Novel mutation affecting the pterin-binding site of PTS gene and review of PTS mutations in Thai patients with 6-pyruvoyltetrahydropterin synthase deficiency.

Tetrahydrobiopterin (BH(4)) deficiency comprises heterogeneous disorders resulting in hyperphenylalaninaemia (HPA) and lack of monoamine neurotransmitters. Among these, 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is the most common disorder. We report a female Thai patient with PTPS deficiency who was initially detected by newborn screening for HPA, and later treated by supplements of BH(4), L-dopa/carbidopa, and 5-hydroxytryptophan. Monitoring of serum prolactin representing dopamine sufficiency is used for optimizing the dosage of L-dopa. She showed a remarkable progress of development despite delayed treatment at 5 months of age. Mutation analysis revealed two heterozygous missense mutations of the PTS gene: c.259C>T (p.P87S) inherited from the father; and c.147T>G (p.H49Q) inherited from the mother. The latter is a novel mutation that affects the pterin-binding site of the PTPS enzyme. This novel mutation expands the mutation spectrum of PTPS deficiency. Notably, some PTS mutations have been reported in both Thai and Chinese patients. Whether these common mutations are the result of a founder effect with common ancestors of Thai and Chinese people or intermarriage between Thai and Chinese descents in Thailand remain unclear. In conclusion, severe neurological impairment from BH(4) deficiency could be prevented by newborn screening for HPA and proper metabolic management. However, pterin analysis for early diagnosis of BH(4) deficiency is still not available in most developing countries.

Molecular analysis of the iduronate-2-sulfatase gene in Thai patients with Hunter syndrome.

Molecular defects in the gene encoding the enzyme iduronate-2-sulfatase (IDS) result in Hunter disease (mucopolysaccharidosis type II, MPS II). To determine the molecular basis of MPS II in Thailand, the IDS gene was analysed in 20 Thai patients with Hunter syndrome from 18 unrelated families. A total of 19 different mutations, including 9 missense mutations, 3 nonsense mutations, 3 splice site alterations, 1 deletion, 2 indels, and 1 rearrangement were identified, 8 of which were novel (p.R101C, p.D148V, p.G224A, p.K227E, p.E254X, p.W337X, c.440_442delinsTT and c.720_731delinsTTTCAGATGTTCTCCCCAG). Evaluation of the IDS activity of two hemizygous variants identified in the same patient, p.R101C and p.R468Q, by expression of IDS with the individual mutations in COS 7 cells indicated that only the p.R468Q mutation affected IDS protein activity. Two exonic mutations, c.257C>T (p.P86L) and c.418G>A, were found to activate multiple cryptic splice sites, resulting in aberrantly spliced transcripts. Thus, MPS II in Thailand is caused by a diverse set of defects affecting both IDS protein production and activity.

IBEM in Thailand.

The incidence of IBEM in Thailand is yet unknown, however, by estimation it is generally accepted to be 1 in 5,000. From a recent survey in 7 medical schools from different parts of the country and a largest pediatric hospital in Bangkok, we found numerous cases of IBEM nationwide. These are amino acids disorders, carbohydrate disorders, urea cycle defects, peroxisomal, lysosomal storage disorders and many others. Since Thais are quite homogeneous in their genetic make-up; it is, therefore, very likely that IBEM is much more prevalent than we realized. With the exception of thalassemias, IBEM is very common in Thailand and other countries in the Asia-Pacific region. It is a real burden, indeed, since the consequences of IBEM are very serious, eg permanent damage to the CNS causing mental retardation, epilepsy, blindness, deafness and shortened life-span of individuals. Newborn screening for IBEM has been initiated at Siriraj and Ramathibodi Hospital Medical Schools of Mahidol University in 1993. There is yet no national screening program, although a pilot program was launched in the North, Northeast and the South by the Ministry of Health in 1991. Main problems we are facing include: only a handful of clinicians and scientists with expertise in IBEM; no well-equipped laboratory facilities; lack of funding and well-organized plan.

Detection of inborn errors of metabolism in Thai infants via gas chromatography and mass spectrometry.

We had studied inherited metabolic disorders at the Department of Pediatrics, Siriraj Hospital Faculty of Medicine, Mahidol University since 1987 using limited resources available and collaboration with other laboratories, both in Thailand and The United States. Since April 1998, we started a collaboration with MILS and Kanazawa Medical University, the Japan, studying inborn errors of metabolism in Asian Countries using urine filter paper and a new GC/MS method. We have since successfully discovered several patients with metabolic disorders. Out of 33 (high-risk) cases we sent for biochemical diagnosis (during April-July 1998), 13 abnormal results were found which is approximately 39.4%. Inherited metabolic disorders identified were as follows: medium-chain acyl CoA dehydrogenase deficiency (MCAD), multiple carboxylase deficiency (MCD), methylmalonic acidemia (MMA), Fanconi syndrome, galactosemia and neuroblastoma.

