Jer Yuarn Wu

ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease

Tandem mass spectrometry was applied to detect derangements in the pathways of amino acid and fatty acid metabolism in N-ethyl-N-nitrosourea-treated (ENU-treated) mice. We identified mice with marked elevation of blood branched-chain amino acids (BCAAs), ketoaciduria, and clinical features resembling human maple syrup urine disease (MSUD), a severe genetic metabolic disorder caused by the deficiency of branched-chain alpha-keto acid dehydrogenase (BCKD) complex. However, the BCKD genes and enzyme activity were normal. Sequencing of branched-chain aminotransferase genes (Bcat) showed no mutation in the cytoplasmic isoform (Bcat-1) but revealed a homozygous splice site mutation in the mitochondrial isoform (Bcat-2). The mutation caused a deletion of exon 2, a marked decrease in Bcat-2 mRNA, and a deficiency in both BCAT-2 protein and its enzyme activity. Affected mice responded to a BCAA-restricted diet with amelioration of the clinical symptoms and normalization of the amino acid pattern. We conclude that BCAT-2 deficiency in the mouse can cause a disease that mimics human MSUD. These mice provide an important animal model for study of BCAA metabolism and its toxicity. Metabolomics-guided screening, coupled with ENU mutagenesis, is a powerful approach in uncovering novel enzyme deficiencies and recognizing important pathways of genetic metabolic disorders.

Association of vitamin D receptor gene BsmI polymorphisms in Chinese patients with systemic lupus erythematosus

The purpose of this study was to evaluate whether vitamin D receptor (VDR) genes BsmI polymorphisms were markers for susceptibility to or severity of systemic lupus erythematosus (SLE) in Chinese patients in Taiwan. The study included 47 Chinese patients with SLE. In addition, 90 unrelated, healthy individuals living in central Taiwan served as control subjects. Each polymorphism was detected as a result of polymerase chain reaction (PCR)-based restriction analysis. A PCR product length was determined to be 580 bp (BB) whereas two fragments of 405 and 175 bp were determined to be excisable lengths (bb) by BsmI endonuclease. The relationship between Bsm polymorphisms and clinical manifestations of SLE was evaluated. We found that BB was significantly more common and bb less common in SLE than in control group (w2 54.2, P 0.0001). In addition, the frequency of B allele was also significantly more common in patients with SLE than in the healthy control subjects (w2 38.7, P 0.0001), giving an odds ratio of 7.14 (95% confidence interval 3.53–14.4). In the SLE patients, we did not detect any associations of VDR genotype with the clinical, laboratory profiles, or lupus nephritis (w2 2.34, P 0.3). This study indicated an increased distribution of VDR BB genotype and B allelic frequencies in the Chinese SLE patients in Taiwan. However, there were no associationsbetween the frequency of VDR allelic variations and clinical manifestations, laboratory profiles, or lupus nephritis.

Smoking and genetic risk variation across populations of European, Asian, and African American ancestry--a meta-analysis of chromosome 15q25.

Recent meta‐analyses of European ancestry subjects show strong evidence for association between smoking quantity and multiple genetic variants on chromosome 15q25. This meta‐analysis extends the examination of association between distinct genes in the CHRNA5‐CHRNA3‐CHRNB4 region and smoking quantity to Asian and African American populations to confirm and refine specific reported associations. Association results for a dichotomized cigarettes smoked per day phenotype in 27 datasets (European ancestry (N = 14,786), Asian (N = 6,889), and African American (N = 10,912) for a total of 32,587 smokers) were meta‐analyzed by population and results were compared across all three populations. We demonstrate association between smoking quantity and markers in the chromosome 15q25 region across all three populations, and narrow the region of association. Of the variants tested, only rs16969968 is associated with smoking (P < 0.01) in each of these three populations (odds ratio [OR] = 1.33, 95% CI = 1.25–1.42, P = 1.1 × 10−17 in meta‐analysis across all population samples). Additional variants displayed a consistent signal in both European ancestry and Asian datasets, but not in African Americans. The observed consistent association of rs16969968 with heavy smoking across multiple populations, combined with its known biological significance, suggests rs16969968 is most likely a functional variant that alters risk for heavy smoking. We interpret additional association results that differ across populations as providing evidence for additional functional variants, but we are unable to further localize the source of this association. Using the cross‐population study paradigm provides valuable insights to narrow regions of interest and inform future biological experiments. Genet. Epidemiol. 36:340–351, 2012.

