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Disorders of sex development (DSD) can be classified as 46,XX DSD, 46,XY DSD, and sex chromosome DSD. Several underlying causes including associated genes have been reported. Steroidogenic factor-1 is encoded by the NR5A1 gene, a crucial regulator of steroidogenesis in the growth of the adrenal and gonadal tissues. It has been discovered to be responsible for 10 to 20% of 46, XY DSD cases. Here, we described a 2-month-old infant who had ambiguous genitalia and 46, XY. Using whole exome sequencing followed by polymerase chain reaction–Sanger sequencing, a novel heterozygous nonsense c.1249C > T (p.Gln417Ter) variant in the NR5A1 gene was identified. It is present in his mother but absent in his father and maternal aunt and uncle. At the age of 7 months, the patient received a monthly intramuscular injection of low-dose testosterone for 3 months in a row. His penile length and diameter increased from 1.8 to 3 cm and from 0.8 to 1.3 cm, respectively. The patient also had normal adrenal reserve function by adrenocorticotropic hormone stimulation test. This study identified a novel causative p.Q417X (c.1249C > T) variant in NR5A1 causing 46,XY DSD in a Thai boy which is inherited from his unaffected mother.

The spectra of underlying genetic variants for various clinical entities including focal segmental glomerulosclerosis (FSGS) vary among different populations. Here we described the clinical and genetic characteristics of biopsy-proven FSGS patients in Thailand. Patients with FSGS pathology, without secondary causes, were included in our study. Clinical laboratory and pathological data were collected. Whole-exome sequencing (WES) was subsequently performed. 53 unrelated FSGS patients were recruited. 35 patients were adults (66.0%), and 51 patients were sporadic cases (96.2%). Clinical diagnosis before kidney biopsy was steroid-resistant nephrotic syndrome (SRNS) in 58.5%, and proteinuric chronic kidney disease in 32.1%. Using WES, disease-associated pathogenic/likely pathogenic (P/LP) variants could be identified in six patients including the two familial cases, making the P/LP detection rate of 11.3% (6/53). Of these six patients, two patients harbored novel variants with one in the COL4A4 gene and one in the MAFB gene. Four other patients carried previously reported variants in the CLCN5, LMX1B, and COL4A4 genes. Four of these patients (4/6) received immunosuppressive medications as a treatment for primary FSGS before genetic diagnosis. All four did not respond to the medications, emphasizing the importance of genetic testing to avoid unnecessary treatment. Notably, the mutation detection rates in adult and pediatric patients were almost identical, at 11.4% and 11.1%, respectively. In conclusion, the overall P/LP variant detection rate by WES in biopsy-proven FSGS patients was 11.3%. The most identified variants were in COL4A4. In addition, three novel variants associated with FSGS were detected.

A heterozygous nonsense variant in the TIGIT gene was identified in a patient in Thailand who had severe COVID-19, resulting in lower TIGIT expression in T cells. The patient's T cells produced higher levels of cytokines upon stimulation. This mutation causes less-controlled immune responses, which might contribute to COVID-19 severity.

To maximize the potential of genomics in medicine, it is essential to establish databases of genomic variants for ethno-geographic groups that can be used for filtering and prioritizing candidate pathogenic variants. Populations with non-European ancestry are poorly represented among current genomic variant databases. Here, we report the first high-density survey of genomic variants for the Thai population, the Thai Reference Exome (T-REx) variant database. T-REx comprises exome sequencing data of 1092 unrelated Thai individuals. The targeted exome regions common among four capture platforms cover 30.04 Mbp on autosomes and chromosome X. 345 681 short variants (18.27% of which are novel) and 34 907 copy number variations were found. Principal component analysis on 38 469 single nucleotide variants present worldwide showed that the Thai population is most genetically similar to East and Southeast Asian populations. Moreover, unsupervised clustering revealed six Thai subpopulations consistent with the evidence of gene flow from neighboring populations. The prevalence of common pathogenic variants in T-REx was investigated in detail, which revealed subpopulation-specific patterns, in particular variants associated with erythrocyte disorders such as the HbE variant in HBB and the Viangchan variant in G6PD. T-REx serves as a pivotal addition to the current databases for genomic medicine.

Objective: To determine the clinicopathologic features and germline tumor protein p53 (TP53) mutation in children with adrenocortical carcinoma (ACC). Material and Methods: This was a retrospective study. From 2009 to 2018, children with ACC from King Chulalongkorn Memorial Hospital and Phramongkutklao Hospital were enrolled into the study. Clinical presentations and hormonal profiles were recorded. Mutation analyses of the TP53 gene were acquired using the next-generation-sequencing method which was performed for germline samples of all patients and the parents of those who tested positive. Result: Two males and six females with ACC were enrolled into this study. The median age at diagnosis was 25.5 months (range 10 to 67 months). All participants had virilization, either virilization only (n=3) or associated with Cushing (n=5). All participants had had surgery to completely remove all of the tumor and three participants had received adjuvant chemotherapy consisting of etoposide, doxorubicin, and cyclophosphamide. All participants had three different known mutations in the TP53 gene: c.1010G>A (p.R337H), c.916C>T (p.R306*) and c.743G>A (p.R248Q). Two of the three participants with TP53 mutations had pulmonary metastasis. One participant had wild-type TP53 and pulmonary recurrence occurred one year after diagnosis. The median follow-up time was 31 months (range 10 to 168 months. As of this writing, seven participants survived without evidence of recurrence. No second malignancy was found in all participants. One participant who had c.916C>T died of pulmonary metastasis 10 months after diagnosis. Conclusion: We successfully identified three different germline TP53 mutations in patients with ACC. Progression to pulmonary metastasis and recurrence were higher among participants with TP53 mutations. The treatment outcome of childhood ACC was good when surgery and adjuvant chemotherapy were used.