Neonatal screening for congenital hypothyroidism and phenylketonuria at Siriraj Hospital, Mahidol University, Bangkok, Thailand--a pilot study.

A newborn screening program for congenital hypothyroidism (CH) and phenylketonuria (PKU), a pilot study, was initiated at the Medical Genetics Unit, Department of Pediatrics, Siriraj Hospital Faculty of Medicine, Mahidol University in Bangkok, Thailand from January 1994 to December 1998, using dried blood spots (DBS). A total of 18,739 infants (out of 85,150 livebirths) were screened (22 % coverage). Three cases of congenital hypothyroidism (CH) were identified (incidence of 1: 6,246, livebirths), by enzyme linked immunosorbent (ELISA) and fluoroimmunoassays using a cut-off level of TSH >20 microlU/ml: the recall rate of 0.24%. The screening for PKU was done by fluorometric (Guthrie) and enzyme linked immunosorbent (ELISA) methods; using cut-off levels of phenylalanine > 4 mg/dl and > 3.6 mg/dl respectively, with a recall rate of 0.13%. There was no PKU found. Our study, a voluntary program, emphasizes the importance of parental education and consent; specimen collection and handling; appropriate follow-up and referral to specialists for treatment and counseling. Routine newborn screening for CH and PKU is being established to ascertain the maximum coverage, using recommendations and guidelines from this pilot study.

Detection of DMD gene deletions in Thai children patients.

For the dystrophin gene, it has been shown that about 65% of DMD/BMD patients have detectable deletions. The majority of deletions are clustered in exons 45-53 and at the 5' terminus. We studied 14 X-linked muscular dystrophy (DMD) Thai child patients for detection of gene deletions by amplification of nine exons plus the promoter of the dystrophin gene in two multiplex polymerase chain reactions that included hot spot region (exons 45-53 and 5' terminus). There were 8 DMD patients who had incomplete gene deletion and most of the deletions were around exon 49. PCR-base assays will allow deletion detection from dry blood spot samples and prenatal diagnosis.

Detection of inherited metabolic disorders via tandem mass spectrometry in Thai infants.

From a retrospective study in Medical Genetics Unit, Department of Pediatrics, Siriraj Hospital Faculty of Medicine, Mahidol University in Bangkok (1983-1988), the estimated pediatric patients with clinically suspected IEM are approximately 2-4% of total annual pediatrics admission of 5,000 or more. This is, a low estimation since survey from all teaching hospitals in the country including the largest Children's Hospital in Bangkok indicated the presence of numerous IEM. However, most IEM were clinically diagnosed with limited laboratory facilities. We started a collaboration with Magee Womens Hospital of Pittsburgh and NeoGen Screening, USA; using tandem mass spectrometry to diagnose high risk infants and children for IEM from July 1993 to March 1998. Of total 146 samples sent, we detected numerous metabolic disorders (11.2%) eg phenylketonuria, organic acidemia, maple syrup urine disease, isovaleric acidemia, methylmalonic acidemia, albinism, translocase/carnitine palmitoyltransferase type II, G6PD deficiency and lysinuric protein intolerance.

Lysosomal storage disorders in Thailand: the Siriraj experience.

Lysosomal storage disorders are a heterogeneous group of biochemical genetic disorders; currently 40-50 are known. The clinical phenotype is determined by the tissue distribution of the storage material and degree of enzyme deficiency. The genetic transmission is mostly autosomal recessive. Lysosomal storage disorders can be divided into three groups according to the major organ system pathology: (1) Primary involvement of the central nervous system without significant somatic or skeletal pathology. Disorders of grey matter, eg gangliosidosis and disorders of white matter eg the leucodystrophy are the most common; (2) Primary involvement of the reticuloendothelial system with or without associated neuropathology, eg Niemann-Pick disease and Gaucher disease; (3) Multisystem involvement in which skeletal manifestations are prominent features. The mucopolysaccharidosis and mucolipidoses are the two major forms with this clinical phenotype. Lysosomal storage disorders identified at Siriraj Hospital are neuronal ceroid lipofuscinosis, GMI gangliosidosis, mucolipidosis II, Maroteaux-Lamy, sialidosis, Sly syndrome, Hunter syndrome, Morquio syndrome, Gaucher disease, Niemann-Pick, Sandhoff disease, Pompe's disease and many more. Most patients came from the provinces where consanguinity is common. Confirmation usually is done by enzyme assays using skin fibroblast culture or leucocytes. Genetic counseling is extremely important and prenatal diagnosis is recommended to high-risk couple.