Mutation analysis of Wilson disease in Taiwan and description of six new mutations

Wilson disease is an autosomal recessive disorder of copper metabolism. Mutation screening in Wilson disease has led to the detection of at least 89 disease‐specific mutations. Some mutations appear to be population specific, while others are common to many populations. In this study, 38 Taiwanese patients with Wilson disease were screened using single‐strand conformation polymorphism analysis, followed by direct DNA sequencing. We found 12 different mutations, six of which were novel. All our detected mutations were found to be in eight exons. Four mutations in three loci (Arg778Gln, Arg778Leu, Gly943Asp, and Pro992Leu) accounted for about 58% of the mutant alleles we detected. Using an RNA transcriptional assay, we confirmed that both of our detected splice‐site mutations resulted in exon skipping. Hum Mutat 12:370–376, 1998.

Interleukin-1β and interleukin-1 receptor antagonist gene polymorphisms in rheumatoid arthritis

The purpose of this study was to evaluate if IL-1β (IL-1β promoter and IL-1β exon 5) and IL-1 receptor antagonist (IL-1Ra) gene polymorphisms act as markers of susceptibility to or severity of RA. The study included 104 RA patients and 103 normal controls. No significant difference was observed in the cytokine allelic frequencies of IL-1β promoter and IL-1β exon 5 between patients with RA and healthy controls. In addition, there was no significant association in the cytokine carriage rates of I and II allele of IL-1Ra between RA patients and healthy controls. In contrast, the IV allele of IL-1Ra was significantly increased in RA patients with low inflammatory activity (P = 0.03). This study indicated that allelic frequency and carriage rate of IL-1β (IL-1β promoter and IL-1β exon 5) and IL-1Ra (I and II allele) do not differ significantly between normal controls and RA patients in Taiwan. However, the carriage rate of IV allele of IL-1Ra was high in the RA patients with low inflammatory activity.The purpose of this study was to evaluate if IL-1beta (IL-1beta promoter and IL-1beta exon 5) and IL-1receptor antagonist (IL-1Ra) gene polymorphisms act as markers of susceptibility to or severity of RA. The study included 104 RA patients and 103 normal controls. No significant difference was observed in the cytokine allelic frequencies of IL-1beta promoter and IL-1beta exon 5 between patients with RA and healthy controls. In addition, there was no significant association in the cytokine carriage rates of I and II allele of IL-1Ra between RA patients and healthy controls. In contrast, the IV allele of IL-1Ra was significantly increased in RA patients with low inflammatory activity (P=0.03). This study indicated that allelic frequency and carriage rate of IL-1beta (IL-1beta promoter and IL-1beta exon 5) and IL-1Ra (I and II allele) do not differ significantly between normal controls and RA patients in Taiwan. However, the carriage rate of IV allele of IL-1Ra was high in the RA patients with low inflammatory activity.

Prenatal diagnosis of Apert syndrome

Apert syndrome (AS) is clinically characterized by typical facial features and symmetrical syndactyly of the digits. AS is inherited as an autosomal dominant trait. Recently, a fibroblast growth factor receptor 2 (FGFR2) mutation, either C934G or C937G, was identified in exon IIIa. Our report documents an affected mother and son in whom one of the two mutations in AS had occurred sporadically in the mother. The diagnosis of AS was based on associated abnormal physical features and on molecular genetic analysis. A C‐to‐G transversion at position 937 of the cDNA resulting in a proline‐to‐arginine substitution at codon 253 was found in the mother. In her second pregnancy, prenatal diagnosis by both restriction analysis and direct sequencing was undertaken and this showed that the female fetus had not inherited the mutation.