Muscular dystrophies and congenital myopathies are heterogeneous groups of inherited muscular disorders. An accurate diagnosis is challenging due to their complex clinical presentations and genetic heterogeneity. This study aimed to determine the utilization of whole exome sequencing (ES) for Thai paediatric patients with muscular disorders. Of 176 paediatric patients suspected of genetic/inherited myopathies, 133 patients received a molecular diagnosis after performing conventional investigations, single gene testing, and gene panels. The remaining 43 patients from 42 families could be classified into three groups: Group 1, MLPA-negative Duchenne muscular dystrophy (DMD) with 9 patients (9/43; 21%), Group 2, other muscular dystrophies (MD) with 18 patients (18/43; 42%) and Group 3, congenital myopathies (CM) with 16 patients (16/43; 37%). All underwent whole exome sequencing which could identify pathogenic variants in 8/9 (89%), 14/18 (78%), and 8/16 (50%), for each Group, respectively. Overall, the diagnostic yield of ES was 70% (30/43) and 36 pathogenic/likely pathogenic variants in 14 genes were identified. 18 variants have never been previously reported. Molecular diagnoses provided by ES changed management in 22/30 (73%) of the patients. Our study demonstrates the clinical utility and implications of ES in inherited myopathies.

Inborn errors of immunity are known to cause not only immunodeficiencies and allergies but also autoimmunity. Leukocyte immunoglobulin-like receptor B1 (LILRB1) is a receptor on leukocytes playing a role in regulating immune responses. No phenotypes have been reported to be caused by germline mutations in LILRB1. We aimed to identify the causative variant in a three-generation family with nine members suffering from one of the three autoimmune diseases—Graves’ disease, Hashimoto's thyroiditis, or systemic lupus erythematosus. Whole-genome linkage study revealed a locus on chromosome 19q13.4 with the maximum LOD score of 2.71. Whole-exome sequencing identified a heterozygous missense variant, c.479G > A (p. G160E) in LILRB1, located within the chromosomal-linked region, in all nine affected members. The variant has never been previously reported. Jurkat cells transfected with the mutant LILRB1, compared with those with the wild-type LILRB1, showed decreased phosphorylation of both LILRB1 and its downstream protein, SHP-1. Flow cytometry was used to study immunophenotype and revealed that LILRB1 was significantly lower on the surface of activated regulatory T lymphocytes (Treg) cells of patients. Single-cell RNA sequencing showed substantially increased M1-like monocytes in peripheral blood mononuclear cells of affected individuals. This study, for the first time, implicates LILRB1 as a new disease gene for autoimmunity.

Neurodevelopmental disorders (NDDs) are a group of conditions that are clinically and etiologically heterogeneous. Biallelic variants inWe identified 2 Thai siblings with NDDs. Clinical and radiologic features of the proband were described. The affected siblings and parents underwent whole-exome sequencing and PCR-Sanger sequencing.Clinical manifestations that have never been previously reported include morbid obesity, pancytopenia with severe infections, diabetes mellitus, cirrhosis, and renal failure, leading to deaths in their early 30s. Molecular studies identified a novel homozygous 1 base-pair duplication (c.360dup; p.Leu121Thrfs*27) in theThis study reported 1 novel single base-pair duplication, expanding the mutational spectrum, and described the clinical features establishing the entity of

The use of rapid DNA sequencing technology in severely ill children in developed countries can accurately identify diagnoses and positively impact patient outcomes. This study sought to evaluate the outcome of Thai children and adults with unknown etiologies of critical illnesses with the deployment of rapid whole exome sequencing (rWES) in Thailand. We recruited 54 unrelated patients from 11 hospitals throughout Thailand. The median age was 3 months (range, 2 days-55 years) including 47 children and 7 adults with 52% males. The median time from obtaining blood samples to issuing the rWES report was 12 days (range, 5-27 days). A molecular diagnosis was established in 25 patients (46%), resulting in a change in clinical management for 24 patients (44%) resulting in improved clinical outcomes in 16 patients (30%). Four out of seven adult patients (57%) received the molecular diagnosis which led to a change in management. The 25 diagnoses comprised 23 different diseases. Of the 34 identified variants, 15 had never been previously reported. This study suggests that use of rWES as a first-tier investigation tool can provide tremendous benefits in critically ill patients with unknown etiology across age groups in Thailand.

Background In approximately half of patients with epilepsy and intellectual disability (ID), the cause is unidentified and could be a mutation in a new disease gene. Patient description To determine the discovery of disease-causing mutation in a female patient with epilepsy and ID, we performed trio whole-exome sequencing, reverse transcription polymerase chain reaction (RT-PCR) followed by Sanger sequencing. Results Trio whole-exome sequencing was performed and revealed a novel de novo heterozygous stop-loss c.467A > T (p.*156Leuext*35) mutation in the ATP6V0C gene. Using RNA from leukocytes, RT-PCR followed by Sanger sequencing showed the existence of the mutant RNA, and real-time PCR demonstrated that the patient's ATP6V0C RNA level was approximately half of that in her parents, suggesting haploinsufficiency as a pathomechanism. Conclusion These findings, along with previous reports of individuals with similar phenotypes and variants in the same gene, substantiate ATP6V0C as a gene causing epilepsy with ID.

Chronic kidney disease of unknown etiology (CKDu) has been a problem in renal practice as indefinite diagnosis may lead to inappropriate management. Here, we report a 54-year-old father diagnosed with CKDu at 33 years old and his 8-year-old son with steroid-resistant nephrotic syndrome. Using whole-exome sequencing, both were found to be heterozygous for c.737G>A (p.Arg246Gln) in LMX1B. The diagnosis of LMX1B-associated nephropathy has led to changes in the treatment plan with appropriate genetic counseling. The previously reported cases with this particular mutation were also reviewed. Most children with LMX1B-associated nephropathy had nonnephrotic proteinuria with normal renal function. Interestingly, our pediatric case presented with steroid-resistant nephrotic syndrome at 8 years old and progressed to ESRD requiring peritoneal dialysis at the age of 15 years. Our report emphasized the need of genetic testing in CKDu for definite diagnosis leading to precise management.

