Biochemical Investigation of Young HospitalizedChinese Children: Results Over a 7-Year Period

C.P. Pang,1* L.K. Law,1 Y.T. Mak,1 C.C. Shek,3 K.L. Cheung,2 T.W.L. Mak,1 C.W. Lam,1 A.Y.W. Chan,4and T.F. Fok2

1Department of Chemical Pathology, Chinese University of Hong Kong, Hong Kong2Department of Paediatrics, Chinese University of Hong Kong, Hong Kong3Department of Chemical Pathology, Queen Elizabeth Hospital, Hong Kong4Department of Pathology, Princess Margaret Hospital, Hong Kong

During the seven years from January 1989 to De-cember 1995, we investigated 2,269 Chinese in-fants and young children for metabolic disordersin Hong Kong. These young patients, all aged un-der 4 years and originated from southern China,were ill with no apparent cause and had clini-cal manifestations suggestive of inherited meta-bolic diseases. A spot urine and a plasma samplewere obtained from each patient for biochemi-cal analysis, including urinary organic acid iden-tification and plasma amino acid analysis. Sixcases of mucopolysaccharidosis, four multiplecarboxylase deficiency, three 2-methylacetoace-tyl CoA thiolase deficiency, two methymalonicaciduria, one glutaric aciduria type I, one glu-taric aciduria type II, one a-oxoglutaric aciduria,and one case of orotic aciduria were detected.There were also single suspected cases of me-dium-chain acyl-CoA dehydrogenase deficiencyand isovaleric aciduria. No primary amino aciddisorder, such as phenylketouria and maplesyrup urine disease, has been detected. Our re-sults suggest that a different pattern of inheritedmetabolic diseases exists in the southern Chi-nese when compared with the Chinese in otherregions of China. Am. J. Med. Genet. 72:417–421,1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: inherited metabolic diseases;southern Chinese

INTRODUCTION

Inherited metabolic diseases (IMD) are a groupof genetically determined disorders involving abnor-malities in a variety of metabolic pathways and struc-tural proteins. Inherited defects have been detectedin the metabolism of all major body substances suchas carbohydrates, amino acids, lipids, metals, puri-nes, and pyrimidines [Beaudet et al., 1995, McKusick,1994]. More than 3,500 IMD have been identifiedand the frequencies of individual diseases varywith ethnicity and geographical location [McKusick1994].

In Asia, screening programs for common IMD havebeen carried out in China, India, Japan, Singapore,and Taiwan [Liu and Zuo, 1986; Chen et al., 1989; Raoet al., 1988; Kaur et al., 1994; Tada et al., 1984; Wong,1978; Hsiao, 1992; Tan et al., 1995]. In the Japanesepopulation, the incidence of maple syrup urine disease(MSUD), phenylketonuria (PKU), and galactosemiaare 1/526,000, 1/109,000, and 1/789,000, respectively,the first one similar to, but the last two about ten timeslower than in Caucasians [Tada et al., 1984]. Amongthe Chinese, incidence of most IMD are unknown ex-cept PKU [Lo, 1987]. Regional screening programs forPKU have documented an incidence of 1/16,500 in Bei-jing (northern China), 1/17,000 in Shanghai (middleChina), and 1/33,000 in Taiwan where the population

Contract grant sponsor: Medicine panel, the Chinese Univer-sity of Hong Kong.

*Correspondence to: Dr. C.P. Pang, Department of ChemicalPathology, The Chinese University of Hong Kong, Prince of WalesHospital, Shatin, N.T., Hong Kong.

Received 28 October 1996; Accepted 14 February 1997

TABLE I. Results of Metabolic Screening Tests

Metabolic dysfunctionNumber

of infants Remarks

Lactic acidosisa 20 Two had amino aciduriaThree had ketosis

Ketoacidosisa 17 One had aminoaciduriaDicarboxylic aciduriaa 8Transient tyrosinemia 17 All had gestation age

<32 weeksMucopolysaccharidosis 6Specific organic acidurias 15Total 83

aThese children did not have other specific biochemical abnormalities ex-cept the specified lactic acidosis, ketoacidosis, dicarboxylic, aciduria, oramino aciduria.

American Journal of Medical Genetics 72:417–421 (1997)

© 1997 Wiley-Liss, Inc.

