2) protein substitute (PS), consuming yolk-free egg substitutes equivalent to 10 eggs/week ( n = 200); and (3) control group (C, ( n = 197)). The outcomes were measured at week 0, 14, and 35. At the baseline, 17% of the students were underweight, 18% were stunted, and 13% were wasted. At week 35, compared to the C group the weight and height difference increased significantly in the WE group (3.6 ± 23.5 kg, p < 0.001; 5.1 ± 23.2 cm, p < 0.001). No significant differences in weight or height were observed between the PS and C groups. Significant decreases in atherogenic lipoproteins were observed in the WE, but not in PS group. HDL-cholesterol tended to increase in the WE group (0.02 ± 0.59 mmol/L, ns ). The bacterial diversity was similar among the groups. The relative abundance of Bifidobacterium increased by 1.28-fold in the WE group compared to the baseline and differential abundance analysis which indicated that Lachnospira increased and Varibaculum decreased significantly. In conclusion, prolonged whole egg supplementation is an effective intervention to improve growth, nutritional biomarkers, and gut microbiota with unaltered adverse effects on blood lipoproteins.

Colorectal cancers (CRC) with KRAS mutations ( KRAS mut ) are frequently included in consensus molecular subtype 3 (CMS3) with profound metabolic deregulation. We explored the transcriptomic impact of KRAS mut , focusing on the tumor microenvironment (TME) and pathways beyond metabolic deregulation. The status of KRAS mut in patients with CRC was investigated and overall survival (OS) was compared with wild-type KRAS ( KRAS wt ). Next, we identified CMS, and further investigated differentially expressed genes (DEG) of KRAS mut and distinctive pathways. Lastly, we used spatially resolved gene expression profiling to define the effect of KRAS mut in the TME regions of CMS3-classified CRC tissues. CRC patients with KRAS mut were mainly enriched in CMS3. Their specific enrichments of immune gene signatures in immunosuppressive TME were associated with worse OS. Activation of TGFβ signaling by KRAS mut was related to reduced pro-inflammatory and cytokine gene signatures, leading to suppression of immune infiltration. Digital spatial profiling in TME regions of KRAS mut CMS3-classified tissues suggested up-regulated genes, CD40 , CTLA4 , ARG1 , STAT3 , IDO , and CD274 , that could be characteristic of immune suppression in TME. This study may help to depict the complex transcriptomic profile of KRAS mut in immunosuppressive TME. Future studies and clinical trials in CRC patients with KRAS mut should consider these transcriptional landscapes.

Background: Though concurrent chemoradiotherapy (CCRT) with cisplatin remains a standard of care for patients with locally advanced nasopharyngeal carcinoma (LA-NPC), carboplatin has alternatively been used without sufficient supportive evidences. Thus, we evaluated an efficacy and tolerability of carboplatin CCRT compared with cisplatin in LA-NPC patients., Methods: Patients with LA-NPC treated with CCRT were identified through the Thai multicenter head and neck cancer database. Patient tolerability and survival were analyzed and compared between regimens. Survivals were calculated by using the Kaplan-Meier method, and compared by the log-rank test. A p-value of <0.05 was considered statistically significant., Results: A total of 135 of 980 patients (13.8%) were treated with carboplatin. Patients treated with carboplatin were significantly associated with older age (p < 0.001), smoking (p = 0.003), more comorbidity (p = 0.014), kidney disease (p = 0.016), and lower baseline creatinine clearance (p < 0.001). Intensity-modulated radiation therapy was used significantly more in the cisplatin group than carboplatin group (p < 0.001). Patients who received carboplatin were associated with delay (p = 0.049) and hospitalization (p = 0.006), whereas cisplatin CCRT had more dose reduction (p = 0.001). Patients treated with cisplatin had CCRT interruption from grade 3-4 mucositis (p = 0.019) more than carboplatin, whereas carboplatin had more grade 3-4 thrombocytopenia (p < 0.001). The 5-year overall survival (OS) of patients treated with cisplatin and carboplatin was 59% and 49%, respectively (p = 0.128). Cisplatin or carboplatin CCRT was not a significant predictor for OS and locoregional recurrence-free survival in multivariate analysis., Conclusions: Carboplatin CCRT provided acceptable efficacy and tolerability profiles in real-world practice. Carboplatin should be considered as an alternative regimen, particularly in cisplatin-ineligible patients with LA-NPC treated with CCRT., (© 2022 John Wiley & Sons Australia, Ltd.)

Background: This study aimed to explore clinical and molecular factors that cause discordance for clinical expression of Leber's hereditary optic neuropathy (LHON) in a pair of identical twins with the 14484 point mutation., Case Summary: Twin patients with the 14484 point mutation were studied for zygosity by using the Short Tandem Repeats Typing system. For the monozygotic twins, the radioactive restriction and densitometric analyses were used to quantitate the heteroplasmy level for the 14484 point mutation. The mitochondrial genome was analyzed to determine influential factors by mitochondrial deoxyribonucleic acid (DNA) sequencing, denaturing high-performance liquid chromatography and next generation sequencing. For the dizygotic twins, the nuclear DNA was analyzed. The twins with 14484 LHON were monozygotic with homoplasmy. No difference in the point mutation in mitochondrial DNA was found. No modifying genes that potentially influenced the disparity in phenotypic expression of LHON were detected in these twins., Conclusion: This 11-year follow-up of monozygotic twins showed additional genetic modifications and epigenetic factors are possibly associated with discordance for LHON., Competing Interests: Conflict-of-interest statement: All authors have no conflict of interest related to the manuscript., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Cyclic vomiting syndrome (CVS) is a functional gastrointestinal disorder characterized by recurrent stereotypic episodes of vomiting. The pathophysiology of CVS remains obscure. Previous studies have supported the hypotheses of mitochondrial dysfunction. However, data on association studies between mitochondrial DNA (mtDNA) polymorphisms and pediatric-onset CVS are limited and inconsistent. The aims of this study were to describe clinical characteristics, evaluate association of mtDNA polymorphisms 16519T and 3010A with pediatric-onset CVS and identify new mtDNA candidate variants., Methods: This study involved Thai patients diagnosed with CVS according to the Rome III or IV criteria before the age of 15 years. Patients' demographic data, clinical characteristics, previous investigations and treatment outcomes were obtained. Blood samples were collected for next-generation (whole exome) sequencing, followed by analysis of chromosome M (mitochondrial. Variants were filtered according to clinical significance using ClinVar and MITOMAP. mtDNA polymorphisms in 148 normal Thai individuals were used as controls., Results: Forty-eight children were enrolled in the clinical study, and 30 participated in the genetic analysis. The median age at onset and median age at diagnosis was 3.0 (1.5-5.6) and 6.3 (3.0-8.6) years, respectively. Maternal history of migraine was positive in 16.7%. About 45.7% (21 of 46) of the patients achieved complete clinical remission, with the mean symptom duration of 5.9 ± 3.3 years. The prevalence of mtDNA variants 16519T and 3010A among the patient group and Thai general population (control) were as follows: 40.0% (12/30) vs. 27.7% ( P = 0.18) and 6.7% (2/30) vs. 0.7% ( P = 0.07), respectively. Five known pathogenic variants were identified in 6 patients, including mtDNA 8528C in one patient who also had infantile hypertrophic cardiomyopathy. Six likely pathogenic variants were found but without statistical significance. We identified 11 variants with significant prevalence in the patient group. Though, these variants were classified as variants of unknown significance (VUS), several of them were located in mt functional regions and therefore they deserve further investigations as new candidates for association with pediatric CVS., Conclusion: There were no associations of mtDNA polymorphisms 16519T and 3010A with CVS in our pediatric cohort. Five pathogenic variants and 11 VUS were found associated with pediatric-onset CVS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Veenin, Wattanasirichaigoon, Suktitipat, Noojarern, Lertrit, Tim-Aroon, Kaewsutthi and Treepongkaruna.)

Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.

Purpose: Prophylactic percutaneous endoscopic gastrostomy (PPEG) is widely used for patients with head and neck cancer undergoing concurrent chemoradiation (CCRT). Nevertheless, the necessity of its use in patients with nasopharyngeal cancer (NPC) is uncertain. This study aimed to evaluate the benefits of PPEG on prevention of weight loss and treatment tolerance in patients with NPC receiving CCRT., Materials and Methods: A retrospective multicenter chart review of 904 patients, 378 in the PPEG group and 526 in the non-PPEG group, was conducted. Baseline characteristics, weight loss, and treatment tolerance were analyzed and compared between the two groups., Results: There was no significant difference in the mean baseline body mass index (BMI) between the groups. At the end of CCRT, no difference in weight loss was found between the 2 groups (non-PPEG group, 6.6%; PPEG group, 5.9%). Nonetheless, the subgroup analysis demonstrated that a baseline BMI < 18.5 kg/m 2 (underweight) and non-intensity-modulated radiation therapy (IMRT) technique were independent factors associated with prevention of weight loss by PPEG. More patients in the PPEG group were able to complete planned cycles of chemotherapy (73.3% vs. 49.0%, P < .0001)., Conclusion: Although the benefits of PPEG on prevention of weight loss were not observed for the entire cohort, we found a potentially protective effect of PPEG in some subgroups of patients. Additionally, PPEG significantly enhanced chemotherapy tolerance. Therefore, PPEG tube insertion should be strongly considered for patients with NPC receiving CCRT, particularly for underweight patients and those undergoing a non-IMRT technique., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The situation of patients developing multiple primary cancers is becoming more frequent and graver. This study investigated the risks of developing second primary cancers that are related to first primary cancers, and the interval times of synchronous and metachronous multiple primary cancers., Patients and Methods: Retrospective data were retrieved from 109,054 patients aged ≥18 who were diagnosed with a first solid cancer and registered at Siriraj Cancer Center between 1991 and 2015. A two-month period between first- and second- primary cancers was used to differentiate metachronous and synchronous multiple primary cancers. The combinations of subsequent cancers and relative risks (RRs) of having multiple primary cancers versus having single primary cancer for the top-ten first and second primary cancers were examined. The RR was adjusted for age of the first primary cancer. A survival analysis of the time to second-primary-cancer development was performed., Results: Multiple primary cancers were found in 1785 (1.63%) patients. Most (70.87%) second primary cancers occurred after 2 months of first breast, skin, colorectal, lung, head and neck, liver, male genital cancer-prostate, thyroid, and female genital cancer-non-uterine cancers, resulting in those cancers being classified as metachronous multiple primary cancer. After adjustment for age at first diagnosis, head and neck cancers had the highest metachronous association with second esophageal cancers (RR, 25.06; 95% CI, 13.41-50.77). Prostate cancer and second colorectal cancer also demonstrated a high metachronous association (RR, 2.00; 95% CI, 1.25-3.05). A strong synchronous association was found between uterine and ovarian cancers (RR, 27.77; 95% CI, 17.97-43.63). The median time from the first uterine cancer to second-cancer development was 55 days., Conclusions: The top-ten most frequent multiple primary cancers were the following: breast; liver; head and neck; colorectal; male genital cancer-prostate; skin; female genital cancer-uterine; thyroid; lung; and female genital cancer-non-uterine. Second primary cancers showed specific associations that depended on the first primary cancer. Physicians should be cognizant of the most common combinations and the interval times of metachronous and synchronous multiple primary cancers., (© 2021. The Author(s).)

Clinacanthus nutans Lindua ( C. nutans ), a strong antiviral traditional medicine, can be used to treat condyloma acuminata (CA) caused by the human papillomavirus (HPV). However, its molecular mechanism for CA elimination is unknown. Herein, we conducted a randomized clinical trial to evaluate the effectiveness of C. nutans and its molecular mechanism compared with podophyllin, the gold standard treatment. Using a randomized block design, six patients were treated with C. nutans and podophyllin for four weeks. Efficacy of drugs was assessed by size reduction of the warts and HPV viral load quantification using droplet digital PCR. The gene expression profiling of CA was analyzed using NanoString Technology. After the podophyllin and C. nutans treatments, CA lesion sizes were reduced to 97.0% and 84.4% clearance, and the HPV viral loads were reduced by 74.0% and 46.6%, respectively. The gene expression pattern of immune profiling showed that 23 genes (i.e., HLA-DPB, CCL3, CXCL2, CXCR2 , and OSM ) were significantly differentially expressed by podophyllin, whereas 2 genes ( IFNL1 and IRF2 ) were remarkably expressed by C. nutans. In inflammatory profiling, 108 genes (i.e., CXCL2 , IL8 , and STAT3 ) were highly expressed by podophyllin, but none of genes were observed to change expression by C. nutans. These results suggested that podophyllin may reduce the HPV infection through a mechanism related to proinflammatory response. In addition, C. nutans was found to suppress the HPV infection through mechanism related to the activation of immune response. This study shows novel therapeutic mechanisms of podophyllin and C. nutans . It is suggested that C. nutans might be used as an alternative treatment for CA treatment., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2021 Jiraporn Jantaravinid et al.)

