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Objective To identify the genetic etiologies of congenital primary hypothyroidism (CH) in Thai patients. Design and methods CH patients were enrolled. Clinical characteristics including age, signs and symptoms of CH, pedigree, family history, screened thyroid-stimulating hormone results, thyroid function tests, thyroid imaging, clinical course and treatment of CH were collected. Clinical exome sequencing by next-generation sequencing was performed. In-house gene list which covered 62 potential candidate genes related to CH and thyroid disorders was developed for targeted sequencing. Sanger sequencing was performed to validate the candidate variants. Thyroid function tests were determined in the heterozygous parents who carried the same DUOX2 or DUOXA2 variants as their offsprings. Results There were 118 patients (63 males) included. Mean (SD) age at enrollment was 12.4 (7.9) years. Forty-five of 118 patients (38%) had disease-causing variants. Of 45 variants, 7 genes were involved (DUOX2, DUOXA2, TG, TPO, SLC5A5, PAX8 and TSHR). DUOX2, a gene causing thyroid dyshormonogenesis, was the most common defective gene (25/45, 56%). The most common DUOX2 variant found in this study was c.1588A>T. TG and TPO variants were less common. Fourteen novel variants were found. Thyroid function tests of most parents with heterozygous state of DUOX2 and DUOXA2 variants were normal. Conclusions DUOX2 variants were most common among Thai CH patients, while TG and TPO variants were less common. The c.1588A>T in DUOX2 gene was highly frequent in this population.

Background Epileptic spasms are a devastating form of early infantile epileptic encephalopathy (EIEE) with various etiologies. Early diagnosis and a shorter lead time to treatment are crucial to stop the seizures and optimize the neurodevelopmental outcome. Genetic testing has become an integral part of epilepsy care that directly guides management and family planning and discovers new targeted treatments. Neuronal differentiation Factor 2 (NEUROD2) variants have recently been a cause of neurodevelopmental disorders (NDDs) and EIEEs with distinctive features. However, there is limited information about the clinical and electroencephalographic response of epileptic spasm treatment in NEUROD2-related NDD syndrome. Case presentation We report a female patient of Southeast Asian ethnicity with global developmental delay and epileptic spasms commencing in the first few months of life. A novel de novo heterozygous pathogenic NEUROD2 variant, p. E130Q, was subsequently identified by whole-exome sequencing. Electroencephalogram before treatment showed multifocal independent spikes predominantly in both posterior head regions and demonstrated marked improvement following combined vigabatrin and high-dose prednisolone treatment. However, multiple courses of relapse occurred after weaning off the antiseizure medication. Conclusions We propose that epileptic spasms related to de novo NEUROD2 pathogenic variant respond well to combined vigabatrin and high-dose prednisolone therapy. These findings may imply the benefit of using combination therapy to treat epileptic spasms in NEUROD2-related NDD syndrome.

Pantothenate kinase-associated neurodegeneration (PKAN) is linked to brain iron accumulation caused by PANK2 gene mutation. Despite the importance of genetic testing to confirm PKAN and identify at risk parents, genetic screening is financially burdensome for developing countries like Thailand. Because genetic screeners are expensive and not reimbursed by the universal health care coverage system, they are not typically performed. To investigate clinical symptoms, radiological findings and mutation analysis for patients based in Thailand with unknown genetic status but suspected PKAN based on clinical symptoms. Genetic testing was performed for cases suspected for PKAN and their biological parents by direct genomic sequencing of PANK2 at Maharat Nakhon Ratchasima Hospital during 2017–2018. Clinical evaluation and documentation were performed by pediatric neurologists. Five children had classical onset form of PKAN. Most presented with gait dystonia. Three patients diagnosed after 4 years showed the eye-of-the-tiger sign in their brain MRI, whereas two younger patients revealed only isolated hyperintensity bilateral globus pallidus. However, PANK2 mutations were identified in all cases: the most common mutation was c.982-1G>C. This mutation was detected in four unrelated individuals but not reported in other studies. Genetic testing is recommended to confirm diagnoses in cases with supporting clinical features of PKAN with or without the classical ‘eye-of-the-tiger-sign’. A novel PANK2 mutation (c.982-1G>C) was identified in South East Asian populations based in Thailand, suggesting that this genetic variant is a founder genotype in this population. Moreover, genetic diagnosis is helpful to provide appropriate genetic counseling to families.