AimsCongenital neutropaenia is a rare inherited disorder that mainly affects neutrophils causing severe infection. Mutations in several genes have been implicated in the disease pathogenesis. The genetic defects may vary in different populations, influenced by ethnicity and geographical location. Here we describe the clinical and genotypic characteristics of seven unrelated Thai cases with congenital neutropaenia.MethodsSeven unrelated patients with congenital neutropaenia were enrolled (5 female and 2 male) at King Chulalongkorn Memorial Hospital, Bangkok, Thailand. Clinical and laboratory data were collected. Whole exome sequencing (WES) analysis was performed in all cases.ResultsWES successfully identified disease-causing mutations in the ELANE gene in all cases, including two novel ones: a heterozygous 12 base pair (bp) inframe insertion (c.289_300dupCAGGTGTTCGCC; p.Q97_A100dup) and a heterozygous 18 bp inframe deletion (c.698_715delCCCCGGTGGCACAGTTTG; p.A233_F238delAPVAQF). Five other previously described ELANE mutations (p.Arg103Pro, p.Gly214Arg, p.Trp241X, p.Ser126Leu and p.Leu47Arg) were also detected.ConclusionsAll Thai patients with congenital neutropaenia in this study harboured causative mutations in the ELANE gene, suggesting it the most common associated with the disease. Two novel mutations were also identified, expanding the genotypic spectrum of ELANE.

Congenital myasthenic syndromes (CMS) comprise a heterogeneous group of genetic disorders of the neuromuscular junction. Next generation sequencing has been increasingly used for molecular diagnosis in CMS patients. This study aimed to identify the disease-causing variants in Thai patients. We recruited patients with a diagnosis of CMS based on clinical and electrophysiologic findings, and whole exome sequencing was performed. Thirteen patients aged from 2 to 54 years (median: 8 years) from 12 families were enrolled. Variants were identified in 9 of 13 patients (69%). Five novel variants and two previously reported variant were found in the COLQ, RAPSN and CHRND gene. The previously reported c.393+1G>A splice site variant in the COLQ gene was found in a majority of patients. Five patients harbor the homozygous splice site c.393+1G>A variant, and two patients carry compound heterozygous c.393+1G>A, c.718-1G>T, and c.393+1G>A, c.865G>T (p.Gly289Ter) variants. The novel variants were also found in RAPSN (p.Cys251del, p.Arg282Cys) and CHRND (p.Met481del). Molecular diagnosis in CMS patients can guide treatment decisions and may be life changing, especially in patients with COLQ mutations.

Benign adult familial myoclonic epilepsy type 1 (BAFME1) in several Japanese and Chinese families has recently been found to be caused by pentanucleotide repeat expansions in SAMD12. We identified a Thai family with six members affected with BAFME. Microsatellite studies suggested a linkage to the BAFME1 region on chromosome 8q24. Subsequently, long-read whole-genome sequencing showed the (TTTTA)(446)(TTTCA)(149) in intron 4 of SAMD12 in an affected member. Repeat-primed PCR and long-range PCR revealed that the pentanucleotide repeat expansions segregated with the disease status. Our Thai family is the first non-Japanese and non-Chinese family with BAFME1. SNP array showed that the aberrant repeats had the same haplotype as those previously determined in Japanese and Chinese patients suggesting a common ancestry. The variant is estimated to arise ~12,000 years ago.

Background GABAA receptors are ligand-gated chloride channels that regulate inhibitory neurotransmission in the central nervous system. Recently, monoallelic de novo mutations in GABRA5 resulting in altered inhibitory synapses were found in three patients with developmental and epileptic encephalopathy. Patient description: We report on a four-year and six-month-old girl with epilepsy and global developmental delay. Serial head growth measurement revealed postnatal onset microcephaly. Results Magnetic resonance imaging (MRI) of the brain was normal at the age of eight months and subsequently showed a decrease in white matter volume and thin corpus callosum at the age of 3 years. Using whole-genome sequencing, we identified the fourth patient harboring a de novo missense mutation in GABRA5. Interestingly, the c.880G > C (p.V294F) affects the same position found in two of the three previously reported patients. Conclusion This study suggests that the nucleotide c.880G is a mutation hotspot. Our patient also demonstrated significant seizure reduction after benzodiazepine. To our knowledge, this is the first case describing the favorable outcome of a GABAergic agent in seizure control for GABRA5-related developmental and epileptic encephalopathy.

Platelet demand has increased around the world. However, the inadequacy of donors, the risk of transfusion-transmitted infections and associated reactions, and the refractory nature of platelet transfusions are among the limitations of allogeneic platelet transfusions. To alleviate these problems, we propose generating platelets in a laboratory that do not induce alloimmunity to human leukocyte antigen (HLA) class I, which is a major cause of immune reaction in platelet transfusion refractoriness. Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) of a healthy Thai woman. We then knocked out the β2-microglobulin (β2m) gene in the cells using paired CRISPR/Cas9 nickases and sequentially differentiated the cells into haematopoietic stem cells (HSCs), megakaryocytes (MKs) and platelets. Silencing of HLA class I expression was observed on the cell surface of β2m-knockout iPSCs, iPSC-derived HSCs, MKs and platelets. The HLA-universal iPSC-derived platelets were shown to be activated, and they aggregated after stimulation. In addition, our in vivo platelet survival experiments demonstrated that human platelets were detectable at 2 and 24 hours after injecting the β2m-KO MKs. In summary, we successfully generated functional iPSC-derived platelets in vitro without HLA class I expression by knocking out the β2m gene using paired CRISPR/Cas9 nickases.

Ablepharon macrostomia syndrome (AMS) is a rare congenital disorder. To our knowledge, only 20 cases have been reported to date, and all in patients from Western countries. We report a case of AMS in a Thai patient, who presented at age 3 months with severe ectropion of both upper and lower eyelids, alopecia totalis, no palpable clitoris, and hypoplasia of both labia minora and labia majora. Trio whole exome sequencing analysis was performed, which revealed a heterozygous missense c.223G>A (p.Glu75Lys) variation in TWIST2. To our knowledge, this is the first reported case of AMS in a patient from Thailand and the first reported case of AMS in Asia.