Background: Osteopetrosis is a rare form of skeletal dysplasia characterized by increased bone density that leads to bone marrow failure, compressive neuropathy, and skeletal dysmorphism. Molecular diagnosis is essential as it guides treatment and prognosis. We report Thai siblings with an ultra-rare form of osteopetrosis., Methods: The older brother and the younger sister presented with chronic mandibular osteomyelitis in their 20s. Since childhood, they had visual impairment, pathological fracture, and skeletal dysmorphism. Quadruplet whole-exome sequencing was performed and confirmed with Sanger sequencing. Novel mutation in TNFSF11 (RANKL) c.842T>G, p.Phe281Cys was identified in a homozygous state in both siblings., Results: Surgical debridement, antibiotic, and hyperbaric oxygen therapy were used and discontinued over a 6-month period with normalization of C-reactive protein. Hematopoietic stem cell transplantation candidacy was excluded by molecular diagnosis., Conclusion: We report a novel mutation in an ultra-rare form of osteopetrosis. Our siblings manifested with a milder phenotype in comparison with nine cases previously published., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Andersen-Tawil syndrome (ATS) is a rare disorder characterized by a triad of ventricular arrhythmia (VA), dysmorphic features, and periodic paralysis. Due to the rarity of this condition, less is known about physiologic effect of pregnancy to ATS and arrhythmia. There is no established guideline for peripartum or postpartum treatment and prevention of arrhythmia in ATS; thus, the clinical management is challenging. We reported two KCNJ2-associated ATS patients who got pregnant and underwent vaginal birth safely. Both individuals had VA, micrognathia without periodic paralysis. β-blocker plus flecainide could be an effective treatment combination when monotherapy failed to control arrhythmia. VA of two pregnant patients with ATS could be controlled by either physiologic changes associated pregnancy or the combination treatment of β-blocker and flecainide., (© 2020 The Authors. Annals of Noninvasive Electrocardiology published by Wiley Periodicals LLC.)

Background: Sandhoff disease (SD) is an autosomal recessive lysosomal storage disorder, resulting in accumulation of GM2 ganglioside, particular in neuronal cells. The disorder is caused by deficiency of β-hexosaminidase B (HEX-B), due to pathogenic variant of human HEXB gene., Method: This study describes clinical features, biochemical, and genetic defects among Thai patients with infantile SD during 2008-2019., Results: Five unrelated Thai patients presenting with developmental regression, axial hypotonia, seizures, exaggerated startle response to noise, and macular cherry red spot were confirmed to have infantile SD based on deficient HEX enzyme activities and biallelic variants of the HEXB gene. In addition, an uncommon presenting feature, cardiac defect, was observed in one patient. All the patients died in their early childhood. Plasma total HEX and HEX-B activities were severely deficient. Sequencing analysis of HEXB gene identified two variants including c.1652G>A (p.Cys551Tyr) and a novel variant of c.761T>C (p.Leu254Ser), in 90 and 10% of the mutant alleles found, respectively. The results from in silico analysis using multiple bioinformatics tools were in agreement that the p.Cys551Tyr and the p.Leu254Ser are likely pathogenic variants. Molecular modelling suggested that the Cys551Tyr disrupt disulfide bond, leading to protein destabilization while the Leu254Ser resulted in change of secondary structure from helix to coil and disturbing conformation of the active site of the enzyme. Genome-wide SNP array analysis showed no significant relatedness between the five affected individuals. These two variants were not present in control individuals. The prevalence of infantile SD in Thai population is estimated 1 in 1,458,521 and carrier frequency at 1 in 604., Conclusion: The study suggests that SD likely represents the most common subtype of rare infantile GM2 gangliosidosis identified among Thai patients. We firstly described a potential common variant in HEXB in Thai patients with infantile onset SD. The data can aid a rapid molecular confirmation of infantile SD starting with the hotspot variant and the use of expanded carrier testing.

Purpose: MYCNOS (MYCN opposite strand) is co-amplified with MYCN in pediatric cancers, including retinoblastoma. MYCNOS encodes several RNA variants whose functions have not been elucidated in retinoblastoma. Thus, we attempted to identify MYCNOS variants in retinoblastoma and aimed to decipher the role of MYCNOS variant 1 (MYCNOS1) on the activity of MYCN-amplified retinoblastoma., Methods: The profiles of MYCNOS variants and MYCN status were determined in 17 retinoblastoma tissues, cell lines, retinas, and retinal organoids. A functional study of MYCNOS1 expression was conducted in patient-derived tumor cells and in retinoblastoma cell lines via short hairpin RNA-mediated gene silencing. We carried out MYCN expression, cell viability, cell cycle, apoptosis, soft agar colony formation, and transwell assays to examine the role of MYCNOS1 in MYCN and cell behaviors. We analyzed a transcriptome of MYCN-amplified retinoblastoma cells deficient for MYCNOS1 and, finally, tested the responses of these cells to chemotherapeutic agents., Results: Expression of MYCNOS1 was associated with the expression and copy number of MYCN. Knockdown of MYCNOS1 caused instability of the MYCN protein, leading to cell cycle arrest and impaired proliferation and chemotaxis-directed migration in MYCN-amplified retinoblastoma cells in which RB1 was intact. MYCNOS1 expression was associated with gene signatures of photoreceptor cells and epithelial-mesenchymal transition. MYCNOS1 silencing enhanced the response of retinoblastoma cells to topotecan but not carboplatin., Conclusions: MYCNOS1 supports progression of retinoblastoma. Inhibition of MYCNOS1 expression may be necessary to suppress MYCN activity when treating MYCN-amplified cancers without RB1 mutation.

MITF is a known gene underlying autosomal dominant hearing loss, Waardenburg syndrome (WS). Biallelic MITF mutations have been found associated with a rare hearing loss syndrome consisting eye abnormalities and albinism; and a more severe type of WS whose heterozygous parents were affected with classic WS in both cases. The aims of this study were to identify a new candidate gene causing autosomal recessive nonsyndromic hearing loss (ARNSHL) and confirm its causation by finding additional families affected with the candidate gene and supporting evidences from functional analyses. By using whole exome sequencing, we identified a homozygous c.1022G>A: p.Arg341His variant of MITF, which co-segregated with the hearing loss in five affected children of a consanguineous hearing couple. Targeted exome sequencing in a cohort of 130 NSHL individuals, using our in-house gene panel revealed a second family with c.1021C>T: p.Arg341Cys MITF variant. Functional studies confirmed that the Arg341His and Arg341Cys alleles yielded a normal sized MITF protein, with aberrant cytosolic localization as supported by the molecular model and the reporter assay. In conclusion, we demonstrate MITF as a new cause of ARNSHL, with heterozygous individuals free of symptoms. MITF should be included in clinical testing for NSHL, though it is rare.

