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We used a combination approach of conventional virus isolation and molecular techniques to detect human metapneumovirus (HMPV) in patients with severe acute respiratory syndrome (SARS). Of the 48 study patients, 25 (52.1%) were infected with HMPV; 6 of these 25 patients were also infected with coronavirus, and another 5 patients (10.4%) were infected with coronavirus alone. Using this combination approach, we found that human laryngeal carcinoma (HEp-2) cells were superior to rhesus monkey kidney (LLC-MK2) cells commonly used in previous studies for isolation of HMPV. These widely available HEp-2 cells should be included in conjunction with a molecular method for cell culture followup to detect HMPV, particularly in patients with SARS.

Background: Recently, much interest has been focused on the quantification of DNA in miscellaneous body fluids. In this study, the application is extended to classifying pleural effusions by measuring cell-free DNA in pleural fluid., Methods: We recruited 50 consecutive patients with pleural effusions with informed consent. Pleural fluids were centrifuged at 13000 g, with supernatants aliquoted for extraction and analysis of beta-globin DNA sequence by quantitative real-time PCR. Serum and pleural fluid biochemistries were performed to classify pleural effusions using the modified criteria of Light et al. (Ann Intern Med 1972;77:507-13). The ROC curve was plotted to determine the cutoff DNA concentration for classifying pleural fluids as transudates or exudates. Indicators of diagnostic accuracy were calculated for both pleural fluid DNA and modified criteria of Light et al., using the discharge, microbiologic, and histologic diagnoses as the reference standard., Results: The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.84-0.99]. At 509 genome-equivalents/mL, pleural fluid DNA alone correctly classified 46 of 50 pleural effusions with 91% sensitivity (95% CI, 76-98%), 88% specificity (95% CI, 64-98%), and positive and negative likelihood ratios of 7.7 (95% CI, 3.1-19.5) and 0.10 (95% CI, 0.04-0.27), respectively. With the modified criteria of Light et al., 43 of 50 pleural effusions were correctly classified with 97% sensitivity (95% CI, 91-100%) and 67% specificity (95% CI, 45-89%). There were significant correlations between cell-free DNA and both lactate dehydrogenase and total protein in pleural fluid, suggesting their common origin., Conclusions: Pleural fluid DNA concentrations are markedly increased in exudative effusions, making it a potential new tool to evaluate the etiologic causes of pleural effusions.

On the basis of previous observations in chromosomes 21 and 22, we hypothesize that there is a tissue-specific organization of cardiovascular gene transcripts in the human genome. To examine the distribution of heart-derived transcripts, we assigned a nonredundant set of 4628 fetal and 3574 adult known and uncharacterized cardiovascular expressed-sequence tags (cvESTs) to 5-Mb chromosomal 'windows' on the basis of publicly available sequence mapping data. On a whole-genome level (36,617 genes), chromosome 17 (19.2% in fetal, 16.5% in adult) contained the highest proportion of cvESTs, whereas chromosome Y (2.0% in fetal and adult) contained the lowest. In total, 50 of the 639 windows contained a significantly higher proportion of cvESTs (P < 0.003) compared with the genome-wide cvEST gene density, particularly on gene-dense chromosomes (that is, 17, 19, 22) as opposed to gene-rich chromosomes (for example, 1, 2, 11). This report provides insight into a possible role for complex tissue-specific gene regulation in the human genome.

Objective: To investigate the role of lipoprotein lipase (LPL) gene on Chinese patients with hypertriglyceridemic type 2 diabetes., Methods: Three subject groups, including hypertriglyceridemic group, normalipidemic type 2 diabetes group and healthy controls, were recruited and screened for sequence changes in LPL gene with PCR, SSCP, restriction analysis and direct DNA sequencing. LPL mass and activity in post-heparin plasma and in in vitro expression were investigated. Comparative modeling was performed via Swiss-PDB Viewer to provide the potential 2-D structures of wildtype and mutant proteins., Results: Four missense mutations, Ala71Thr, Val18Ile, Gly188Glu and Glu242Lys, were identified in patients with hypertriglyceridemic type 2 diabetes, and not in both normalipidemic diabetes and the control subjects. The four missense mutations were located in the highly conserved amino acid sites, which are involved in highly conserved exon 3, 5, or 6 regions. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed the differences to a great extent between the mutant and wide-type molecules., Conclusion: These results indicated that the 4 missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. The LPL deficiency predispose a progressive diabetic pathway to those affected individuals. LPL gene is one of susceptibility gene for hypertriglyceridemic type 2 diabetes.