Background: The signal transducer and activator of transcription 6 (STAT6) signaling pathway plays a central role in allergic inflammation. To date, however, there have been no descriptions of STAT6 gain-of-function variants leading to allergies in humans. Objective: We report a STAT6 gain-of-function variant associated with early-onset multiorgan allergies in a family with 3 affected members. Methods: Exome sequencing and immunophenotyping of T-helper cell subsets were conducted. The function of the STAT6 protein was analyzed by Western blot, immunofluorescence, electrophoretic mobility shift assays, and luciferase assays. Gastric organoids obtained from the index patient were used to study downstream effector cytokines. Results: We identified a heterozygous missense variant (c.1129G>A;p.Glu377Lys) in the DNA binding domain of STAT6 that was de novo in the index patient's father and was inherited by 2 of his 3 children. Severe atopic dermatitis and food allergy were key presentations. Clinical heterogeneity was observed among the affected individuals. Higher levels of peripheral blood TH2 lymphocytes were detected. The mutant STAT6 displayed a strong preference for nuclear localization, increased DNA binding affinity, and spontaneous transcriptional activity. Moreover, gastric organoids showed constitutive activation of STAT6 downstream signaling molecules. Conclusions: A germline STAT6 gain-of-function variant results in spontaneous activation of the STAT6 signaling pathway and is associated with an early-onset and severe allergic phenotype in humans. These observations enhance our knowledge of the molecular mechanisms underlying allergic diseases and will potentially contribute to novel therapeutic interventions.

Background Gaucher disease (GD) is a rare lysosomal storage disorder, characterized by hepatosplenomegaly and pancytopenia, with or without neurologic involvement. The disorder is categorized into three phenotypes: GD type 1 or nonneuronopathic GD; GD type 2 or acute neuronopathic GD; and GD type 3 or chronic neuronopathic GD. The purposes of this study were to describe clinical characteristics of Thai GD in patients diagnosed and/or followed up during 2010–2018 and to perform re-genotyping including analysis of GBA recombinant alleles which had not been investigated in Thai patients before. Results There were 27 patients from seven medical centers, enrolled in the study. All the cases had pediatric onset. GD3 (44.5%) was the most common phenotype, followed by GD2 (40.7%) and GD1 (14.8%), with one case of neonatal GD. The median age of onset for GD1, GD2, and GD3 was 72, 4 and 12 months, respectively, suggesting relatively earlier onset of GD1 and GD3 in Thai patients. All patients with GD1 and most patients with GD3 received ERT. Four patients with GD3 had ERT followed by HSCT. Patients with GD3 who received no or late ERT showed unfavorable outcomes. We identified 14 variants including two novel (p.S384F and p.W533*) and 12 reported pathogenic variants: p.L483P, p.N409S, p.R159W, p.P305A, p.A175G, p.D448H, p.V414L, IVS2+1G>A, IVS6-1G>C, IVS7+1G>C, IVS9-3C>G, and Rec1a. The p.L483P was the most prevalent allele found in this study, at 66% (33/50 alleles), followed by IVS2+1G>A, Rec1a, and IVS6-1G>C. Twenty-four percent of patients were reassigned with validated genotypes, most of whom (4 of 6) were patients with GD2. The [p.S384F + p.W533*] being compounded with p.L483P, was found in the patient with neonatal GD, suggesting that the p.S384F could potentiate the deleterious effect of the p.W533*, and/or vice versa. Conclusions Neuronopathic GD was strikingly prevalent among Thai affected population. Homozygous p.L483P was the most common genotype identified in Thai patients. Recombinant allele Rec1a and splicing mutations were associated with GD2 and severe cases of GD3. Mutation spectrum could be useful for designing stepwise molecular analysis, genetic screenings in population, and new therapeutic research for neuronopathic GD.

Background Inborn errors of immunity (IEI) comprise more than 400 rare diseases with potential life-threatening conditions. Clinical manifestations and genetic defects are heterogeneous and diverse among populations. Here, we aimed to characterize the clinical, immunological and genetic features of Thai pediatric patients with IEI. The use of whole exome sequencing (WES) in diagnosis and clinical decision making was also assessed. Methods 36 unrelated patients with clinical and laboratory findings consistent with IEI were recruited from January 2010 to December 2020. WES was performed to identify the underlying genetic defects. Results The median age of disease onset was 4 months (range; 1 month to 13 years) and 24 were male (66.7%). Recurrent sinopulmonary tract infection was the most common clinical presentation followed by septicemia, and severe pneumonia. Using WES, we successfully identified the underlying genetic defects in 18 patients (50%). Of the 20 variants identified, six have not been previously described (30%). According to the International Union of Immunological Societies (IUIS), 38.9% of these detected cases (7/18) were found to harbor variants associated with genes in combined immunodeficiencies with associated or syndromic features (Class II). Conclusion The diagnostic yield of WES in this patient cohort was 50%. Six novel genetic variants in IEI genes were identified. The clinical usefulness of WES in IEI was demonstrated, emphasizing it as an effective diagnostic strategy in these genetically heterogeneous disorders.

ObjectivesNeurodevelopmental disorders (NDDs) are a group of conditions that are clinically and etiologically heterogeneous. Biallelic variants inACBD6were previously reported in 7 patients with NDDs. Unfortunately, their clinical information remains very limited with descriptions of only their neurologic and craniofacial features. The purpose of this report is to expand the clinical phenotype of theACBD6-associated NDDs.MethodsWe identified 2 Thai siblings with NDDs. Clinical and radiologic features of the proband were described. The affected siblings and parents underwent whole-exome sequencing and PCR-Sanger sequencing.ResultsClinical manifestations that have never been previously reported include morbid obesity, pancytopenia with severe infections, diabetes mellitus, cirrhosis, and renal failure, leading to deaths in their early 30s. Molecular studies identified a novel homozygous 1 base-pair duplication (c.360dup; p.Leu121Thrfs*27) in theACBD6gene.DiscussionThis study reported 1 novel single base-pair duplication, expanding the mutational spectrum, and described the clinical features establishing the entity ofACBD6-associated NDDs.