Background: Chemoradiotherapy (CRT) with high cumulative doses (CDs) of cisplatin has been considered the standard of care for non-metastatic nasopharyngeal carcinoma (NPC). However, given most patients' inability to tolerate high CDs due to cisplatin-related toxicities, the optimal CD of cisplatin during CRT remains undetermined., Methods: Patients with non-metastatic NPC who received CRT with cisplatin between 2007 and 2017 were identified through the Thai head and neck cancer multicenter database and then categorized according to cisplatin CD (mg/m 2 ) received. All complications and cisplatin-related toxicities during CRT were recorded., Results: We identified 779 non-metastatic NPC patients receiving low (≤150; n = 97), intermediate (151-250; n = 411), and high (> 250; n = 271) CDs of cisplatin. Low CD patients had significantly lower mean actual radiation dose (p < 0.001) and more radiotherapy delay (p = 0.010), while intermediate CD patients had the least hospitalization (p < 0.001). Overall, 39.3% of the patients experienced cisplatin-related toxicity, which was associated with poor overall survival (OS) (p = 0.001). Acute kidney injury was observed in 7% in all patients, which was highest among low CD patients (15.5%; p = 0.002). Intermediate CD patients had significantly longer median OS than the low and high groups (64 vs. 49.8 vs. 53.2, respectively; p = 0.015). Univariate, but not multivariate, analysis showed that CD of cisplatin was significantly associated with OS., Conclusion: CD of cisplatin during CRT was not an independent prognostic factor for OS. An intermediate CD induced minimal toxicity without compromising survival and should be considered the optimal CD. Nonetheless, a randomized phase 3 study evaluating the optimal CD of cisplatin is warranted.

Patients with head and neck squamous cell carcinoma are at increased risk of developing a second primary malignancy, which is associated with poor prognosis and early death. To help improve clinical outcome, we aimed to identify biomarkers for second primary malignancy risk prediction using the routinely obtained formalin-fixed paraffin-embedded tissues of the index head and neck cancer. Liquid chromatography-tandem mass spectrometry was initially performed for candidate biomarker discovery in 16 pairs of primary cancer tissues and their matched normal mucosal epithelia from head and neck squamous cell carcinoma patients with or without second primary malignancy. The 32 candidate proteins differentially expressed between head and neck cancers with and without second primary malignancy were identified. Among these, 30 selected candidates and seven more from literature review were further studied using NanoString nCounter gene expression assay in an independent cohort of 49 head and neck cancer patients. Focusing on the p16-negative cases, we showed that a multivariate logistic regression model comprising the expression levels of ITPR3, KMT2D, EMILIN1, and the patient's age can accurately predict second primary malignancy occurrence with 88% sensitivity and 75% specificity. Furthermore, using Cox proportional hazards regression analysis and survival analysis, high expression levels of ITPR3 and DSG3 were found to be significantly associated with shorter time to second primary malignancy development (log-rank test P = 0.017). In summary, we identified a set of genes whose expressions may serve as the prognostic biomarkers for second primary malignancy occurrence in head and neck squamous cell carcinomas. In combination with the histopathologic examination of index tumor, these biomarkers can be used to guide the optimum frequency of second primary malignancy surveillance, which may lead to early diagnosis and better survival outcome.

Background: Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by absence of both hormonal receptors and human epithelial growth factor receptor 2 (HER2). TNBC accounts for 15-20% of breast cancer. TNBC is associated with more aggressive disease and worse clinical outcome. Though the underlying mechanism of TNBC is currently unclear, the heterogeneity of clinical characteristics in various population may relate to the difference in tumor mutational profile. There were studies on TNBC gene mutations in various ethnic groups but the tumor genome data on Thai TNBC patients is currently unknown. This study aims to investigate mutational profile of Thai TNBC., Methods: The patients were Thai individuals who were diagnosed with primary breast carcinoma between 2014 and 2017. All surgically removed primary tumor tissues were carefully examined by pathologists and archived as formalin-fixed paraffin-embedded tumor. TNBC was defined by absence of hormonal receptors and HER2 by immunohistochemistry. Genomic DNA was extracted, enriched and sequenced of all exomes on the Illumina HiSeq. Genomic data were then processed through bioinformatics platform to identify genomic alterations and tumor mutational burden., Results: A total of 116 TNBC patients were recruited. Genomic analysis of TNBC samples identified 81,460 variants, of which 5,906 variants were in cancer-associated genes. The result showed that Thai TNBC has higher tumor mutation burden than previously reported data. The most frequently mutated cancer-associated gene was TP53 similar to other TNBC cohorts. Meanwhile KMT2C was found to be more commonly mutated in Thai TNBC than previous studies. Mutational profile of Thai TNBC patients also revealed difference in many frequently mutated genes when compared to other Western TNBC cohorts., Conclusion: This result supported that TNBC breast cancer patients from various ethnic background showed diverse genome alteration pattern. Although TP53 is the most commonly mutated gene across all cohorts, Thai TNBC showed different gene mutation frequencies, especially in KMT2C . In particular, the cancer gene mutations are more prevalent in Thai TNBC patients. This result provides important insight on diverse underlying genetic and epigenetic mechanisms of TNBC that could translate to a new treatment strategy for patients with this disease., Competing Interests: The authors declare that they have no competing interests.

