Comprehensive identification of Vibrio vulnificus genes required for growth in human serum.

Vibrio vulnificus can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent V. vulnificus lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the V. vulnificus essential gene set; comparisons of these genes with similar TIS-based classification of underrepresented genes in other vibrios enabled the compilation of a common Vibrio essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum suggested that genes involved in capsule biogenesis are critical for YJ016 complement resistance. Notably, homologues of two genes required for YJ016 serum-resistance and capsule biogenesis were not previously linked to capsule biogenesis and are largely absent from other V. vulnificus strains. The relative abundance of mutants after exposure to heat inactivated serum was compared with the findings from the serum screen. These comparisons suggest that in both conditions the pathogen relies on its Na+ transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive/proliferate within the metabolic constraints of serum. Collectively, our findings reveal the potency of comparative TIS screens to provide knowledge of how a pathogen overcomes the diverse limitations to growth imposed by serum.

Novel B3GALTL mutations in classic Peters plus syndrome and lack of mutations in a large cohort of patients with similar phenotypes.

Peters plus syndrome (PPS) is a rare autosomal-recessive disorder characterized by Peters anomaly of the eye, short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable other systemic abnormalities. In this report, we describe screening of 64 patients affected with PPS, isolated Peters anomaly and PPS-like phenotypes. Mutations in the coding region of B3GALTL were identified in nine patients; six had a documented phenotype of classic PPS and the remaining three had a clinical diagnosis of PPS with incomplete clinical documentation. A total of nine different pathogenic alleles were identified. Five alleles are novel including one frameshift, c.168dupA, p.(Gly57Argfs*11), one nonsense, c.1234C>T, p.(Arg412*), two missense, c.1045G>A, p.(Asp349Asn) and c.1181G>A, p.(Gly394Glu), and one splicing, c.347+5G>T, mutations. Consistent with previous reports, the c.660+1G>A mutation was the most common mutation identified, seen in eight of the nine patients and accounting for 55% of pathogenic alleles in this study and 69% of all reported pathogenic alleles; while two patients were homozygous for this mutation, the majority had a second rare pathogenic allele. We also report the absence of B3GALTL mutations in 55 cases of PPS-like phenotypes or isolated Peters anomaly, further establishing the strong association of B3GALTL mutations with classic PPS only.

Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk.

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.

DNA haplotype analysis of CAG repeat in Taiwanese Huntington's disease patients.

We studied the expanded CAG repeat and adjacent CCG repeat in 53 Huntington's disease (HD) patients and 172 unrelated normal subjects matched to the patients for ethnic origin. The range of the CAG repeat varied from 38 to 109 in the HD patients and from 10 to 29 in the control group. A significant negative correlation was found between the age at onset and the CAG expansion, with no significant influence of the adjacent CCG repeat on the age at onset by multiple regression analysis. Allelic association using CCG repeat and 2 flanking dinucleotide repeat markers within 150 kb of the HD gene revealed linkage disequilibrium for 2 of 3 markers. Haplotype analysis of 24 HD families using these markers identified 3 major haplotypes underlying 87.5% of HD chromosomes. The data suggested frequent haplotypes in the Taiwanese population on which one or more mutational events leading to the disease occurred.

Cloning, characterization and physical mapping of three cervid satellite DNA families in the genome of the Formosan muntjac (Muntiacus reevesi micrurus).

Due to their high sequence diversity even among closely related species, satellite DNA sequences can be a useful molecular marker for phylogenetic and taxonomic analyses. To characterize the satellite DNA in the genome of a native muntjac species of Taiwan, the Formosan muntjac, satellite DNA clones representing three different cervid satellite DNA families from this species were isolated and analyzed. Genomic organization study of these satellite DNAs was also undertaken. Three Formosan muntjac satellite DNA clones were obtained and designated as FM-satI (1,391 bp), FM-satII (1,143 bp) and FM-satIV (1,103 bp), and found to share approximately 82, 81 and 98% sequence homology with the Chinese muntjac satellite I clone (C5), Indian muntjac satellite II clone (Mmv-0.7) and Chinese muntjac satellite IV clone (MR-1.0), respectively. These three satellite DNA families are organized in a pter<--FM-satII-FM-satIV-FM-satI-->qter orientation in the centromeric region with satII closely associated with the telomeric sequences. Satellite DNA sequence comparison, in combination with chromosome data concludes that the Formosan muntjac is likely a subspecies of M. reevesi, closely related to the Chinese muntjac. With the kinetochore satellite II DNA co-localizing with the telomeric sequences, the Formosan muntjac chromosomes could be truly telocentric.