TABLE II. Clinical and Biochemical Findings in Patients With Biochemical Lesions: Fifteen Cases of Specific Organic Aciduriasand Six Mucopolysaccharidoses*

Patient Sex/ageClinical/biochemical

abnormalities Urinary organic acids Biochemical lesions

LTY M/2y Metabolic acidosis 3-OH-isovaleric acid Multiple carboxylase deficiencySkin rash 3-methylcrotonylglycineNormal serum Tiglyglycine

biotinidase activity Methylcitric acidWHT F/12m Aminoacidemia Tiglyglycine Multiple carboxylase deficiency

Coma Methylcitric acidMetabolic acidosis 3-OH-propionic acidDied 3-OH-isovaleric acid

3-methylcrotonyglycineWLY F/18m Reye syndrome, ketosis 3-OH-isovaleric acid Multiple carboxylase deficiency

Methylcitric acid3-Methylcrotonylglycine

propionic acidLTK F/30m Lactic acidosis, died Lactic acid Multiple carboxylase deficiency

3-OH-isovaleric acid3-OH-propionic acidMethylcitric acid3-methylcrotonylglycine

CTM F/12m Metabolic acidosis, died 2-methyl-3-OH butyric acid 2-methylacetoacetyl3-OH-2-phenylbutyric acid CoA thiolase deficiency4-OH-phenylacetic acidTiglyglycine

HSY F/10m Hypotonia 3-OH-2-methylbutyric acid 2-methylacetoacetylMetabolic acidosis, died Tiglyglycine CoA thiolase deficiency

WCY M/2y Metabolic acidosis 2-methyl-3-OH-butyric acid 2-methylacetoacetylReye syndrome, died CoA thiolase deficiency

CLY F/2y Gross developmental Acetoacetic acid Methylmalonic aciduriaencephalopathy Adipic acid

Drowsiness, vomiting Suberic acidSebacic acidMethylcitric acidMethylmalonic acid3-OH-butyric acid3-OH-propionic acid

LKK M/4y Lactic acid Methylmalonic aciduriaMethylcitric acidMethymalonic acid3-OH-propionic acid

LKP M/2m Minor anomalies 2-oxoglutaric acid a-oxoglutaric aciduriaMicrocephaly

SPY F/4m Failure to thrive Orotic acid Orotic aciduriaHeart failureNormal plasma NH3

NKS M/5m Epilepsy Glutaric acid Glutaric aciduria type I3-OH-glutaric acid

CYK F/2m Epilepsy Adipic acid Glutaric aciduria type IISuberic acid (multiple acyl CoA dehydrogenaseMethylmalonic acid deficiency?)2-OH-glutaric acid

KSK F/3d Metabolic acidosis Lactic acid Isovaleric aciduria?Respiratory distress 3-OH-butyric acidDied of septicaemia at 3d 3-OH-isovaleric acid

LHM F/6m Reye syndrome Adipic acid MCAD deficiency?Plasma NH3 200 mmol/L Suberic acidHypoglycemia Sebacic acid

1,12-dodecanedioic acidHexanoylglycine (trace)

LKH M/4y Large head, minor Glycosaminoglycans MucopolysaccharidosisFacial anomalies 37 mmol/mmol creatinine Type IHepatosplenomegaly (Hurler disease)Typical dysostosis multiplexDeficiency in leukocyte

a-iduronidase activity

418 Pang et al.

is more related to the Chinese of middle and northernChina than those of southern China [Liu and Zuo,1986; Chen et al., 1989; Hsiao, 1992]. These incidencesare similar to those in Caucasians but higher than inthe Japanese. The incidence of PKU in the southernChinese is not known. No PKU has been documented inthe Chinese population in Hong Kong [Davies, 1992],which is located at the southernmost coast of Chinaand where the population is predominantly from south-ern China. It appears that a different pattern of IMDexists in southern Chinese.

To obtain an overview of the occurrence of metabolicdisorders in the southern Chinese, we have conducteda surveillance program on hospitalized pediatric pa-tients in Hong Kong.