Background: For decades, various cardiovascular symptoms have been relieved by the use of Ya-Hom Navakot, which is a formulation comprising 54 herbal medicines. The Thailand Ministry of Public Health listed Ya-Hom Navakot's nine active principle and nomenclative herbal ingredients and termed them 'Phikud Navakot' (PN). Several reports have confirmed that PN has cardiovascular benefits similar to Ya-Hom Navakot. However, whether PN facilitates lipid-lowering activity remains unclear., Methods: The present study investigated an in vitro model for examining the gene expression levels of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMGCR) and low-density lipoprotein receptor (LDL-R) in HepG2 cells using qRT-PCR. The ethanol and water extractions of Ya-Hom Navakot, PN and Ya-Hom Navakot without PN were compared., Results: One mg/ml of both NYEF and NYWF were found to significantly lower cholesterol by either the up-regulation of LDL-R or down-regulation of HMGCR compared with negative controls and 1 mg/ml simvastatin (p < 0.05). PNEF also up-regulated LDL-R gene expression, even more than NYEF (p < 0.05). In addition, the ethanol and water extracts of PN significantly down-regulated HMGCR gene expression compared with those of Ya-Hom Navakot without PN (p < 0.05)., Conclusion: The use of Ya-Hom Navakot or PN may provide an alternative treatment to lower cholesterol through HMGCR gene inhibition and LDL-R gene enhancement.

Background: Imputation involves the inference of untyped single nucleotide polymorphisms (SNPs) in genome-wide association studies. The haplotypic reference of choice for imputation in Southeast Asian populations is unclear. Moreover, the influence of SNP annotation on imputation results has not been examined., Methods: This study was divided into two parts. In the first part, we applied imputation to genotyped SNPs from Southeast Asian populations from the Pan-Asian SNP database. Five percent of the total SNPs were removed. The remaining SNPs were applied to imputation with IMPUTE2. The imputed outcomes were verified with the removed SNPs. We compared imputation references from Chinese and Japanese haplotypes from the HapMap phase II (HMII) and the complete set of haplotypes from the 1000 Genomes Project (1000G). The second part was imputation accuracy and yield in Thai patient dataset. Half of the autosomal SNPs was removed to create Set 1. Another dataset, Set 2, was then created where we switched which half of the SNPs were removed. Both Set 1 and Set 2 were imputed with HMII to create a complete imputed SNPs dataset. The dataset was used to validate association testing, SNPs annotation and imputation outcome., Results: The accuracy was highest for all populations when using the HMII reference, but at the cost of a lower yield. Thai genotypes showed the highest accuracy over other populations in both HMII and 1000G panels, although accuracy and yield varied across chromosomes. Imputation was tested in a clinical dataset to compare accuracy in gene-related regions, and coding regions were found to have a higher accuracy and yield., Conclusions: This work provides the first evidence of imputation reference selection for Southeast Asian studies and highlights the effects of SNP locations respective to genes on imputation outcome. Researchers will need to consider the trade-off between accuracy and yield in future imputation studies.

Introduction: Sudden unexpected death syndrome (SUDS) is an important cause of death in young healthy adults with a high incident rate in Southeast Asia; however, there are no molecular autopsy reports about these victims. We performed a combination of both a detailed autopsy and a molecular autopsy by whole exome sequencing (WES) to investigate the cause of SUDS in Thai sudden death victims., Materials and Methods: A detailed forensic autopsy was performed to identify the cause of death, followed by a molecular autopsy, in 42 sudden death victims who died between January 2015 and August 2015. The coding sequences of 98 SUDS-related genes were sequenced using WES. Potentially causative variants were filtered based on the variant functions annotated in the dbNSFP database. Variants with inconclusive clinical significance evidence in ClinVar were resolved with a variant prediction algorithm, metaSVM, and the frequency data of the variants found in public databases, such as the 1000 Genome Project, ESP6500 project, and the Exome Aggregation Consortium (ExAc) project., Results: Combining both autopsy and molecular autopsy enabled the potential identification of cause of death in 81% of the cases. Among the 25 victims with WES data, 72% (18/25) were found to have potentially causative SUDS mutations. The majority of the victims had at a mutation in the TTN gene (8/18 = 44%), and only one victim had an SCN5A mutation., Conclusions: WES can help to identify the genetic causes in victims of SUDS and may help to further guide investigations into their relatives to prevent additional SUDS victims.

Background: The clinical syndrome of disseminated nontuberculous mycobacterial (NTM) infection in patients who were previously healthy is now well recognized to be associated with an acquired autoantibody to Interferon gamma (Anti IFN- γ autoantibody). However, the risk factors of this syndrome remain unknown., Method: We performed an unmatched case control study among patients with NTM diseases who were diagnosed and treated at Siriraj Hospital, Bangkok, Thailand. Anti-IFN autoantibody was detected by enzyme-linked immunosorbent assay (ELISA) method. Cases were patients with NTM diseases and detectable anti IFN- γ autoantibody. Controls were randomly selected from those with undetectable anti IFN- γ autoantibody. Data from both groups including demographic data, clinical presentation, laboratory results, other risk factors and HLA genotypes were collected. Univariate and multivariate analyses were performed to identify independent risk factors for this syndrome., Results: 70 cases (mean age 50 ± 11 years) and 70 controls (mean age 58 ± 18 years) were enrolled into the study. Mycobacterial abscessus was the most common NTM pathogen found in both groups (72.9% in cases and 41.4% in controls respectively). However, disseminated NTM disease was significantly more common in cases (92.9%) than in the controls (14.3%, p<0.001). Binary logistic regression analysis showed that previous OIs (adjusted OR14.87, 95% CI 2.36-93.86), birthplace outside Central region (adjusted OR 19.19, 95% CI 3.86-95.35), lack of comorbidities lead to immunosuppression, such as HIV infection or diabetes mellitus (adjusted OR 23.68, 95% CI 4.01-139.94), and presence of HLA DRB1*15/16 (adjusted OR 153.28, 95% CI 16.87-139.88) were independent factors associated with this syndrome., Conclusion: Patients with NTM disease associated with anti IFN- γ autoantibody are almost always previously healthy and HIV negative. Most of these patients presented with disseminated NTM disease with generalized lymphadenitis and often with reactive skin lesions. Factors associated with detectable anti IFN- γ autoantibody are HLA-DRB1 and DQB1 alleles, and history of previous OIs in patients without comorbidity that leads to immunosuppression. Further studies are needed to better understand these associations and to improve the treatment outcome.

Genome-wide association studies (GWAS) for nonsyndromic cleft lip with or without cleft palate (CL/P) have identified multiple genes as important in the etiology of this common birth defect. We performed a candidate gene/pathway analysis explicitly considering gene-gene (G × G) interaction to further explore the etiology of CL/P. Animal models have shown the WNT signaling pathway plays an important role in mid-facial development, and various genes in this pathway have been associated with nonsyndromic CL/P in previous studies. We propose a combined approach to search for possible G × G interactions using machine learning and regression-based methods to test for interactions between genes in the WNT family, and between these genes and other genes identified by GWAS in case-parent trios. Using this combined approach of regression-based and machine learning methods in CL/P case-parent trios, we found robust evidence of G × G interaction between markers in WNT5B and MAFB (empiric P-values = 0.0076 among Asian trios and P-values = 0.018 among European trios). Additional evidence for epistatic interaction between markers in WNT5A, IRF6, and C1orf107 was seen among Asian trios, and markers in the 8q24 region and WNT5B among European trios., (© 2015 WILEY PERIODICALS, INC.)