Background: Primary immunodeficiency diseases (PIDs) comprise more than 400 rare diseases with potential life-threatening conditions. Clinical manifestations and genetic defects are heterogeneous and diverse among populations. Here, we aimed to characterize the clinical, immunological and genetic features of Thai pediatric patients with PIDs. The use of whole exome sequencing (WES) in diagnosis and clinical decision making was also assessed. Methods: 36 unrelated patients with clinical and laboratory findings consistent with PIDs were recruited from January 2010 to December 2020. WES was performed to identify the underlying genetic defects. Results: The median age of disease onset was 4 months (range; 1 month to 13 years) and 24 were male (66.7%). Recurrent sinopulmonary tract infection was the most common clinical presentation followed by septicemia, and severe pneumonia. Using WES, we successfully identified the underlying genetic defects in 18 patients (50%). Of the 20 variants identified, six have not been previously described (30%). According to the International Union of Immunological Societies (IUIS), 38.9% of these detected cases (7/18) were found to harbor variants associated with genes in combined immunodeficiencies with associated or syndromic features (Class II). Conclusion: The diagnostic yield of WES in this patient cohort was 50%. Six novel genetic variants in PID genes were identified. The clinical usefulness of WES in PIDs was demonstrated, emphasizing it as an effective diagnostic strategy in these genetically heterogeneous disorders.

Purpose Our meta-analysis of several ethnic groups (Japanese, Korean, Indian, Brazilian) revealed a significant genome-wide association between cold medicine-related SJS/TEN (CM-SJS/TEN) with severe ocular complications (SOC) and IKZF1 SNPs, suggesting that IKZF1 might be a potential marker for susceptibility to CM-SJS/TEN with SOC. In this study, we examined the association between CM-SJS/TEN with SOC and the IKZF1 SNPs in the Thai population. Methods 57 CM-SJS/TEN with SOC and 171 control samples were collected at Chulalongkorn University and Mahidol University. Genomic DNA samples were genotyped for the IKZF1 SNPs at Kyoto Prefectural University of Medicine in Japan using the TaqMan SNP genotyping assay. Results The four SNPs previously reported to be associated with CM-SJS/TEN with SOC in the Japanese were examined in the Thai samples. Although the number of Thai cases (n = 57) was small, a significant association between CM-SJS/TEN with SOC and IKZF1 SNPs which included rs4917014 (T vs G, OR = 2.9, p = 0.0012, Pc = 0.0049), rs4917129 (T vs C, OR = 2.8, p = 0.0026, Pc = 0.010) and rs10276619 (G vs A, OR = 1.8, p = 0.012, Pc = 0.048) was identified. Conclusion In addition to the Japanese, Korean and Indian populations, Thai cases with CM-SJS/TEN and SOC were significantly associated with IKZF1 SNPs. With our previous report of the critical role of IKZF1 in mucocutaneous inflammation, these results suggest that IKZF1 is important in the pathogenesis of CM-SJS/TEN with SOC.

Graphical abstract, Highlights • The mutations in two different genes should be sought in the patients with complex phenotypes. • The c.1531G>T in COL1A2 leading to OI and c.364C>T (p.R122W) in LHX4 to CPHD were found in a Thai boy. • The incomplete penetrance and loss-of-function are the features of p.R122W mutation in LHX4. • The mutation spectra of COL1A2 and LHX4 and pathomechanism of LHX4 are expanded., Genetic disorders have been shown to co-occur in individual patient. A Thai boy with features of osteogenesis imperfecta (OI) and combined pituitary hormone deficiency (CPHD) was identified. The causative mutations were investigated by whole exome and Sanger sequencing. Pathogenicity and pathomechanism of the variants were studied by luciferase assay. The proband was found to harbor a novel de novo heterozygous missense mutation, c.1531G > T (p.G511C), in COL1A2 leading to OI and a heterozygous missense variant, c.364C > T (p.R122W), in LHX4. The LHX4 p.R122W has never been reported to cause CPHD. The variant was predicted to be deleterious and found in the highly conserved LIM2 domain of LHX4. The luciferase assays revealed that the p.R122W was unable to activate POU1F1, GH1, and TSHB promoters, validating its pathogenic effect in CPHD. Moreover, the variant did not alter the function of wild-type LHX4, indicating its hypomorphic pathomechanism. In conclusion, the novel de novo heterozygous p.G511C mutation in COL1A2 and the heterozygous pathogenic p.R122W mutation in LHX4 were demonstrated in a patient with OI and CPHD. This study proposes that the mutations in two different genes should be sought in the patients with clinical features unable to be explained by a mutation in one gene.

PURPOSE: To evaluate the association of single nucleotide polymorphisms (SNPs) and the intronic expansion of a trinucleotide repeat (TNR) in the TCF4 gene with Fuchs endothelial corneal dystrophy (FECD) in a Thai population. METHODS: In total, 54 Thai FECD patients and 54 controls were recruited for the study. Five SNPs (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), previously reported to be associated with FECD, were genotyped by direct sequencing. The repeat length was determined by direct sequencing of PCR-amplified DNA (a short tandem repeat; STR assay) and by triplet repeat primed PCR (TP-PCR). RESULTS: Only one of the 54 patients with FECD harboured rs613872 (1.9%). Four SNPs (rs2123392, rs17089887, rs1452787, and rs1348047), which are not rare polymorphisms in the Thai population, were found in approximately half of the patients. Of the 54 patients, 21 (1 homozygous and 20 heterozygous patients; 39%) harboured a TNR ≥ 40, while 33 patients (61%) harboured a TNR

Epilepsy is a common neurological disorder and identification of its causes is important for a better understanding of its pathogenesis. We previously studied a Thai family with a type of epilepsy, benign adult familial myoclonic epilepsy type 4 (BAFME4), and localized its gene to chromosome 3q26.32-q28. Here, we used single-molecule real-time sequencing and found expansions of TTTTA and insertions of TTTCA repeats in intron 1 of YEATS2 in one affected member of the family. Of all the available members in the family-comprising 13 affected and eight unaffected-repeat-primed PCR and long-range PCR revealed the co-segregation of the TTTCA repeat insertions with the TTTTA repeat expansions and the disease status. For 1116 Thai control subjects, none were found to harbour the TTTCA repeats while four had the TTTTA repeat expansions. Therefore, our findings suggest that BAFME4 is caused by the insertions of the intronic TTTCA repeats in YEATS2. Interestingly, all four types of BAFMEs for which underlying genes have been found (BAFMEs 1, 4, 6 and 7) are caused by the same molecular pathology, suggesting that the insertions of non-coding TTTCA repeats are involved in their pathogenesis.