MATERIALS AND METHODSClinical Specimens

The study was carried out at the Prince of WalesHospital, which is the only general hospital in a districthaving a population of about 1,000,000. During the sur-veillance period from January 1989 to December 1995,all infants and children admitted with one or more pre-sentation of IMD were investigated for metabolic dis-eases unless they had an obvious alternative diagnosisto account for the presentations. Manifestations thatwould lead to IMD screening included failure to thrive,convulsion, persistent hypoglycemia, peculiar odor,metabolic acidosis, hypotonicity, and hepatospleno-megaly [Beaudet et al., 1995]. From each infant, bloodand spot urine were obtained for the following analysis.

General biochemical investigations. In theurine specimens, osmolality, creatinine, pH, and gly-cosaminoglycan (GAG) were measured while reducingsubstances and ketoacids were determined qualita-tively. Plasma electrolytes, calcium, glucose, ammonia,lactic acid, pyruvic acid, and uric acid were measured.Also determined were blood gases, pH, and cell count.Thyroid function was assessed by a two-site chemilu-minometric immunoassay for serum thyroid stimulat-

ing hormone (Ciba Corning Diagnostics Corp., Med-field, MA).

Amino acid analysis. The amino acid residues inplasma or urine were derivatized to substituted isoin-doles with o-phthalaldehyde and b-mercaptoethanolprior to analysis by reversed phase high-performanceliquid chromatography [Van Eijk et al., 1988].

Organic acid identification. The organic acidresidues in acidified urine samples saturated with so-dium chloride were extracted by ethyl acetate and de-rivatized with N,O,bis-(trimethylsilyl)trifluoracet-amide to form trimethylsilyl ethers for analysis by gaschromatography mass fragmentography (GCMS)[Chalmers and Lawson, 1982]. For keto-acids their ox-ime derivatives were prepared by alkaline methoxyl-amine prior to GCMS analysis. Perfluorotributylaminewas used as the standard for mass fragmentation andphenylisobutyric acid the internal standard.

RESULTSOf the 76,532 pediatric admissions during the 7-year

period from January 1989 to December 1995, 2,269 in-fants or young children under age 4 years were inves-tigated for IMD. Among them, 83 (3.66%) were found tohave metabolic abnormalities (Table I). The childrenfound to have lactic acidosis, ketoacidosis, and dicar-boxylic aciduria were not investigated further, becausetheir clinical conditions improved quickly. No conclu-sion was drawn on the cause of the dicarboxylic acid-uria. Transient neonatal tyrosinemia was detected in17 infants age <4 weeks and having gestation age <32weeks. In their urine, N-acetyl-tyrosine, 4-hydroxy-phenyllactic acid and 4-hydroxyphenylacetic acid werecommonly found. The tyrosinemia subsided after 1–2weeks’ hospitalization without any specific treatment.No congenital hypothyroidism (CHT) was detected,probably because the condition would have been de-tected at birth by a territory-wide umbilical cord bloodCHT screening program. None of the patients investi-gated had persistent hyperuricemia or reducing sugarsin urine.

TABLE II. Continued

Patient Sex/ageClinical/biochemical

abnormalities Urinary organic acids Biochemical lesions

LKW M/4m No clinical signs MucopolysaccharidosisYounger brother of LKH Type IDeficiency in leukocyte

a-iduronidase activity(Hurler disease)

HKT M/4y “Coarse” facial appearance Glycosaminoglycans MucopolysaccharidosisHepatomegaly 117 mmol/mmol creatinine Type IIDeficiency in serum and Hunter disease

leukocyte iduronatesulphatase activity

WCH M/2y Deficiency in leukocyte Glycosaminoglycans Mucopolysaccharidosisand fibroblast 73 mmol/mmol creatinine Type VIarylsulphatase B activity Maroteaux-Lamy disease

CTK M/16m Developmental delay Glycosaminoglycans Mucopolysaccharidosis33 mmol/mmol creatinine

LSP M/3y Developmental delay Glycosaminoglycans Mucopolysaccharidosis259 mmol/mmol creatinine

*In the local Chinese population, urinary glycosaminoglycans excretion was lower than 24.5 and 20 (mmol/mmol creatinine) for children aged 1 and 5years, respectively (97.5th centile, Ngai and Ho 1996, unpublished data).