Introduction Pheochromocytomas and paragangliomas (PPGLs) are highly heritable tumours, with up to 40% of cases carrying germline variants. Current guidelines recommend genetic testing for all patients with PPGLs. Next-generation sequencing (NGS) enables accurate, fast, and inexpensive genetic testing. This study aimed to compare the costs related to PPGL genetic testing between the sequential testing using the decisional algorithm proposed in the 2014 Endocrine Society guidelines and targeted NGS gene panels. Methods Patients with proven PPGLs were enrolled. A gene list covering 17 susceptibility genes related to hereditary PPGLs was developed for targeted sequencing. Validation was carried out by Sanger sequencing. We simulated the diagnostic workflow to examine the anticipated costs based on each strategy for genetic testing. Results Twenty-nine patients were included, among whom a germline variant was identified in 34.5%. A total of 22.7% with apparently sporadic PPGL carried a variant. Five genes were involved (RET, n = 3; SDHB, n = 3; SDHD, n = 2; EGLN1, n = 1; and NF1, n = 1). According to the diagnostic workflow, the average cost of the targeted NGS (534.7 US dollars per patient) is lower than that of the sequential testing (734.5 US dollars per patient). The targeted NGS can also reduce the number of hospital visits from 4.1 to 1 per person. The cost can be further reduced to 496.24 US dollars per person (32% reduction) if we apply a new syndromic-driven diagnostic algorithm to establish priorities for specific genetic testing for syndromic and selected cases, and targeted NGS for non-syndromic patients. Conclusions Targeted NGS can reduce both the cost of PPGL genetic testing and the number of hospital visits, compared with the conventional approach. Our proposed algorithm is the preferred approach due to its significant reduction of the cost of genetic testing.Key messagePheochromocytomas and paragangliomas are highly heritable neoplasms.The targeted next-generation sequencing (NGS) gene panels have proven to be fast, accurate, and inexpensive for the genetic analysis.According to this cost analysis, it is economically reasonable to use targeted NGS gene panels for genetic screening.

Genomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

Highlights • There were multiple introduction events of SARS-CoV-2 into Thailand. • One lineage, designated A/Thai-1, has expanded and has become a predominant and unique lineage in Thailand. • A major frame-shift mutation was found at the gene encoding ORF7a, a putative host antagonizing factor of the virus., Coronavirus Disease 2019 (COVID-19) is a global public health threat. Genomic surveillance of SARS-CoV-2 was implemented in March of 2020 at a major diagnostic hub in Bangkok, Thailand. Several virus lineages supposedly originated in many countries were found, and a Thai-specific lineage, designated A/Thai-1, has expanded to be predominant in Thailand. A virus sample in the SARS-CoV-2 A/Thai-1 lineage contains a frame-shift deletion at ORF7a, encoding a putative host antagonizing factor of the virus.

Coronavirus Disease 2019 (COVID-19) is a global public health threat. Genomic surveillance of SARS-CoV-2 was implemented during March 2020 at a major diagnostic hub in Bangkok, Thailand. Several virus lineages supposedly originated in many countries were found, and a Thai-specific lineage, designated A/Thai-1, has expanded to be predominant in Thailand. A virus sample in the SARS-CoV-2 A/Thai-1 lineage contains a frame-shift deletion at ORF7a, encoding a putative host antagonizing factor of the virus.