Background: We have previously shown that platelet aggregation has higher heritability in African Americans than European Americans. However, a genome-wide association study (GWAS) of platelet aggregation in African Americans has not been reported. We measured platelet aggregation in response to arachidonic acid, ADP, collagen, or epinephrine by optical aggregometry. The discovery cohort was 825 African Americans from the GeneSTAR study. Two replication cohorts were used: 119 African Americans from the Platelet Genes and Physiology Study and 1221 European Americans from GeneSTAR. Genotyping was conducted with Illumina 1 M arrays. For each cohort, age- and sex-adjusted linear mixed models were used to test for association between each SNP and each phenotype under an additive model., Results: Six SNPs were significantly associated with platelet aggregation (P<5×10(-8)) in the discovery sample. Of these, three SNPs in three different loci were confirmed: 1) rs12041331, in PEAR1 (platelet endothelial aggregation receptor 1), replicated in both African and European Americans for collagen- and epinephrine-induced aggregation, and in European Americans for ADP-induced aggregation; 2) rs11202221, in BMPR1A (bone morphogenetic protein receptor type1A), replicated in African Americans for ADP-induced aggregation; and 3) rs6566765 replicated in European Americans for ADP-induced aggregation. The rs11202221 and rs6566765 associations with agonist-induced platelet aggregation are novel., Conclusions: In this first GWAS of agonist-induced platelet aggregation in African Americans, we discovered and replicated, novel associations of two variants with ADP-induced aggregation, and confirmed the association of a PEAR1 variant with multi-agonist-induced aggregation. Further study of these genes may provide novel insights into platelet biology.

Recently a newly identified clinical syndrome of disseminated non-tuberculous mycobacterial diseases (with or without other opportunistic infections in adult patients who were previously healthy, has been recognized in association with an acquired autoantibody to interferon-gamma. This syndrome is emerging as an important cause of morbidity and mortality, especially among people of Asian descent. Trigger for the production of this autoantibody remains unknown, but genetic factors are strongly suspected to be involved. We compared HLA genotyping between 32 patients with this clinical syndrome, and 38 controls. We found that this clinical syndrome was associated with very limited allele polymorphism, with HLA-DRB1 and DQB1 alleles, especially HLA-DRB1*15:01, DRB1*16:02, DQB1*05:01 and DQB1*05:02. Odds ratio of DRB1*15:01, DRB1*16:02, DQB1*05:01 and DQB1*05:02 were 7.03 (95% CI, 2.18-22.69, P<0.0001, 9.06 (95% CI, 2.79-29.46, P<0.0001), 6.68 (95% CI, 2.29-19.52, P = 0.0004), and 6.64 (95% CI, 2.30-19.20, P = 0.0004), respectively. Further investigation is warranted to provide better understanding on pathogenesis of this association.

Hyperbaric Oxygen (HBO, HBO2) Therapy is a non-invasive therapy. It has been applied as adjuvant treatment in many medical conditions over the past 50 years. Different treatment protocols have been proven effective for specifically indicated conditions. To evaluate the clinical effectiveness of Hyperbaric Oxygen (HBO) Therapy as an adjunctive treatment for patients with complex wounds. In this prospective cohort study, 40 patients with complex wounds were included. All patients received HBO. HBO was delivered with 100% oxygen for 90 min at 2.0-2.4 ATA. Wound sizes were assessed by one wound surgeon before, during, and every 2 weeks for a total of 12 months after HBO. An analysis of demographic data, wound size and wound photography was performed. Over the 22-month period ending October 31, 2013, 40 patients (21 men and 19 women) with a mean age of 59.73 (range, 29-88) with complex wounds were included. All complex wounds studied were at least 6 months old. The mean wound size was 16.72 cm(2) in diameter. Thirty-one patients with complex wounds healed after the completion of a series of HBO treatments (77.5%). Two orocutaneous fistulas were completely closed without further surgery. After 5 HBO treatments, the wound size reduced by 29.7% on average (p = 1.24 × 10(-6)). After 10 HBO treatments, the wound size statistically significantly reduced by an additional 16.9% (p = 0.0002). There were no complications in this study. Wound healing process was accelerated by HBO. Significant wound size reduction was noted after 5 HBO treatments. Because the biggest reduction in wound size occurred within the first 10 HBO treatments, it is important to conduct these first treatments without interruption. HBO is an effective and safe treatment modality for complex wounds.

Clopidogrel is an antiplatelet prodrug that is recommended to reduce the risk of recurrent thrombosis in coronary artery disease (CAD) patients. Paraoxonase 1 (PON1) is suggested to be a rate-limiting enzyme in the conversion of 2-oxo-clopidogrel to active thiol metabolite with inconsistent results. Here, we sought to determine the associations of CYP2C19 and PON1 gene polymorphisms with clopidogrel response and their role in ADP-induced platelet aggregation. Clopidogrel response and platelet aggregation were determined using Multiplate aggregometer in 211 patients with established CAD who received 75 mg clopidogrel and 75-325 mg aspirin daily for at least 14 days. Polymorphisms in CYP2C19 and PON1 were genotyped and tested for association with clopidogrel resistance. Linkage disequilibrium (LD) and their epistatic interaction effects on ADP-induced platelet aggregation were analysed. The prevalence of clopidogrel resistance in this population was approximately 33.2% (n = 70). The frequencies of CYP2C19*2 and *3 were significantly higher in non-responder than those in responders. After adjusting for established risk factors, CYP2C19*2 and *3 alleles independently increased the risk of clopidogrel resistance with adjusted ORs 2.94 (95%CI, 1.65-5.26; p<0.001) and 11.26 (95%CI, 2.47-51.41; p = 0.002, respectively). Patients with *2 or *3 allele and combined with smoking, diabetes and increased platelet count had markedly increased risk of clopidogrel resistance. No association was observed between PON1 Q192R and clopidogrel resistance (adjusted OR = 1.13, 95%CI, 0.70-1.82; p = 0.622). Significantly higher platelet aggregation values were found in CYP2C19*2 and *3 patients when compared with *1/*1 allele carriers (p = 1.98 × 10(-6)). For PON1 Q192R genotypes, aggregation values were similar across all genotype groups (p = 0.359). There was no evidence of gene-gene interaction or LD between CYP2C19 and PON1 polymorphisms on ADP-induced platelet aggregation. Our findings indicated that only CYP2C19*2 and *3 alleles had an influence on clopidogrel resistance. The risk of clopidogrel resistance increased further with smoking, diabetes, and increased platelet count.