Bernard–Soulier syndrome (BSS) is a hereditary macrothrombocytopenia caused by defects in the glycoprotein (GP) Ib-IX-V complex. The mechanism of giant platelet formation remains undefined. Currently, megakaryocytes (MKs) can be generated from induced pluripotent stem cells (iPSCs) to study platelet production under pharmacological or genetic manipulations. Here, we generated iPSC lines from two BSS patients with mutations in different genes (GP1BA and GP1BB: termed BSS-A and BSS-B, respectively). The iPSC-derived MKs and platelets were examined under electron microscopy and stained by immunofluorescence to observe proplatelet formation and measure platelet diameters which were defined by circumferential tubulin. BSS-iPSCs produced abnormal proplatelets with thick shafts and tips. In addition, compared with the normal iPSCs, the diameters were larger in platelets derived from BSS-A and BSS-B with the means ± standard deviations of 4.34 ± 0.043 and 3.88 ± 0.045 µm, respectively (wild-type iPSCs 2.61 ± 0.025 µm, p

Intellectual disabilities (ID) are etiologically heterogeneous. Advanced molecular techniques could be helpful in identification of the underlying genetic defects. We aimed to characterize clinical and molecular features of three Thai patients with ID. Patient 1 had ID, hypotonia and lactic acidosis. Patient 2 had ID and growth failure. Patient 3 had ID, seizure, diarrhea and hypoglycemia. Whole exome sequencing found that Patient 1 was homozygous for a nonsense, c.1303C>T (p.Arg435Ter), mutation in FBXL4, a gene responsible for encephalomyopathic mitochondrial DNA depletion syndrome-13 (MTDPS13). Patient 2 was compound heterozygous for two novel mutations, c.3226C>T (p.Arg1076Ter) and c.3205C>T (p.Arg1069Ter), in UNC80, a known gene of infantile hypotonia with psychomotor retardation and characteristic facies-2 (IHPRF2). Patient 3 was homozygous for a novel missense, c.427T>C (p.Cys143Arg), mutation in ADK, a known gene of adenosine kinase deficiency leading to hypermethioninemia. This study expands the mutational spectra of ID genes.

Background Macrodactyly is a rare congenital deformity characterized by gigantism of all structures of the digits. Previous reports showed that the mosaic PIK3CA variants were associated with overgrowth syndromes including macrodactyly. Objectives To determine the genetic alteration in a Thai patient with enlarged soft tissue of the left middle and left fourth fingers with abnormal enlarged phalanges. Method A nerve and a skin piece were separated from a therapeutically surgically removed part of the enlarged digit. Skin fibroblasts were cultured from the removed skin piece. DNA was isolated from the nerve tissue, cultured skin fibroblasts, and peripheral blood leukocytes. Polymerase chain reaction (PCR) followed by Sanger sequencing of PIK3CA was performed. Results Mutation analysis revealed the c.3140A>T (p.(H1047L)) variant of PIK3CA in the nerve tissue and the cultured dermal fibroblasts but not in leukocytes from the peripheral blood. Conclusion The somatic c.3140A>T (p.(H1047L)) variant of PIK3CA was found in a Thai patient with isolated macrodactyly, the same as those previously identified in other populations.

Infantile Cushing’s syndrome is potentially found as part of McCune-Albright syndrome (MAS) which is caused by postzygotic somatic mutations of the GNAS gene. MAS is typically characterized by a triad of polyostotic fibrous dysplasia, café-au-lait skin pigmentation, and precocious puberty or other endocrine hyperfunction. Here, we describe a 2-month-old female infant with features of Cushing’s syndrome without café au lait spots, polyostotic fibrous dysplasia, and clinical evidence of other endocrine hyperfunction. Investigations demonstrated adrenocorticotropic hormone-independent Cushing’s syndrome with bilateral adrenal gland enlargement. Whole-exome sequencing of leukocytes identified a de novo heterozygous novel missense mutation (c.521G>A, p.Cys174Tyr) in the GNAS gene. The patient experienced clinical improvement of Cushing’s syndrome during ketoconazole treatment. Her clinical course was complicated by Pneumocystis jiroveci pneumonia. She also had shortened activated partial thromboplastin time indicating a hypercoagulable state and resulting in pulmonary embolism. She eventually manifested gonadotropin-independent precocious puberty at the age of 13 months after ketoco­nazole was discontinued. This patient demonstrated that Cushing syndrome can be the presenting sign of MAS in infancy. A high index of suspicion followed by genetic analysis is essential in order to establish a diagnosis.

Hereditary thrombocytopenia comprises extremely diverse diseases that are difficult to diagnose by phenotypes alone. Definite diagnoses are helpful for patient (Pt) management. To evaluate the role of whole exome sequencing (WES) in these Pts. Cases with unexplained long-standing thrombocytopenia and/or suggestive features were enrolled to the observational study. Bleeding scores and blood smear were evaluated. The variant pathogenicity from WES was determined by bioinformatics combined with all other information including platelet aggregometry, flow cytometry, and electron microscopy (EM). Seven unrelated Pts were recruited. All were female with macrothrombocytopenia. Clinical bleeding was presented in four Pts; extra-hematological features were minimal and family history was negative in every Pt. WES successfully identified all the 11 responsible mutant alleles; of these, four have never been previously reported. Pt 1 with GNE-related thrombocytopenia showed reduced lectin binding by flow cytometry, increased glycogen granules by EM and a novel homozygous mutation in GNE. Pts 2 and 3 had phenotypic diagnoses of Bernard Soulier syndrome and novel homozygous mutations in GP1BB and GP1BA, respectively. Pt 4 had impaired microtubule structures, concomitant delta storage pool disease by EM and a novel heterozygous TUBB1 mutation. Pt 5 had sitosterolemia showing platelets with reduced ristocetin responses and a dilated membrane system on EM with compound heterozygous ABCG5 mutations. Pts 6 and 7 had MYH9 disorders with heterozygous mutations in MYH9. This study substantiates the benefits of WES in identifying underlying mutations of macrothrombocytopenia, expands mutational spectra of four genes, and provides detailed clinical features for further phenotype-genotype correlations.