Genetic skeletal dysplasia in Thailand: the Siriraj experience.

Genetic skeletal dysplasias are a heterogeneous group of genetic disorders associated with abnormalities in the skeletal system frequently presenting with disproportionate short stature. There are over 100 distinct skeletal dysplasias which have been classified primarily on the basis of the clinical or radiographic characteristics. We have identified many genetic skeletal dysplasia disorders at Department of Pediatrics, Siriraj Hospital, Bangkok, Thailand. We have cases of achondroplasia, hypochondroplasia, pseudoachondroplasia, atelosteogenesis, pyknodysostosis, spondyloepiphyseal dysplasia (SED) congenita, spondylometaepiphyseal dysplasia (SMED), osteogenesis imperfecta type I, II and III, Ellis-van Creveld syndrome, cleidocranial dysostosis, thanatophoric dysplasia, rhizomelic chondrodysplasia punctata, trichorhinophalangeal syndrome, mucopolysaccharidosis I, II, IV and VI, mucolipidosis II, osteopetrosis, camptomelic dysplasia, metaphyseal dysplasia with spine involvement (Kozlowski type), Langer-Gideon syndrome and hypophosphatemic rickets. We have established a Genetic Skeletal Dysplasia Clinic at Siriraj Hospital since 1992, and see referrals from around the country. Genetic counseling is provided, including prenatal diagnosis and a multidisciplinary approach.

Plasma amino acid and urine organic acid analyses of methylmalonic acidemia in a Thai infant.

Methylmalonic acidemia is an inborn error of organic acid metabolism resulting from defects in methylmalonyl CoA mutase. Analysis of plasma free amino acids in a 15-month-old Thai infant by HPLC showed marked elevation of glycine. HPLC analysis of urinary organic acids showed high levels of methylmalonic acid.

Congenital generalized lipodystrophy, a case report.

Generalized lipodystrophy is a rare condition which can be divided into congenital and acquired types, based on the age at presentation and pattern of inheritance. The congenital type of generalized lipodystrophy or Lawrence-Seip syndrome presents in first two years of life and is inherited in an autosomal recessive pattern. The diagnosis is made on the basis of loss of body fat, muscular hypertrophy, acanthosis nigricans, hirsutism, hepatomegaly with fatty liver, hyperlipidemia and hyperglycemia with insulin resistance. A 2 1/2-year-old Thai girl with the clinical features of Lawrence-Seip syndrome is reported. Abnormal platelet function was detected in this girl.

Inherited metabolic disorders in Thailand--Siriraj experience.

The incidence of inborn errors of metabolism (IEM) in Thailand is yet unknown. However, by estimation it is generally accepted to be 1 in 5,000. From a survey in 7 medical schools from different parts of the country and a large pediatric hospital in Bangkok, we found numerous cases of IEM nationwidc. Thesc were amino acid disorders, carbohydrate disorders, urea cycle defects, peroxisomal, lysosomal storage disorders, and many others. Since Thais are quite homogeneous in their genetic make-up; it is, therefore, very likely that IEM is much more prevalent than we realized. With the exception of thalassemias, IFAM is probably very common in Thailand and other countries in the Asia-Pacific region. IEM identified were amino acid disorders eg phenylketonuria, maple syrup urine disease: urea cycle disorders eg ornithine transcarbamylase deficincy (OTC), argininosuccinic lyase deficiency (ALD), argininosuccinic acid synthetasc deficiency (ASD); glycogen storage disorders eg Pompe's discase, Von Gierkc's; organic acid disorders eg, isovaleric acidemia, methylmalonic acidemia. Lysosmal storagc disorders identified were GM1 gangliosidosis, mucolipidosis II, Hurler, Hunter, Maroteaux-Lamy, Sialidosis (neuraminidase deficicncy), Sly, Scheie, Gaucher, Niemann-Pick, Sandhoff and many other neurodegeneraative disorders identified were rhizomelic chondrodysplasia punctata (RCDP) and Zellweger. Recently fatty acid oxidation disorders: MCAD, translocase deficiency and multiple carbosxylase deficiency 9biotinidase deficiency) were also identified.

Plasma amino acid analyses in two cases of maple syrup urine disease.

Maple syrup urine disease is a rare inborn error of metabolism, characterized by elevated plasma levels of branched chain amino acids and urinary excretion of branched chain keto acids. Plasma amino acid levels in two subjects were followed by deproteinizing plasma, derivatizing the free amino acids with phenylisothiocyanate, and analysis by HPLC. The results indicate that valine, leucine and isoleucine are elevated in Maple syrup urine disease, and that leucine remains high even after dietary treatment.

