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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 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.

BackgroundPompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase (EC. 3.2.1.20) due to mutations in humanGAAgene. The objective of the present study was to examine clinical and molecular characteristics of infantile-onset Pompe disease (IOPD) in Thailand.MethodsTwelve patients with infantile-onset Pompe disease (IOPD) including 10 Thai and two other Asian ethnicities were enrolled. To examine the molecular characteristics of Pompe patients,GAAgene was analyzed by PCR amplification and direct Sanger-sequencing of 20 exons coding region. The novel mutations were transiently transfected in COS-7 cells for functional verification. The severity of the mutation was rated by study of the GAA enzyme activity detected in transfected cells and culture media, as well as the quantity and quality of the proper sized GAA protein demonstrated by western blot analysis. The GAA three dimensional structures were visualized by PyMol software tool.ResultsAll patients had hypertrophic cardiomyopathy, generalized muscle weakness, and undetectable or G (p.Tyr292X), c.1226insG (p.Asp409GlyfsX95), c.1538G > A (p.Asp513Gly), c.1895 T > G (p.Leu632Arg), and a previously reported rare allele of unknown significance: c.781G > A (p.Ala261Thr) were identified. The rating system ranked p.Tyr292X, p. Asp513Gly and p. Leu632Arg as class “B” and p. Ala261Thr as class “D” or “E”. These novel mutations were located in the N-terminal beta-sheet domain and the catalytic domain.ConclusionsThe present study provides useful information on the mutations ofGAAgene in the underrepresented population of Asia which are more diverse than previously described and showing the hotspots in exons 14 and 5, accounting for 62% of mutant alleles. Almost all mutations identified are in class A/B. These data can benefit rapid molecular diagnosis of IOPD and severity rating of the mutations can serve as a partial substitute for cross reactive immunological material (CRIM) study.

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.

Joubert syndrome and related disorders (JSRD) are a heterogeneous group of ciliopathies defined based on the mid-hindbrain abnormalities that result in the characteristic “molar tooth sign” on brain imaging. The core clinical findings of JSRD are hypotonia, developmental delay, abnormal eye movements and breathing abnormalities. To date, more than 30 JSRD genes that encode proteins important for structure and/or function of cilia have been identified. Here, we present 2 siblings with Joubert syndrome associated with growth hormone deficiency. Whole exome sequencing of the family identified compound heterozygous mutations in KIAA0753, i.e., a missense mutation (p.Arg257Gly) and an intronic mutation (c.2359-1G>C). The intronic mutation alters normal splicing by activating a cryptic acceptor splice site in exon 16. The novel acceptor site skips nine nucleotides, deleting three amino acids from the protein coding frame. KIAA0753 (OFIP) is a centrosome and pericentriolar satellite protein, previously not known to cause Joubert syndrome. We present comprehensive clinical descriptions of the Joubert syndrome patients as well as the cellular phenotype of defective ciliogenesis in the patients’ fibroblasts.

Background WNT1 mutations cause bone fragility as well as brain anomalies. There are some reported cases of WNT1 mutations with normal cognition. Genotype and phenotype correlation of WNT1 mutations has not been established. Case presentation Here we present two female siblings with osteogenesis imperfecta (OI) born to a consanguineous couple. Both sustained severe bone deformities. However, only the younger had severe brain anomalies resulting in an early death from pneumonia, while the older had normal intellectual development. Next generation sequencing showed a homozygous mutation, c.6delG, p.Leu3Serfs*36 in WNT1. To our knowledge, it is the most 5′ truncating mutation to date. Conclusion This report emphasizes the intrafamilial variability of brain anomalies found in this OI type and suggests that WNT1 may not be necessary for normal human cognitive development.