Copy number variation (CNV) is a major genetic polymorphism contributing to genetic diversity and human evolution. Clinical application of CNVs for diagnostic purposes largely depends on sufficient population CNV data for accurate interpretation. CNVs from general population in currently available databases help classify CNVs of uncertain clinical significance, and benign CNVs. Earlier studies of CNV distribution in several populations worldwide showed that a significant fraction of CNVs are population specific. In this study, we characterized and analyzed CNVs in 3,017 unrelated Thai individuals genotyped with the Illumina Human610, Illumina HumanOmniexpress, or Illumina HapMap550v3 platform. We employed hidden Markov model and circular binary segmentation methods to identify CNVs, extracted 23,458 CNVs consistently identified by both algorithms, and cataloged these high confident CNVs into our publicly available Thai CNV database. Analysis of CNVs in the Thai population identified a median of eight autosomal CNVs per individual. Most CNVs (96.73%) did not overlap with any known chromosomal imbalance syndromes documented in the DECIPHER database. When compared with CNVs in the 11 HapMap3 populations, CNVs found in the Thai population shared several characteristics with CNVs characterized in HapMap3. Common CNVs in Thais had similar frequencies to those in the HapMap3 populations, and all high frequency CNVs (>20%) found in Thai individuals could also be identified in HapMap3. The majorities of CNVs discovered in the Thai population, however, were of low frequency, or uniquely identified in Thais. When performing hierarchical clustering using CNV frequencies, the CNV data were clustered into Africans, Europeans, and Asians, in line with the clustering performed with single nucleotide polymorphism (SNP) data. As CNV data are specific to origin of population, our population-specific reference database will serve as a valuable addition to the existing resources for the investigation of clinical significance of CNVs in Thais and related ethnicities.

Overexpression of cyclin D1 and its catalytic partner, CDK4, is frequently seen in human cancers. We constructed cyclin D1 and CDK4 protein interaction network in a human breast cancer cell line MCF7, and identified novel CDK4 protein partners. Among CDK4 interactors we observed several proteins functioning in protein folding and in complex assembly. One of the novel partners of CDK4 is FKBP5, which we found to be required to maintain CDK4 levels in cancer cells. An integrative analysis of the extended cyclin D1 cancer interactome and somatic copy number alterations in human cancers identified BAIAPL21 as a potential novel human oncogene. We observed that in several human tumor types BAIAPL21 is expressed at higher levels as compared to normal tissue. Forced overexpression of BAIAPL21 augmented anchorage independent growth, increased colony formation by cancer cells and strongly enhanced the ability of cells to form tumors in vivo. Lastly, we derived an Aggregate Expression Score (AES), which quantifies the expression of all cyclin D1 interactors in a given tumor. We observed that AES has a prognostic value among patients with ER-positive breast cancers. These studies illustrate the utility of analyzing the interactomes of proteins involved in cancer to uncover potential oncogenes, or to allow better cancer prognosis.

Mangosteen extracts (ME) contain high levels of polyphenolic compounds and antioxidant activity. Protective effects of ME against β-amyloid peptide (Aβ), induced cytotoxicity have been reported. Here, we further studied the protective effects of ME against oxidative stress induced by hydrogen peroxide (H2O2) and polychlorinated biphenyls (PCBs), and demonstrated the protection against memory impairment in mice. The cytoprotective effects of ME were measured as cell viability and the reduction in ROS activity. In SK-N-SH cell cultures, 200 μg/ml ME could partially antagonize the effects of 150 or 300 µM H2O2 on cell viability, ROS level and caspase-3 activity. At 200, 400 or 800 µg/ml, ME reduced AChE activity of SK-N-SH cells to about 60% of the control. In vivo study, Morris water maze and passive avoidance tests were used to assess the memory of the animals. ME, especially at 100 mg/kg body weight, could improve the animal's memory and also antagonize the effect of scopolamine on memory. The increase in ROS level and caspase-3 activity in the brain of scopolamine-treated mice were antagonized by the ME treatment. The study demonstrated cytoprotective effects of ME against H2O2 and PCB-52 toxicity and having AChE inhibitory effect in cell culture. ME treatment in mice could attenuate scopolamine-induced memory deficit and oxidative stress in brain.

Platelet aggregation is heritable, and genome-wide association studies have detected strong associations with a common intronic variant of the platelet endothelial aggregation receptor1 (PEAR1) gene both in African American and European American individuals. In this study, we used a sequencing approach to identify additional exonic variants in PEAR1 that may also determine variability in platelet aggregation in the GeneSTAR Study. A 0.3 Mb targeted region on chromosome 1q23.1 including the entire PEAR1 gene was Sanger sequenced in 104 subjects (45% male, 49% African American, age = 52±13) selected on the basis of hyper- and hypo- aggregation across three different agonists (collagen, epinephrine, and adenosine diphosphate). Single-variant and multi-variant burden tests for association were performed. Of the 235 variants identified through sequencing, 61 were novel, and three of these were missense variants. More rare variants (MAF<5%) were noted in African Americans compared to European Americans (108 vs. 45). The common intronic GWAS-identified variant (rs12041331) demonstrated the most significant association signal in African Americans (p = 4.020×10(-4)); no association was seen for additional exonic variants in this group. In contrast, multi-variant burden tests indicated that exonic variants play a more significant role in European Americans (p = 0.0099 for the collective coding variants compared to p = 0.0565 for intronic variant rs12041331). Imputation of the individual exonic variants in the rest of the GeneSTAR European American cohort (N = 1,965) supports the results noted in the sequenced discovery sample: p = 3.56×10(-4), 2.27×10(-7), 5.20×10(-5) for coding synonymous variant rs56260937 and collagen, epinephrine and adenosine diphosphate induced platelet aggregation, respectively. Sequencing approaches confirm that a common intronic variant has the strongest association with platelet aggregation in African Americans, and show that exonic variants play an additional role in platelet aggregation in European Americans.