Elevated plasma triglyceride and nonesterified fatty acid concentrations may cause insulin resistance. Lipoprotein lipase (LPL) is a rate-determining enzyme in lipid metabolism. To investigate the role of the LPL gene in Chinese patients with hypertriglyceridemic type 2 diabetes, 277 patients with type 2 diabetes and 241 healthy control subjects were recruited and screened for sequence changes in the LPL gene by PCR, SSCP, restriction analysis and direct DNA sequencing. Ten mutations were identified: four missense mutations, Ala71Thr, Val181Ile, Gly188Glu and Glu242Lys; one nonsense mutation Ser447Ter; and five silent mutations. Ser447Ter was found in both patients and controls with no significant difference in frequency. The four missense mutations were located in the highly conserved exon 3, 5, and 6 regions and in highly conserved amino acid sites. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed major differences between the mutant and wildtype molecules. These results indicated that the four missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. Based on our study and published data, a putative pathogenic pathway was suggested: LPL enzyme deficiency causes elevated plasma triglyceride level and subsequent insulin resistance; both increased free fatty acids and insulin resistance promote gluconeogenesis and hyperglycaemia, a vicious circle leading to type 2 diabetes., (Copyright 2003 Wiley-Liss, Inc.)

We describe a Chinese patient with glycogen storage disease type 1b presenting with failure to thrive and protuberant abdomen. The neutropenia was mild and the patient did not have fasting hypoglycemia. Direct DNA sequencing of the G6PT1 gene revealed the patient to be a compound heterozygote of a novel missense mutation, Y24H, and another missense mutation, P191L, which we had described previously. The mother is heterozygous for the Y24H mutation and the father is heterozygous for the P191L mutation. Y24H and P191L may be ethnic-specific mutations as they have not been reported in other populations. The DNA-based diagnosis of GSD 1b will enable us to make an accurate determination of carrier status and to perform prenatal diagnosis of this disease.

We screened 160 unrelated Chinese hypertriglyceridemic subjects for sequence alterations in the promoter and the 10 exons of the lipoprotein lipase (LPL) gene. We identified one reported mutation (L252R), one common polymorphism (S447X), and six novel mutations: V181I, C283Y, S298R and S338F (found in single individuals), L252V (in two individuals), and A71T (in three individuals). Screening of family members of the above probands revealed a total of 19 mutation carriers, most of whom, though not all, displayed reduced LPL activity and mass when compared to normolipidemic control subjects. In in vitro expression studies, A71T, V181I, L252R, L252V and C283Y decreased the specific activity of the gene product. Interestingly, S298R had no effect on the catalytic activity while S338F increased it. A71T and C283Y reduced the secretion of the mutant proteins significantly while V181I, S298R and S338F had mild effects only. The total LPL mass of all the mutant constructs was reduced compared to that of the wild type construct, probably due to the instabilities of the mutant mRNA or the mutant protein. The heterogeneity in phenotypic effects of these mutations is a likely consequence of their variable effects on proteoglycan binding, conformation and interactions with other secondary genetic or environmental factors., (Copyright 2002 Wiley-Liss, Inc.)

A case-control study was conducted on 1986 Hong Kong women to assess the risk of human papillomavirus (HPV) type 16 variants for cervical neoplasia. In total, 255 women were HPV-16 positive and were analyzed for E6 and E7 sequence variation. Two novel substitutions at E6 (T86I and Q116E) and 1 at E7 (R66W) were found. Most HPV-16 variants were of Asian (50.6%) or European (44.3%) lineage, and both lineages showed similar risk associations for high-grade and invasive cervical neoplasia. No increased risk was observed for the subclasses European variant and European 350G, which carry a higher risk for invasive cancer in some Western populations. The E7 N29S substitution, reported to have a higher risk in Korean women, was found equally distributed among normal and various degrees of neoplasia. The epidemiology and risk implication of HPV-16 variant infection in Hong Kong differ markedly from other parts of the world.

Human papillomavirus (HPV) type 58 has been found to be prevalent among Chinese patients with cervical cancer. This study examined the oncogenic risk of HPV58 variants in Hong Kong, a southern part of China. Altogether, 1924 women were studied: 42.8% with a normal cervix, 16.2% with cervical intraepithelial neoplasia (CIN) I, 12.7% with CIN II, 20.8% with CIN III, and 7.6% with invasive cervical cancer (ICC). The overall prevalence of HPV58 was 11.4% (220) and increased statistically significantly with the severity of neoplasia (P(trend)<.001, chi(2) test for trend). Among HPV58-positive women, the occurrence of E7 632C-->T (T20I) and E7 760G-->A (G63S) variants (T20I/G63S) showed a positive trend of association with the severity of neoplasia (P(trend)<.001, chi(2) test for trend). HPV58 variants carrying these two substitutions showed an odds ratio (OR) for ICC of 26.79 (95% confidence interval = 10.14 to 74.72), and this OR was 6.9-fold higher than the ORs of variants without these substitutions. Patients with CIN III or ICC who were also infected with T20I/G63S variants had a statistically significant younger age at diagnosis than those infected with other variants (median age = 37 years versus 48 years; P =.038, two-sided Mann-Whitney U test). Thus, HPV58 variants carrying E7 T20I/G63S substitutions may be associated with an increased risk for cervical cancer.