Gaucher's disease;thirty-two years experience at Siriraj Hospital.

Gaucher's disease, a lysosomal disorder, is not a common disease in Thailand. During the period 1966-1998 we saw 20 patients with Gaucher's disease at the Department of Pediatrics. Siriraj Hospital. The patients came from different regions of the country but mostly from the central part of Thailand. There were 8 males and 12 females from 13 families of Thai, Thai-Chinese, Thai-Laos and Chinese-Chinese in origin. A history of consanguinity was present in 2 families. The age of onset was 2 months-4 years and the age when they were diagnosed was 4 months-15 years. The most common clinical features included splenomegaly, hepatomegaly, growth retardation, pallor, bleeding disorders and neurological abnormalities. The diagnosis was made by the clinical manifestations, hematologic complications and demonstration of Gaucher cells in the bone marrow and/or other tissues. In one family, the diagnosis was confirmed by evaluation of glucocerebrosidase activities in skin fibroblasts. The management of these patients was symptomatic ie packed red cell and platelet transfusion, splenectomy and other supportive measures. Most patients died of bleeding or infection at an early age.

Normal plasma free amino acid levels in Thai children.

Analysis of plasma free amino acid levels is important for diagnosis of inborn errors of metabolism. Traditionally, this is performed using commercially available dedicated amino acid analyzers, but few such instruments are available in Thailand, and many are not used in routine operations. Here, the authors describe the analysis of plasma free amino acid levels in 57 normal children by reverse-phase HPLC and pre-column derivatization with phenylisothiocyanate. Plasma free amino levels are reported as mean +/- SD and 95 per cent confidence interval of mean for each of 5 age groups: 0-6 months; 6-12 months; 1-3 years; 3-6 years; 6-12 years. Mean amino acid levels were generally similar in all age groups (p > or = 0.01), except that hydroxyproline tended to be higher in the 0-6 months age group compared to other age groups (p<0.01). Comparisons were made between the present data with the normal free plasma amino acid levels in children of similar age groups reported both in Thailand and overseas in terms of both mean +/- SD and maximum and minimum values. Overall, our methodology involving HPLC can identify 35 amino acid derivatives, including all the major amino acids except for cysteine, which is substantially more than the number reported in earlier work on plasma free amino acid levels in normal Thai children. Moreover, the present methodology gives mean +/- SD values similar to an overseas report. For these reasons, HPLC should be considered as an alternative approach in laboratories, where demand does not justify the need for dedicated amino acid analyzers. However, there can be substantial variations between the results from different laboratories, and each laboratory should establish its own normal values.

An infant with cartilage-hair hypoplasia due to a novel homozygous mutation in the promoter region of the RMRP gene associated with chondrodysplasia and severe immunodeficiency.

Cartilage-hair hypoplasia (CHH) is a rare autosomal-recessive disorder characterized by short-limbed dwarfism, sparse hair, and immune deficiency. It is caused by mutations in the RMRP gene, which encodes the RNA component of the mitochondrial RNA-processing ribonuclease (RNase MRP). Several mutations have been identified in its promoter region or transcribed sequence. However, homozygous mutations in the promoter region have been only reported in a patient with primary immunodeficiency without other features of CHH. We report on a Thai girl who first presented with chronic diarrhea, recurrent pneumonia, and severe failure to thrive, without apparently disproportionate dwarfism. The diagnosis of CHH was made after the severe wasting was corrected, and disproportionate growth became noticeable. The patient had the typical features of CHH, including sparse hair and metaphyseal abnormalities. The immunologic profiles were consistent with combined immune deficiency. Mutation analysis identified a novel homozygous mutation, g.-19_-25 dupACTACTC, in the promoter region of the RMRP gene. Identification of the mutation enabled us to provide a prenatal diagnosis in the subsequent pregnancy. This patient is the first CHH case with the characteristic features due to the homozygous mutation in the promoter region of the RMRP gene. The finding of severe immunodeficiency supports that promoter mutations markedly disrupt mRNA cleavage function, which causes cell-cycle impairment.

Supernumerary small chromosomal anomaly: report of three cases including one with a familial inversion of chromosome 5.

Three cases of a small supernumerary chromosomal anomaly of essentially unknown origin associated with wide variability of clinical expression are reported. Case II has, in addition, a pericentric inversion of chromosome 5, which enables us to propose a mechanism, involving an unsuccessful crossing over, for the origin of the supernumerary chromosome.