Thalassemia and hemoglobinopathy is a group of hereditary blood disorder with diverse clinical manifestation inherited by autosomal recessive manner. The Beta thalassemia/Hemoglobin E disease (HbE/βthal) causes a variable degree of hemolysis and the most severe form of HbE/βthal disease develop a lifelong transfusion-dependent anemia. Preimplantation genetic testing (PGT) is an established procedure of embryo genetic analysis to avoid the risk of passing on this particular condition from the carrier parents to their offspring. Preimplantation genetic testing for chromosomal aneuploidy (PGT-A) also facilitates the selection of embryos without chromosomal aberration resulting in the successful embryo implantation rate. Herein, we study the clinical outcome of using combined PGT-M and PGT-A in couples at risk of passing on HbE/βthal disease. The study was performed from January 2016 to December 2017. PGT-M was developed using short tandem repeat linkage analysis around the beta globin gene cluster and direct mutation testing using primer extension-based mini-sequencing. Thereafter, we recruited 15 couples at risk of passing on HbE/βthal disease who underwent a combined total of 22 IVF cycles. PGT was performed in 106 embryos with a 3.89% allele drop-out rate. Using combined PGT-M and PGT-A methods, 80% of women obtained satisfactory genetic testing results and were able to undergo embryo transfer within the first two cycles. The successful implantation rate was 64.29%. PGT accuracy was evaluated by prenatal and postnatal genetic confirmation and 100% had a genetic status consistent with PGT results. The overall clinical outcome of successful live birth for couples at risk of producing offspring with HbE/βthal was 53.33%. Conclusively, combined PGT-M and PGT-A is a useful technology to prevent HbE/βthal disease in the offspring of recessive carriers.

e16176 Background: The systemic treatment option of unresectable and advanced cholangiocarcinoma (CCA) is limited. The mainstay of treatment is gemcitabine and 5-FU based chemotherapy. Recently, there is the role of promising immunotherapy in MSI-HI CCA. This research aims to study molecular alterations and tumor microenvironment in Thai CCA patients in order to explore the novel treatment and the clinical correlations. Methods: Thirty-six CCA patients in Ramathibodi Hospital between year of 2018 and 2020 with adequate tissue were enrolled. NGS by ThermoFisher (comprehensive panel) for molecular profile and immunohistochemistry (IHC) staining for tumor microenvironment protein expression (CD3, CD4, CD8, CD20, CD138, CD163, FOXP3), and PD-L1 were performed. Clinical data was retrieved from electronic medical record. Survival analysis, univariate and multivariate analysis were performed by Cox-regression analysis. Stata version 16.0 was used for statistical analysis. Results: Intrahepatic CCA was the most common type of CCA in our population (81%). The top 10 common mutations were TP53 (69%), KMT2D 58%), KMT2B (52%), ARID1A (50%), FAT1 (50%), NOTCH3 (44%), FANCA (42%), SMAD4 (42%), EPHA2 (39%), and ARID1B (39%). We also found the interesting targetable genes in our CCA patients, such as, KRAS (33%), MTOR (33%), HER2 (31%), EGFR (31%), FGFR2 (28%), BRCA2 (25%), PIK3CA (22%), NTRK1 (19%), BRCA1 (17%), NTRK3 (11%), and BRAF (8%). Three patients had ALK fusion gene, 1 patient had NTRK1 fusion and the other one had NTRK3 fusion gene. Twelve out of 36 patients (33%) had MSI-HI and 56% of patients had TMB ≥ 10 mutations/megabase. The clinical factors significantly correlated with poor overall survival (OS) were unresectable disease, stage IV, bilirubin > 1.2, and CEA ≥ 22.5. The NF1, MTOR, HER2 mutations, and FOXP3-negative in stromal cells were significantly associated with longer OS, whereas EGFR, APC, FGFR2, and BRCA1 mutations might correlated with better OS. Conclusions: Our study demonstrated the unique molecular profile of Thai CCA patients. We found high prevalence of MSI-HI and high TMB in our population, together with the promising potential of targetable genes for developing targeted therapy in the future. FOXP3 expression was probably one of potential predictive biomarker for immunotherapy. Larger cohort should be explored.