Background Mucopolysaccharidosis type I (MPS I) is a rare autosomal-recessive disorder caused by defects in alpha-L-iduronidase (IDUA), a lysosomal enzyme encoded by the IDUA gene. Herein, we characterized IDUA mutations underlying mucopolysaccharidosis type I intermediate form (Hurler-Scheie syndrome) and its molecular pathogenic mechanisms. Methods Clinical data, activity of the IDUA enzyme in leukocytes, and a mutation of the IDUA gene were analyzed. Pathogenesis associated with an IDUA mutation was further investigated by evaluating the mutant cDNA sequence, protein expression and activity in COS-7 cells. Results Five unrelated patients were identified to have clinical diagnosis of intermediate form of MPS I (Hurler-Scheie) and exhibited low-to-absent levels of leukocyte IDUA activity. Genetic analysis revealed homozygous c.*1T>C (p.X654R) mutation in two patients and compound heterozygosity between the c.*1T>C and another allele including c.265G>A (p.R89Q), c.935G>A (p.W312X), or c.1138 C>T (p.Q380X), each in a single patient. Sequencing the 3'RACE product of the c.*1T>C (p.X654R) allele indicated a 38-amino acids elongation of the mutant protein. COS-7 cells expressing IDUA with the mutations exhibited extremely low levels or complete absence of enzyme activity compared to wild-type IDUA. Western blot analysis detected no protein in p.W312X and p.Q380X samples, while an elevated molecular mass and a different pattern of protein bands were found in p.X654R specimen compared with the wild type IDUA. Conclusions Mutational spectrum underlying intermediate MPS I is expanded. Our data suggest that the p.X654R is an intermediate IDUA mutant allele with residual enzyme activity. It can lead to intermediate or milder form of MPS I depending on another associated allele.

© 2015 Thiboonboon et al. Background: Inborn errors of metabolism (IEM) are a rare group of genetic diseases which can lead to several serious long-term complications in newborns. In order to address these issues as early as possible, a process called tandem mass spectrometry (MS/MS) can be used as it allows for rapid and simultaneous detection of the diseases. This analysis was performed to determine whether newborn screening by MS/MS is cost-effective in Thailand. Method: A cost-utility analysis comprising a decision-tree and Markov model was used to estimate the cost in Thai baht (THB) and health outcomes in life-years (LYs) and quality-adjusted life year (QALYs) presented as an incremental cost-effectiveness ratio (ICER). The results were also adjusted to international dollars (I$) using purchasing power parities (PPP) (1 I$ = 17.79 THB for the year 2013). The comparisons were between 1) an expanded neonatal screening programme using MS/MS screening for six prioritised diseases: phenylketonuria (PKU); isovaleric acidemia (IVA); methylmalonic acidemia (MMA); propionic acidemia (PA); maple syrup urine disease (MSUD); and multiple carboxylase deficiency (MCD); and 2) the current practice that is existing PKU screening. A comparison of the outcome and cost of treatment before and after clinical presentations were also analysed to illustrate the potential benefit of early treatment for affected children. A budget impact analysis was conducted to illustrate the cost of implementing the programme for 10 years. Results: The ICER of neonatal screening using MS/MS amounted to 1,043,331 THB per QALY gained (58,647 I$ per QALY gained). The potential benefits of early detection compared with late detection yielded significant results for PKU, IVA, MSUD, and MCD patients. The budget impact analysis indicated that the implementation cost of the programme was expected at approximately 2,700 million THB (152 million I$) over 10 years. Conclusion: At the current ceiling threshold, neonatal screening using MS/MS in the Thai context is not cost-effective. However, the treatment of patients who were detected early for PKU, IVA, MSUD, and MCD, are considered favourable. The budget impact analysis suggests that the implementation of the programme will incur considerable expenses under limited resources. A long-term epidemiological study on the incidence of IEM in Thailand is strongly recommended to ascertain the magnitude of problem. Copyright