Despite similar clinical endpoints, heart failure resulting from dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) appears to develop through different remodeling and molecular pathways. Current understanding of heart failure has been facilitated by microarray technology. We constructed an in-house spotted cDNA microarray using 10,272 unique clones from various cardiovascular cDNA libraries sequenced and annotated in our laboratory. RNA samples were obtained from left ventricular tissues of precardiac transplantation DCM and HCM patients and were hybridized against normal adult heart reference RNA. After filtering, differentially expressed genes were determined using novel analyzing software. We demonstrated that normalization for cDNA microarray data is slide-dependent and nonlinear. The feasibility of this model was validated by quantitative real-time reverse transcription-PCR, and the accuracy rate depended on the fold change and statistical significance level. Our results showed that 192 genes were highly expressed in both DCM and HCM (e.g., atrial natriuretic peptide, CD59, decorin, elongation factor 2, and heat shock protein 90), and 51 genes were downregulated in both conditions (e.g., elastin, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase). We also identified several genes differentially expressed between DCM and HCM (e.g., alphaB-crystallin, antagonizer of myc transcriptional activity, beta-dystrobrevin, calsequestrin, lipocortin, and lumican). Microarray technology provides us with a genomic approach to explore the genetic markers and molecular mechanisms leading to heart failure.

Basal cell nevus syndrome (BCNS) is an autosomal dominant disease characterized by the presence of multiple basal cell carcinomas, odontogenic keratocysts, palmoplantar pits, and calcification in the falx cerebri caused by mutational inactivation of the PTCH gene. To investigate the molecular basis of BCNS in Chinese, we did a mutational analysis of the PTCH gene by performing denaturing high-performance liquid chromatography in three BCNS families. In this study, three novel mutations, two 1-bp frameshift insertions, i.e., 1468insA and 2392insC, and one 8-bp deletion, i.e., IVS5 + 1delGTAAGTGT, affecting a donor splice site, were identified. All the mutations cause a shift of the open reading frames and lead to premature termination of PTCH protein translation. Our results showed that mutational inactivation of the PTCH gene causes BCNS in Chinese.

Narcolepsy is a lifelong, crippling sleep disorder. Although the discovery of the hypocretin system has been a breakthough in genetics, the epidemiological aspects of narcolepsy remain elusive. Ethnic predisposition was suggested to partially account for the 2,500-fold difference in the reported prevalence rates of narcolepsy between Japanese (0.59%) and Israeli Jews (0.00023%). We carried out a general population study, conducting a random telephone survey with a structured questionnaire, which included a validated screening instrument (a Chinese version of the Ullanlinna Narcolepsy Scale). It was followed by clinical-polysomnographic-HLA confirmation of the subjects determined to be positive for narcolepsy based on the questionnaire. Of 9,851 subjects interviewed, 28 subjects (0.28%, 58% female) were screened positive. Ninety percent had a second detailed interview, 64% had HLA typing, and over half of them had a sleep assessment. Only three subjects were found to have genuine narcolepsy. The most common nonnarcolepsy diagnoses were sleep apnea syndrome and sleep-wake schedule disorder. The prevalence rate of narcolepsy in Southern (Hong Kong) Chinese was found to be 0.034% (95% confidence interval = 0.010-0.117%). All available narcoleptic subjects were HLA DRB1-1501 positive and 50% were DQB1-0602 positive. The prevalence rate of narcolepsy among Chinese is comparable to the rates for other populations in studies with stringent epidemiological designs, suggesting that major cross-ethnic differences in the prevalence rates of narcolepsy previously reported likely resulted from methodological limitations.