We aim to present a case of a healthy infant born after intracytoplasmic sperm injection-in vitro fertilization (ICSI-IVF) with a preimplantation genetic diagnosis (PGD) for pantothenate kinase-associated neurodegeneration (PKAN) due to PANK2 mutation.ICSI-IVF was performed on a Thai couple, 34-year-old female and 33-year-old male, with a family history of PKAN in their first child. Following fertilization, each of the embryos were biopsied in the cleavage stage and subsequently processed for whole-genome amplification. Genetic status of the embryos was diagnosed by linkage analysis and direct mutation testing using primer extension-based mini-sequencing. Comprehensive chromosomal aneuploidy screening was performed using a next-generation sequencing-based strategy.Only a single cycle of ICSI-IVF was processed. There were seven embryos from this couple-two were likely affected, three were likely carriers, one was likely unaffected, and one failed in target genome amplification. Aneuploidy screening was performed before making a decision on embryo transfer, and only one unaffected embryo passed the screening. That embryo was transferred in a frozen thawed cycle, and the pregnancy was successful. The diagnosis was confirmed by amniocentesis, which presented with a result consistent with PGD. At 38 weeks of gestational age, a healthy male baby was born. Postnatal genetic confirmation was also consistent with PGD and the prenatal results. At the age of 24 months, the baby presented with normal growth and development lacking any neurological symptoms.We report the first successful trial of PGD for PKAN in a developing country using linkage analysis and mini-sequencing in cleavage stage embryos.

To investigate the cause(s) of a Thai male proband presenting low oxygen saturation by pulse oximetry (SpOAs Hb variant was suspected, Hb typing was determined by high-performance liquid chromatography and capillary electrophoresis, and subsequently Hb variant was identified by DNA sequencing. Complete blood counts were performed using automated blood cell counter and oxygen saturation was measured by pulse oximetry.Proband was compound heterozygous for Hb Louisville [β42(CD1)Phe→Leu] and Hb La Desirade [β129(H7)Ala→Val]. Of the proband's two sons, one was compound heterozygous for Hb Louisville and Hb E and the other for Hb La Desirade and Hb E. The former son had similar clinical features and laboratory findings with those of the proband while the latter showed had no abnormal clinical manifestations.This the first report of compound heterozygosity of Hb Louisville and Hb La Desirade in an individual of Southeast Asian ethnicity. Hb variant identification is crucial for genetic counseling and appropriate treatment in regions where hemoglobinopathies are common.

We report, for the first time, hemoglobin (Hb) Lansing-Ramathibodi [α87(F8)His → Gln; CACCAG (HBA1: c.264CG)] in four members of a Thai family presented with low measured oxygen saturation by pulse oximetry (SpO2), with discrepancy between low SpO2 and normal calculated oxygen saturation by arterial blood gas analysis, and no cyanosis or methemoglobinemia. The causative mutation is located in HBA1 whereas in previous reports of Hb Lansing the mutation is on HBA2, including that in a Japanese individual. The index and a male sibling also co-inherited Hb Pakse, a non-deletional α-thalassemia 2, resulting in mild reticulocytosis. Correct Hb identification is crucial for genetic counselling and, thereby, avoiding unnecessary investigation and treatment for spurious hypoxemia.

Background: Expanded carrier screening (ECS) is the process of screening individuals for hundreds of autosomal recessive (AR) and X-linked recessive (XLR) diseases. ECS result is beneficial for couples who want to make an informed reproductive choice. Though next-generation sequencing allows thousand of genes to be sequenced at once, the ideal ECS gene panel should only include diseases that its carriers are common in the population (1% or higher), and the prenatal diagnosis of the disease is allowed. However, designing the ECS panel for Thais can be difficult because the carrier frequency for most genetic diseases is unknown. Therefore, we retrospectively review our exome sequencing data from Thai patient to get an estimation of the carrier frequency. Method: Variants in 540 genes associated with AR and XLR from exome sequencing data of 178 unrelated Thai patients are filtered and matched against known pathogenic mutations in reputable mutation databases (OMIM, Clinvar, and HGMD). Result: Pathogenic mutations in SLC26A4 gene associated with Pendred syndrome has the highest carrier frequency of 2.81%. Usher syndrome (USH2A) are the second-most common with the carrier frequency of 2.25%. 1.69% of the patients also have pathogenic mutations in the gene related to impaired vision and impaired hearing such as OCA2 OTOF CLCN1 BSND and AHI1. Only 1.12% or less are the carrier of severe diseases such as myotonia congenita (CLCN1), lactic acidosis (POLG) and molybdenum cofactor deficiency (MOCS1). Conclusion: Inherited diseases that involved the impairment of hearing and vision is somewhat common among Thais. The ethical issue could arise when these diseases are included the ECS panel because they are not severe enough to allow for prenatal diagnosis. More discussion about ethics of ECS and more data on carrier frequency is needed to offer ECS ethically.