Variance components analysis (VCA), the traditional method for handling correlations within families in genetic association studies, is computationally intensive for genome-wide analyses, and the computational burden of VCA increases with family size and the number of genetic markers. Alternative approaches that do not require the computation of familial correlations are preferable, provided that they do not inflate type I error or decrease power. We performed a simulation study to evaluate practical alternatives to VCA that use regression with generalized estimating equations (GEE) in extended family data. We compared the properties of linear regression with GEE applied to an entire extended family structure (GEE-EXT) and GEE applied to nuclear family structures split from these extended families (GEE-SPL) to variance components likelihood-based methods (FastAssoc). GEE-EXT was evaluated with and without robust variance estimators to estimate the standard errors. We observed similar average type I error rates from GEE-EXT and FastAssoc compared to GEE-SPL. Type I error rates for the GEE-EXT method with a robust variance estimator were marginally higher than the nominal rate when the minor allele frequency (MAF) was <0.1, but were close to the nominal rate when the MAF was ≥0.2. All methods gave consistent effect estimates and had similar power. In summary, the GEE framework with the robust variance estimator, the computationally fastest and least data management-intensive approach, appears to work well in extended families and thus provides a reasonable alternative to full variance components approaches for extended pedigrees in a genome-wide association study setting., (Copyright © 2012 S. Karger AG, Basel.)

Adult height is a classic polygenic trait of high heritability (h(2) approximately 0.8). More than 180 single nucleotide polymorphisms (SNPs), identified mostly in populations of European descent, are associated with height. These variants convey modest effects and explain approximately10% of the variance in height. Discovery efforts in other populations, while limited, have revealed loci for height not previously implicated in individuals of European ancestry. Here, we performed a meta-analysis of genome-wide association (GWA) results for adult height in 20,427 individuals of African ancestry with replication in up to 16,436 African Americans. We found two novel height loci (Xp22-rs12393627, P = 3.4×10(-12) and 2p14-rs4315565, P = 1.2×10(-8)). As a group, height associations discovered in European-ancestry samples replicate in individuals of African ancestry (P = 1.7×10(-4) for overall replication). Fine-mapping of the European height loci in African-ancestry individuals showed an enrichment of SNPs that are associated with expression of nearby genes when compared to the index European height SNPs (P<0.01). Our results highlight the utility of genetic studies in non-European populations to understand the etiology of complex human diseases and traits., Competing Interests: The authors have declared that no competing interests exist.

Genetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10⁻⁸) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10⁻²⁷) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10⁻⁵). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10⁻⁶) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype.

Purpose: To investigate the role of mitochondrial DNA (mt DNA) background on the expression of Leber hereditary optic neuropathy (LHON) in Southeast Asian carriers of the G11778A mutation., Methods: Complete mtDNA sequences were analyzed from 53 unrelated Southeast Asian G11778A LHON pedigrees in Thailand and 105 normal Thai controls, and mtDNA haplogroups were determined. Clinical phenotypes were tested for association with mtDNA haplogroup, with adjustment for potential confounders such as sex and age at onset., Results: mtDNA subhaplogroup B was significantly associated with LHON. Follow-up analysis narrowed the association down to subhaplogroup B5a1 (P = 0.008). Survival analyses with Cox's proportional hazards modeling on 469 samples (91 affected and 378 unaffected), adjusted for sex and heteroplasmy, revealed that haplogroup B5a1 tended to increase the risk of visual loss, but the trend was not statistically significant. Conversely, haplogroup F, the second most common haplogroup in the control population, was the least frequent haplogroup in LHON. This negative association was narrowed down to subhaplogroup F1 (P = 0.00043), suggesting that haplogroup F1 confers a protective effect. The distributions of sex, age at onset and heteroplasmy were not significantly different among haplogroups., Conclusions: The specific mtDNA background B5a1 was significantly associated with Southeast Asian G11778A LHON and appeared to modify the risk of visual loss.

A 58 kb region on chromosome 9p21.3 has consistently shown strong association with coronary artery disease (CAD) in multiple genome-wide association studies in populations of European and East Asian ancestry. In this study, we sought to further characterize the role of genetic variants in 9p21.3 in African American individuals. Apparently healthy African American siblings (n = 548) of patients with documented CAD < 60 years of age were genotyped and followed for incident CAD for up to 17 years. Tests of association for 86 single-nucleotide polymorphisms (SNPs) across the 9p21.3 region in a generalized estimating equation logistic framework under an additive model adjusting for traditional risk factors, family, follow-up time and population stratification were performed. A single SNP within the CDKN2B gene met stringent criteria for statistical significance, including permutation-based evaluations. This variant, rs3217989, was common (minor allele (G) frequency 0.242), conveyed protection against CAD (odds ratio (OR) = 0.19, 95% confidence interval (CI): 0.07 to 0.50, P = 0.0008) and was replicated in a combined analysis of two additional case/control studies of prevalent CAD/MI in African Americans (n = 990, P = 0.024, OR = 0.779, 95% CI: 0.626-0.968). This is the first report of a CAD association signal in a population of African ancestry with a common variant within the CDKN2B gene, independent from previous findings in European and East Asian ancestry populations. The findings demonstrate a significant protective effect against incident CAD in African American siblings of persons with premature CAD, with replication in a combination of two additional African American cohorts.

Objectives: We sought to replicate the association between the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism (rs20455), and clinical coronary artery disease (CAD)., Background: Recent prospective studies suggest that carriers of the 719Arg allele in KIF6 are at increased risk of clinical CAD compared with noncarriers., Methods: The KIF6 Trp719Arg polymorphism (rs20455) was genotyped in 19 case-control studies of nonfatal CAD either as part of a genome-wide association study or in a formal attempt to replicate the initial positive reports., Results: A total of 17,000 cases and 39,369 controls of European descent as well as a modest number of South Asians, African Americans, Hispanics, East Asians, and admixed cases and controls were successfully genotyped. None of the 19 studies demonstrated an increased risk of CAD in carriers of the 719Arg allele compared with noncarriers. Regression analyses and fixed-effects meta-analyses ruled out with high degree of confidence an increase of ≥2% in the risk of CAD among European 719Arg carriers. We also observed no increase in the risk of CAD among 719Arg carriers in the subset of Europeans with early-onset disease (younger than 50 years of age for men and younger than 60 years of age for women) compared with similarly aged controls as well as all non-European subgroups., Conclusions: The KIF6 Trp719Arg polymorphism was not associated with the risk of clinical CAD in this large replication study., (Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)