Objective: To investigate the role of a potential diabetes-related mitochondrial region, which includes two previously reported mutations, 3243A-->G and 3316G-->A, in Chinese patients with adult-onset type 2 diabetes., Methods: A total of 277 patients and 241 normal subjects were recruited for the study. Mitochondrial nt 3116 - 3353, which spans the 16S rRNA, tRNA(leu(UUR)) and the NADH dehydrogenase 1 gene, were detected using polymerase chain reaction (PCR), direct DNA sequencing, PCR-restriction fragment length polymorphism and allele-specific PCR. Variants were analyzed by two-tailed Fisher exact test. The function of the variants in 16S rRNA were predicted for minimal free energy secondary structures by RNA folding software mfold version 3., Results: Four homoplasmic nucleotide substitutions were observed, 3200T-->C, 3206C-->T, 3290T-->C and 3316G-->A. Only the 3200T-->C mutation is present in the diabetic population and absent in the control population. No statistically significant associations were found between the other three variants and type 2 diabetes. The 3200T-->C and 3206C-->T nucleotide substitutions located in 16S rRNA are novel variants. The 3200T-->C caused a great alteration in the minimal free energy secondary structure model while the 3206C-->T altered normal 16S rRNA structure little., Conclusions: The results suggest that the 3200T-->C mutation is linked to the development of type 2 diabetes, but that the other observed mutations are neutral. In contrast to the Japanese studies, the 3316G-->A does not appear to be related to type 2 diabetes.

We studied the in vivo cerebral metabolites and documented the presence of MECP2 gene mutations in six Chinese females with Rett syndrome. Magnetic resonance spectroscopy spectra from the frontal lobe (gray and white matter) and deep gray nuclei (basal ganglia and thalamus) of either side were obtained. N-acetylaspartate/total creatine, choline/total creatine, and N-acetylaspartate/choline ratios were analyzed and compared with six healthy age-matched female control subjects. MECP2 gene mutation was identified in four patients; one patient had polymorphism and one patient did not have gene mutation. N-acetylaspartate/total creatine of the frontal lobe of all patients (mean: 2.63, S.D. = 0.33) was decreased compared with age-matched control subjects (mean: 3.15, S.D. = 0.27), and the difference was statistically significant (P = 0.017) with a mean difference of 0.52 (95% CI = 0.68-0.36). The difference in all other metabolite ratios in the frontal lobe and deep gray nuclei were not statistically significant compared with age-matched control subjects. Mild frontal lobe and anterior temporal lobe atrophy was present in three patients. Proton-magnetic resonance spectroscopy is a sensitive method capable of detecting the biochemical changes in Rett syndrome and is able to detect changes before conventional magnetic resonance imaging. Our preliminary results suggest that reduction in N-acetylaspartate/total creatine ratio may not be related to the MECP2 mutation.

In a patient with sitosterolemia, we found two different mutations of the ATP-binding cassette, subfamily G, member 5 (ABCG5) gene. The first is a missense mutation that changes the amino acid residue at position 419 from arginine to histidine, i.e., R419H. The second is a novel splicing mutation affecting the invariant guanine at the first base of the donor splice site of intron 12, i.e., IVS12 + 1G --> A. The father of the patient is heterozygous for the missense mutation, and the mother is heterozygous for the splicing mutation. No mutations were found in the sister of the patient. Up until now, the missense mutation has only been found in Japanese patients with sitosterolemia. We believe that R419H in our Chinese patient may have the same origin as the mutation in the Japanese patients with sitosterolemia.

Isolated sulfite oxidase deficiency is a rare autosomal recessive disease, characterized by severe neurological abnormalities, seizures, mental retardation, and dislocation of the ocular lenses, that often leads to death in infancy. There is a special demand for prenatal diagnosis, since no effective treatment is available for isolated sulfite oxidase deficiency. Until now, the cDNA sequence of the sulfite oxidase (SUOX) gene has been available, but the genomic sequence of the SUOX gene has not been published. In this study, we have performed a DNA-based diagnosis of isolated sulfite oxidase deficiency in a Chinese patient. To do so, we designed oligonucleotide primers for amplification of the predicted exons and intron-exon boundaries of the SUOX gene obtained from the completed draft version of the human genome. Using overlapping PCR products, we confirmed the flanking intronic sequences of the coding exons and that the entire 466-residue mature peptide is encoded by the last exon of the gene. We then performed mutation detection using denaturing high-performance liquid chromatography (DHPLC). The DHPLC chromatogram of exon 2b showed the presence of heteroduplex peaks only after mixing of the mutant DNA with the wild-type DNA, indicating the presence of a homozygous mutation. Direct DNA sequencing showed a homozygous base substitution at codon 160, changing the codon from CGG to CAG, which changes the amino acid from arginine to glutamine, i.e., R160Q. The DNA-based diagnosis of isolated sulfite oxidase deficiency will enable us to make an accurate determination of carrier status and to perform prenatal diagnosis of this disease. The availability of the genomic sequences of human genes from the completed draft human genome sequence will simplify the development of molecular genetic diagnoses of human diseases from peripheral blood DNA., ((C)2002 Elsevier Science (USA).)