Biochemical Investigations of Chinese Children 419

Five male and ten female patients were diagnosed tohave specific organic acidurias, which is 1/151 (0.7%) ofthe patients investigated and 1/5,102 (0.02%) of thetotal pediatric admissions during the surveillance pe-riod (Table II). Among the four cases of multiple car-boxylase deficiency, enzyme assays in cultured skin fi-broblasts confirmed one case to be due to holocarbox-ylase deficiency and not biotinidase deficiency. Theother cases remained unclassified because of refusedconsent for skin biopsy to culture fibroblasts for en-zyme assays.

Six male cases of mucopolysaccharidosis (MPS) wereidentified (Table II). Two patients (LKH and hisbrother LKW) could be classified as type I, and onepatient (HKT) was type II and one patient (WCH) wastype VI. The remaining two MPS cases have not beenclassified by measurement of leukocyte enzyme activi-ties.

Including MPS, inherited metabolic diseases wereidentified in 21 children who constituted 1/108 (0.9%)of all children studied and 1/3,644 (0.03%) of all thepediatric admissions. None of these patients were fromconsanguineous marriages. All of them were unrelated,except LKH and LKW who both suffered from Hurler’sdisease.

DISCUSSION

China is a large country with a population of about1.2 × 109 people. Hong Kong is located in the southern-most part of China and most of her 6,000,000 popula-tion were the southern Chinese. In this surveillancestudy, 15 out of 2,269 babies investigated for metabolicdisorders have been shown to have specific organic ac-idurias indicative of congenital metabolic derange-ment. This is 0.02% of the total pediatric admissionsand is not a low percentage when compared with simi-lar investigations in hospitalized children in Cauca-sians [Galjaard, 1980]. Two disorders, multiple carbox-ylase and ketothiolase deficiencies whose frequenciesin the Chinese are not known and are rare among theCaucasian and Japanese, occurred more frequentlythan the other IMD in our patients. Meanwhile, fourcases of MSUD were identified over a period of 10 years[Feng et al., 1986; Pang and Ng, 1987; Pang et al.,1994]. Some cases were possibly overlooked, sincethere is no IMD screening program in Hong Kong.Without a territory-wide screening program, we cannotascribe the frequencies of these inborn enzyme defi-ciencies to be high or low.

We detected no amino acid disorder in this surveil-lance study. So far there is no reported case of PKU inHong Kong [Davies, 1992]. Our observation contrastssharply with that in northern and middle China wherethe incidence of PKU are at similarly high level as theCaucasian. Results of screening programs for PKU inmainland China and Taiwan are consistent [Liu andZuo, 1986; Hsiao, 1992]. Interestingly, the occurrenceof PKU in the Chinese in Singapore, who probably orig-inated from southern China, is reputed to be rare, al-though the actual incidence is unknown [Tan et al.,1995]. Meanwhile, an R413P mutation in the phenyl-alanine hydroxylase gene, which is caused by a G→C

substitution at the second base of codon 413 and leadsto classic phenylketonuria, was shown to be carried bya founder effect in the northern Chinese but not thesouthern Chinese [Wang et al., 1991]. These findingssupport a historical belief that the northern and south-ern Chinese are of two different origins, the formerbeing descendants of an indigenous population in theYellow River basin, and the latter the offsprings of an-cient settlers in the Yangtze River valley [Zhao andLee, 1989].

MPS is a relatively common condition in China[Zhou, 1995], although the actual incidence has notbeen determined We detected 6 cases of at least threetypes of MPS, i.e., Hurler, Hunter, and Maroteaux-Lamy syndromes. In the Chinese in Singapore, there isprobably more occurrence of MPS than organic acid-urias and amino acid disorders, although the actualfrequencies are not known [Tan et al., 1995]. In order toascertain a genetic pattern of this group of diseases inHong Kong, all existing MPS patients should be clas-sified by enzyme analysis. Detailed molecular geneticstudies should also be carried out to identify the seg-regation of gene mutations.

CONCLUSIONS

Our findings suggest that the pattern of IMD insouthern Chinese is different not only from other eth-nic groups but also from other regions in China. It isprobably a diversified pattern. Thus, genetics of meta-bolic diseases in this population deserves further ex-ploration including the establishment of their true in-cidence by territory-wide screening programs.

ACKNOWLEDGMENTS

We thank Dr. Anthony Fensom for performing theenzyme assays for the MPS patients.

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