Congenital neutropaenia is a rare inherited disorder that mainly affects neutrophils causing severe infection. Mutations in several genes have been implicated in the disease pathogenesis. The genetic defects may vary in different populations, influenced by ethnicity and geographical location. Here we describe the clinical and genotypic characteristics of seven unrelated Thai cases with congenital neutropaenia.Seven unrelated patients with congenital neutropaenia were enrolled (5 female and 2 male) at King Chulalongkorn Memorial Hospital, Bangkok, Thailand. Clinical and laboratory data were collected. Whole exome sequencing (WES) analysis was performed in all cases.WES successfully identified disease-causing mutations in theAll Thai patients with congenital neutropaenia in this study harboured causative mutations in the

Hemoglobin (Hb) H/Constant Spring disease is a common nondeletional Hb H disease, typically causing a more severe phenotype than the deletional Hb H disease counterpart. Hb Tak, resulting from a dinucleotide insertion (+AC) at codon 146 of beta-globin gene, has an increased oxygen affinity and usually presents with polycythemia. We studied a case of a 4-year-old Thai boy with a severe, early-onset anemia. To our knowledge, he is the first reported patient with Hb H/Constant Spring disease and heterozygous Hb Tak. Trio-whole-exome sequencing does not identify other genetic variants that may contribute to the severity of anemia. The observation suggests that coinherited Hb H/Constant Spring and heterozygous Hb Tak lead to severe hemolytic anemia.

Context DAX1 (NR0B1) mutations cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH) in affected male patients. Affected individuals typically present with early-onset adrenal insufficiency and develop HH during puberty. Rare cases can present with late-onset adrenal insufficiency or other unusual phenotypes. Objectives We sought to identify and functionally characterize DAX1 mutations in seven Thai male subjects in six families with X-linked AHC. Patients and Methods Six patients had classic phenotypes with early-onset adrenal failure. One patient presented with late-onset Addison disease at 17 years. In the early-onset group, one patient had GnRH-independent sexual precocity at 3 years of age, and another patient had growth hormone deficiency. The DAX1 gene was sequenced from all patients, and the transcriptional activities of the identified mutations were assessed in vitro using luciferase assays. Results DAX1 mutations were identified in all patients, including three novel mutations [c.363delG (p.Gly122Valfs*142), c.1062delC (p.Ala355Profs*17), and c.1156C>T (p.Leu386Phe)] and three known mutations [c.1148_1149delGG (p.Gly383Aspfs*5), c.501_502insG (p.Ala170Argfs*15), and c.805_807delGTC (p.Val269del)]. Functional studies showed that the DAX1 mutants had lower levels of repressor activity on the StAR gene promoter compared with the wild-type DAX-1 protein. Conclusions This study describes unusual phenotypes and three novel mutations, extending the phenotypic and mutational spectra of DAX1 mutations.

Metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria (MC-HGA) (OMIM 614875) is a severe chondrodysplasia combined with a urinary excretion of D-2-hydroxyglutaric acid. Here, we reported the tenth case of this disease. A 15-year-old boy had symmetric radiolulencies in the metaphyses of the long bones suggesting enchondromatosis and platyspondyly. Remarkably, he manifested widespread cavernous hemangiomas including scalp, lips, tongue, larynx, and prepuce, with the onset of 3 years of age. Hemangiomas at the larynx had caused dyspnea and those in the oral cavity led to recurrent bleeding, requiring several surgical removals. These multiple and debilitating hemangiomas have never been previously reported in patients with MC-HGA. Mutation analyses including Sanger sequencing of genes involving in enchondromatosis and the metabolic pathway of D-2-hydroxyglutarate including PTHR1, D2HGDH, HOT, and IDH1, as well as whole-exome sequencing for proband-parent trio analysis and paired blood versus hemangioma studies showed no pathogenic variants. In summary, we reported the tenth patient with MC-HGA who manifested widespread and debilitating hemangiomas in several organs, expanding the clinical spectrum of MC-HGA.

BackgroundPolymorphisms in human leucocyte antigen (HLA) class I genes have been found to be associated with cold medicine (CM)-related Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) with severe ocular complications (SOC). Because ethnic differences in genetic predisposition to SJS/TEN among different populations have been proposed, we focused on Thai patients and investigated the association between HLA class I genotypes and CM-SJS/TEN with SOC.MethodsThis multicentre case–control study was conducted between September 2014 and August 2017. Seventy-one Thai patients with SJS/TEN with SOC and 159 healthy Thai controls were enrolled. HLA typing was performed. Genetic relationships were analysed using Fisher’s exact test.ResultsOf 71 patients with SJS/TEN with SOC (28 male, 43 female), 49 (69%) had a history of taking cold medications prior to SJS/TEN onset. The mean age of onset was 26.7±17.1 years (range, 2–77 years). HLA-B*44:03 (OR, 7.2, p=5.5×10-6, pc=1.1×10-4) and HLA-C*07:01 (OR, 6.1, p=7.1×10-6, pc=1.1×10-4) showed significant positive associations with Thai patients with CM-SJS/TEN with SOC. Additionally, 17 of 49 patients with CM-SJS/TEN with SOC (34.7%) significantly harboured the HLA-B*44:03 and HLA-C*07:01 haplotype compared with only 11 of 159 healthy controls (6.9%) (OR=7.1, p=5.5×10-6).ConclusionsHLA-B*44:03-HLA-C*07:01 haplotype is a potential risk factor for CM-SJS/TEN with SOC in the Thai population. This study supports that HLA-B*44:03 might be a common marker for CM-SJS/TEN with SOC in Eurasia populations, including European, Indian, Japanese and Thai.