To analyze the clinical presentation and SLC39A4 mutations in zinc deficiency patients.The authors conducted a cross-sectional study on all cases of zinc deficiency treated at Queen Sirikit National Institute of Child Health between January 2004 and December 2012. Demographic data, clinical manifestations, laboratory results, treatment and outcome were analyzed. Genetic, SLC39A4 for acrodermatitis enteropathic (AE), mutation analysis was performed in all cases.There were 15 cases, 10 males and 5 females. The age of onset was between 2 and 10 months (median 3 months). Duration of the disease ranged between 3 days and 17 months (median 2 months). Acral and periorificial dermatitis, diarrhea and alopecia were present in 15 cases (100%), 12 cases (80%) and 8 cases (53%) respectively. The characteristic triad of acral and periorificial dermatitis, diarrhea and alopecia was observed in only 6 patients (40%). Serum zinc level ranged between 10 and 111 mcg/dl (mean 49.69 ± 33.87 mcg/100 ml). Low serum zinc level was observed in 10 cases (67%). All of the patients were treated with zinc sulfate 5 mg/kg/day. All cutaneous lesions and diarrhea had resolved within 7 days of starting therapy. A genetic study of SLC39A4 gene in our 15 patients revealed that 3 patients had homozygous c.1878_1879ins21 (p.G627_T633dup) in exonl2. These three patients have to receive lifelong zinc supplementation to prevent recurrence of the disease. The other twelve patients, who did not carry the gene mutation, did not have symptoms after discontinuance of oral zinc therapy. This is the first report of genetically confirmed acrodermatitis enteropathic in Thailand.Acrodermatitis enteropathica is a rare disease, which needs lifelong zinc supplementation. A genetic study of SLC39A4 gene will confirm the diagnosis. Most of patients presenting with characteristic triad of acral and periorificial dermatitis, diarrhea and alopecia in Thailand were acquired zinc deficiency. Early recognition and treatment of the disease will decrease morbidity and mortality.

Mucopolysaccharidosis (MPS) can be classified into 7 types according to the enzyme defects. Several countries use enzyme replacement therapy (ERT) as treatment for types 1, 2 and 6. ERT is very expensive:--therefore, to determine if this treatment could be made available in Thailand, it is important to know the numbers of the patients with MPS.To investigate the number and clinical profiles of MPS patients who visited the Queen Sirikit National Institute of Child Health (QSNICH) to determine the incidence of MPS in Thailand.Review of MPS patients' medical records with confirmed diagnosis by enzyme tests, who visited QSNICH from January 1999 to December 2013.Medical records showed that 22 MPS patients visited QSNICH during the past 15 years. Of these patients, 5 were MPS 1 patients (intermediate type or Hurler-Scheie syndrome), 8 were MPS2 patients (severe form), 1 was a MPS3 patient, 2 were MPS4 patients and 6 were MPS6 patients (severe form). The first clinical sign observed in MPS1 is joint contracture, whereas in MPS2 is delayed development. For MPS2, all except one patient had macrocephaly (head circumference is more than 90 percentile). Other growth parameters, including weight and height, in MPS2 patients were higher than average (50 percentile).MPS2 is the most common type of MPS in this study, followed by type 6 and 1. The difference in growth parameters seen in MPS2 suggest that it may be a factor in the development of MPS2.

Deaths attributed to birth defects are a major cause of infant and under-five mortality as well as lifetime disabilities among those who survive. In Thailand, birth defects contribute to 21% of neonatal deaths. There is currently no systematic registry for congenital anomalies in Thailand. Queen Sirikit National Institute of Child Health has initiated a Thailand Birth Defects Registry to capture birth defects among newborn infants.To establish the national birth defects registry in order to determine the burden of birth defects in Thailand.The birth defects data come from four main sources: National Birth Registry Database; National Health Security Office's reimbursement database; Online Birth Defect Registry Database designed to capture new cases that were detected later; and birth defects data from 20 participated hospitals. All data are linked by unique 13-digit national identification number and International Classification of Diseases (ICD)-10 codes. This registry includes 19 common structural birth defects conditions and pilots in 20 hospitals. The registry is hospital-based, hybrid reporting system, including only live births whose information was collected up to 1 year of age.3,696 infants out of 67,813 live births (8.28% of total live births in Thailand) were diagnosed with congenital anomalies. The prevalence rate of major anomalies was 26.12 per 1,000 live births. The five most common birth defects were congenital heart defects, limb anomalies, cleft lip/cleft palate, Down syndrome, and congenital hydrocephalus respectively.The present study established the Birth Defects Registry by collecting data from four databases in Thailand. Information obtained from this registry and surveillance is essential in the planning for effective intervention programs for birth defects. The authors suggest that this program should be integrated in the existing public health system to ensure sustainability.