No Association of Vitamin D Receptor Gene BsmI Polymorphisms with Calcium Oxalate Stone Formation

BACKGROUND AND PURPOSE The formation of urinary stones is reported to be associated with the vitamin D receptor (VDR). As the most frequently seen polymorphism within the VDR gene is BsmI, it has been used as a genetic marker in searching for the cause of urolithiasis. We aimed to evaluate the association between calcium stone disease and the BsmI polymorphisms. MATERIALS AND METHODS A control group of 90 healthy people and a group of 124 patients with calcium oxalate stones were examined. The polymorphism was detected using polymerase chain reaction (PCR)-based restriction analysis. A PCR product length was determined to be 580 bp (BB) whereas two fragments of 405 bp and 175 bp were determined to be excisable (bb) by BsmI endonuclease. Associations between calcium stone disease and BsmI polymorphisms were evaluated. RESULTS AND CONCLUSIONS The results revealed no significant difference between normal individuals and stone patients (P = 0.891). The allelic distribution of B and b were similar within both the normal group and the stone patients. Therefore, the BsmI polymorphism of the VDR gene at intron 8 is not a suitable genetic marker for urinary stone disease.

Mutation in the FGFR2 gene in a Taiwanese patient with Beare–Stevenson cutis gyrata syndrome

The present authors report the first case of Beare–Stevenson syndrome in Taiwan. The patient shares several clinical characteristics of Beare–Stevenson syndrome such as cutis gyrata, cloverleaf skull, prominent eyes, cleft palate, ear defects and a protruding umbilical stump. Molecular genetic analysis of the FGFR2 gene in this patients DNA revealed a missense A → G mutation on nucleotide 1303 of the FGFR2 cDNA. This mutation leads to a Tyr → Cys substitution at residue 375 located at the N‐terminal end of the transmembrane domain of FGFR2. The present results are in accordance with other previously published reports and strengthen the importance of the FGFR2 gene in the pathogenesis of Beare–Stevenson syndrome.

Molecular analysis of Wilson disease in Taiwan: Identification of one novel mutation and evidence of haplotype-mutation association

AbstractWilson disease (WND) is caused by a deficiency of the copper-transporting enzyme, P-type ATPase (ATP7B). Twelve different mutations have previously been identified in Taiwan Chinese with Wilson disease. We, herein, report another 4 missense mutations, 1 of which is novel. We did haplotype analysis of Taiwanese WND chromosomes, using three well characterized short tandem repeat markers (haplotype was assigned in the order of D13S314-D13S301-D13S316). Association correlation was found between the mutations and their respective haplotypes. Haplotype-deduced pedigree analysis was shown to be helpful in the mutation analysis of WND chromosomes and in the molecular assessment of both pre-symptomatic WND patients and carriers. Given the complexity and heterogeneity of the mutation spectrum of ATP7B, we suggest that haplotype analysis should be performed before full-scale mutation analysis.

Molecular cloning of acid α-glucosidase cDNA of Japanese quail(Coturnix coturnix japonica)and the lack of its mRNA in acid maltase deficient quails

Acid alpha-glucosidase (GAA) hydrolyzes alpha-1, 4 and alpha-1, 6 glucosidic linkages of oligosaccharides and degrades glycogen in the lysosomes. The full-length GAA I cDNA, pQAM8, was isolated from a cDNA library derived from Japanese quail liver. The cDNA is 3569 base pairs long and has an open reading frame capable of coding 932 amino acids. The deduced amino acid sequence shares 52% identity with human GAA. Transfection of expression vector pETAM8 into COS-7 cells or acid maltase deficient (AMD) quail embryonic fibroblasts increased the level of GAA 20-50-fold. Compared to normal quail, the levels of GAA I mRNA were significantly reduced in the muscle, liver, heart, and brain of AMD quails, suggesting the GAA deficiency in AMD quail is due to a lack of GAA I mRNA. A second GAA II cDNA was identified after probing the cDNA library from the ovarian large follicles of quails with a PCR product derived from cultured quail skin fibroblasts. This clone having 3.1 kb insert, has GAA activity as well (3 to 10 fold increase). This cDNA, designated GAA II, predicted an 873 amino acid polypeptide showing 63% identity to human GAA and 51% identity to the GAA I. The RT-PCR analysis demonstrated that GAA II mRNAs were barely detectable in normal tissues, while they were enhanced to higher levels in AMD tissues. These results suggest that GAA II expression is up-regulated at the transcription levels, and quail GAA gene redundancy performs the same function of satisfying GAA demand at the two different phases represented by normal and AMD.