Dentin sialophosphoprotein (DSPP) plays an important role in the mineralization of both dentin and bones. The Dspp null mice developed periodontal diseases. Patients with DSPP mutations have dentinogenesis imperfecta (DGI), but very little is known about their bone characteristics. This study aims to characterize alveolar bone cells of a DGI patient with DSPP mutation. Pathogenic variants were identified by whole exome and sanger sequencing. Cells isolated from the alveolar bones of a DSPP patient were investigated for their characteristics including cell morphology, attachment, spreading, proliferation, colony formation, mineralization, and osteogenic differentiation. We identified a Thai family with three members affected with autosomal dominant DGI harboring a heterozygous pathogenic missense mutation, c.50C > T, p.P17L, in exon 2 of the DSPP gene. The patients’ phenotypes presented deteriorated opalescent teeth with periapical lesions, thickening of lamina dura, furcation involvement, alveolar bone loss, and bone exostoses. The alveolar bone cells isolated from DSPP patient exhibited compromised proliferation and colony formation. Scanning electron microscope revealed altered cellular morphology and spreading. The DSPP cells showed deviated mRNA levels of OCN, ALP, and COL1 but maintained in vitro mineralization ability compared to the control. We demonstrate that the DSPP p.P17L mutant alveolar bone cells had compromised cell spreading, proliferation, colony formation, and osteogenic induction, suggesting abnormal bone characteristics in the patient with DGI caused by DSPP mutation. DSPP mutation can induce the behavior alterations of alveolar bone cells.

Objective To investigate physical characteristics and behaviours of dental pulp cells of teeth isolated from a dentinogenesis imperfecta (DGI) patient with a novel DSPP mutation. Subjects And Methods Whole exome and Sanger sequencing were employed to identify mutations. Physical characteristics of the teeth were examined. Pulp cells’ behaviors including cell proliferation, colony forming unit, osteogenic differentiation, pluripotent markers, and mesenchymal stem cell markers were investigated. Results The proband had opalescent brown primary teeth with extensive loss of enamel. Mutation analysis revealed a novel heterozygous 4-bp deletion, c.1915_1918delAAGT (p.K639QfsX674), in exon 5 of the DSPP associated with DGI. Analysis of the extracted primary incisor demonstrated a decrease in brightness but an increase in yellow and red chroma. The dentin showed reduced mineral density. The dentinal tubules were present in the predentin, but progressively collapsed in the dentin. The pulp cells exhibited markedly reduced CD105 expression, decreased cell proliferation, and smaller colony forming units. Conclusions We identified a novel mutation in the DSPP gene which disturbed dentin characteristics and pulp cells’ behaviours. Our study expands the mutation spectrum and understanding of pathologic dentin phenotypes related to the frameshift deletion in the DPP region of the DSPP gene. This article is protected by copyright. All rights reserved.

Objectives Glanzmann thrombasthenia (GT) is an autosomal recessive platelet disorder, caused by defects of the platelet integrin αIIbβ3 (GPIIb/IIIa) resulting from pathogenic mutations in either ITGA2B or ITGB3. It is characterized by spontaneous mucocutaneous bleeding. The molecular features of GT in Thailand have not been identified. This study aimed to determine the clinical and molecular features of unrelated Thai patients with GT. Methods Four patients with clinically suspected GT were recruited at the Division of Pediatric Hematology/Oncology, King Chulalongkorn Memorial Hospital. The diagnosis was based on clinical and hematological parameters as well as genetic analysis. Whole exome sequencing (WES) was performed in all cases. Results Of the four patients studied, the median age at first suspicion of GT was 2.5 years. All presented with severe bleeding symptoms (WHO bleeding scale 3). Flow cytometry to assess the surface GPIIb/IIIa complex showed reduced expression. By WES, we successfully identified seven mutant alleles in ITGA2B. One alteration, the c.2915dup (p.Leu973Alafs*63) was detected in two unrelated families. One patient was homozygous for the c.617T>A (p.Val206Asp). Of the five different mutations, three have never been previously described. These include a missense, c.617T>A (p.Val206Asp), a deletion, c.1524_1533del (p.Gln508Hisfs*3), and a nonsense, c.2344C>T (p.Arg782Ter). Conclusion This study reported three novel mutations expanding the genotypic spectrum of ITGA2B causing GT. This article is protected by copyright. All rights reserved.

Skeletal dysplasias are a complex group of more than 350 disorders with phenotypic and genotypic heterogeneity affecting bone and cartilage growth. We studied a 2-year-old girl and her 21-year-old mother with disproportionate short stature. In addition to typical features of hypochondroplasia found in both patients, the child had deformities of the extremity bones, metaphyseal flares, and bilateral transverse (Bowdler) fibular spurs with overlying skin dimples detected at birth. Intravenous pamidronate was started in the child since the age of 17 days, and then every two months. Exome sequencing revealed that the girl was heterozygous for a missense mutation (c.1651A>G, p.Ile538Val) in exon 13 of FGFR3, a known mutation for hypochondroplasia, inherited from her mother. Interestingly, the child also harbored compound heterozygous missense mutations in exon 12 of ALPL, c.1460C>T (p.Ala487Val) inherited from her mother and c.1479C>A (p.Asn493Lys) inherited from her healthy father. The former mutation was previously reported in perinatal hypophosphatasia while the latter was novel. Constantly reduced serum alkaline phosphatase levels including the one before the pamidronate administration and a substantially elevated level of plasma pyridoxal 5'-phosphate detected at age 28 months supported the diagnosis of hypophosphatasia. After a definite diagnosis was achieved, pamidronate was withdrawn at the age of 28 months. No adverse events were observed during pamidronate therapy. In conclusion, we describe a unique case with monoallelic FGFR3 and biallelic ALPL mutations leading to features of both hypochondroplasia and hypophosphatasia.

Objective To characterize clinical features and identify genetic causes of a patient with oculodentodigital dysplasia (ODDD). Subjects and methods Clinical, dental, radiological features were obtained. DNA was collected from an affected Thai family. Whole-exome sequencing was employed to identify the disease-causing mutation causing ODDD. The presence of the identified variant was confirmed by Sanger sequencing. Results The proband suffered with extensive enamel hypoplasia, polysyndactyly and clinodactyly of the 3rd–5th fingers, microphthalmia, and unique facial characteristics of ODDD. Mutation analysis revealed a novel missense mutation, c. 31C>A, p.L11I, in the GJA1 gene which encodes gap junction channel protein connexin 43. Bioinformatics and structural modeling suggested the mutation to be pathogenic. The parents did not harbor the mutation. Conclusions This study identified a novel de novo mutation in the GJA1 gene associated with severe tooth defects. These results expand the mutation spectrum and understanding of pathologic dental phenotypes related to ODDD.