The enzyme 6-pyruvoyl-tetrahydropterin synthase (PTPS, gene symbol: PTS) is involved in the second step of the de novo biosynthesis of tetrahydrobiopterin (BH4), which is a vital cofactor of nitric oxide synthases and three types of aromatic amino acid hydroxylases; the latter are important enzymes in the production of neurotransmitters. We conducted a study of PTS mutations in East Asia, including Taiwan, Mainland China, Japan, South Korea, the Philippines, Thailand and Malaysia. A total of 43 mutations were identified, comprising 22 previously reported mutations and 21 new discovered mutations. Among these, the c.155A>G, c.259C>T, c. 272A>G, c.286G>A and c.84-291A>G mutations were the most common PTS mutations in East Asia, while the c.58T>C and c.243G>A mutations were, respectively, specific to Filipinos and Japanese originating from Okinawa. Further studies demonstrated that each of the mutations listed above was in linkage disequilibrium to a specific allele of polymorphic microsatellite marker, D11S1347. These results suggest the presence of founder effects that have affected these frequent mutations in East Asia populations. In this context, D11S1347 should become one of the most reliable polymorphic markers for use in prenatal diagnosis among PTPS deficient families, especially where mutations are yet to be identified.

Reported here is a novel presentation of late onset glutaric aciduria type 2 in two Thai siblings. A 9-year-old boy presented with gradual onset of proximal muscle weakness for 6 weeks. The initial diagnosis was postviral myositis, and then polymyositis. Electromyography and nerve conduction velocity testing indicated a myopathic pattern. Muscle biopsy revealed excessive accumulation of fat. Acylcarnitine profiling led to the diagnosis of glutaric aciduria type 2. Immunoblot analysis of electron-transferring-flavoprotein and its dehydrogenase electron-transferring-flavoprotein dehydrogenase led to mutation analysis of the ETFDH gene, which revealed two different pathogenic mutations in both alleles and confirmed the diagnosis of glutaric aciduria type 2 caused by electron-transferring-flavoprotein dehydrogenase deficiency. The boy recovered completely after treatment. Later, his younger sibling became symptomatic; the same diagnosis was confirmed, and treatment was similarly effective. Acylcarnitine profiling was a crucial investigation in making this diagnosis in the presence of normal urine organic acid findings. Late onset glutaric aciduria type 2, a rare cause of muscle weakness in children, should be included in the differential diagnosis of myopathy.

Background Disorders of organic acid (OA) metabolism are generally detected by qualitative analysis of urine organic acids by gas chromatography/mass spectrometry (GC/MS) which was well established in developed countries since 1980s. Confirmation of the diagnosis of organic acid disorders by OA analysis, enzyme analysis and molecular study is a difficult task in developing countries. Methods During 2001–2004, we had analysed 442 urine samples in 365 patients and identified 12 cases of organic acid disorders. Results We identified the following disorders: alkaptonuria (ALK) = 1, isovaleric acidemia (IVA) = 3, propionic acidemia (PA) = 2, methylmalonic acidemia (MMA) = 3, glutaric aciduria, type I (GA-I) = 1, multiple carboxylase deficiency (MCD) = 1, and glutaric acidemia, type II (GA-II) = 1. Conclusions OA disorders had never been diagnosed in Thailand before, until GC/MS technology was introduced to Thailand in 2001. Urine OA analysis also provided a diagnostic clue to other inborn errors of metabolism including amino acid disorders, urea cycle disorders, disorders of carbohydrate metabolism, and mitochondrial fatty acid oxidation disorders. Since then, we were able to diagnose numerous disorders, which led to prompt treatment and better outcome in our patients.

Background Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia leading to a susceptibility to fractures. OI can be caused by mutations in several genes including BMP1. It encodes two isoforms, bone morphogenetic protein 1 (BMP1) and mammalian tolloid (mTLD); both have proteolytic activity to remove the C-propeptide from procollagen. Case presentation We report a Thai OI patient who had his first fracture at the age of three months. Using next generation sequencing, we successfully identified two novel compound heterozygous BMP1 mutations. One mutation, c.796_797delTT (p.Phe266Argfs*25) affects both BMP1 and mTLD isoforms, while the other, c.2108-2A > G, affects only the BMP1 isoform. Preservation of the mTLD may explain the relatively less severe clinical phenotype in this patient. Intravenous bisphosphonate was given from the age of 8 months to 5 years. He was free from fractures for 9 months before discontinuation. Conclusion This case expands the mutation spectrum of BMP1, strengthens the correlation between genotype and phenotype, and supports the benefits of bisphosphonate in OI patients with BMP1 mutations.