Prenatal diagnosis of der(11)t(11;18)(q24;q21.3) due to paternal balanced translocation and both parents are carriers of alpha-thalassemia-1--a case report.

A couple were identified as alpha-thalassemia-1 carriers (father: alpha-thal-1 of Filipino type, mother: alpha-thal-1 of SEA type). Amniocentesis was done at 19 weeks of gestation by a local obstetrician. Molecular study of amniotic fluid presented a non-thalassemia fetus, but the cytogenetic study revealed a karyotype of 46,XX,der(11)t(11;18)(q24;q21.3), resulting from a paternal balanced reciprocal translocation and unbalanced adjacent 1 segregation. The pregnancy was terminated at 23 weeks of gestation. The gross of fetus revealed bilateral cleft lip and palate, hypertelorism, flat nasal bridge, frontal bossing, micrognathia, low set ears, short neck with cystic hygroma, overlapping fingers, prominent heels, and limited hip abduction. The chromosome complement of the present case was partial monosomy for 11q24-qter and partial trisomy for 18q21.3-qter. This is the first prenatal diagnosis of unbalanced translocation with der(11)t(11;18)(q24;q21.3) pat due to paternal balanced translocation and both parents being carriers of alpha-thalassemia-1.

GAD65 antibody prevalence and association with thyroid antibodies, HLA-DR in Chinese children with type 1 diabetes mellitus.

Persistent humoral autoimmunity to the enzyme glutamic acid decarboxylase (GAD) has been described in a substantial proportion of patients with type 1 diabetes mellitus. Higher prevalence of GAD antibody in diabetes patients using a new radioligand-binding assay with recombinant human GAD65 antibodies (GAD65Ab) has been seen in several studies. Using this method, we have reassessed the prevalence of GAD65Ab and investigated the association of GAD65Ab with HbA1C values, C-peptide values, HLA-DR typing and thyroid autoimmune antibody in 70 Chinese children with type 1 diabetes mellitus (mean age of onset 8.21+/-3.84 years, mean duration 3.39+/-2.54 years). Our result revealed that GAD65 antibodies were present in 54.3% (38/70) of diabetes children. There was no significant difference in gender, diabetes onset and duration, HbA1c, C-peptide concentration and frequencies of HLA DR3, DR4, DR9, DR3/DR4, DR3/DR9 and DR4/DR9 genotypes between GAD65Ab+ and GAD65Ab- groups. There was no negative correlation between GAD65Ab values and duration of diabetes in those with GAD65Ab positivity (r=-0.239, P>0.05). The frequencies of antimicrosomal and anti-thyroglobulin antibodies in GAD65Ab+ (13.5,8.1%, respectively) were not different from GAD65- patients (9.4,12.5%, respectively).

Coexistent insulin dependent diabetes mellitus and hyperthyroidism in a patient with Down's syndrome.

The prevalence of thyroid disease is increased in Down's syndrome. Compared with adults, thyroid dysfunction in children with Down's syndrome is less frequently reported. Insulin dependent diabetes mellitus is also uncommon in Down's syndrome children. Coexistent insulin dependent diabetes mellitus and hyperthyroidism in Down's syndrome was only reported once previously in literature. We report an 8-year-old girl with Down's syndrome that had polyuria, polydipsia, abdominal pain and urinary incontinence one and half a month prior to admission. Physical examination revealed typical face of Mongolism and tachycardia. Thyroid glands were not palpable. Laboratory data revealed diabetic ketoacidosis with plasma glucose: 860 mg/dl. She had thyroid hyperfunction with TSH: < 0.1 microU/ml, T3: 219.7 ng/dl, T4: 15 micrograms/dl. Thyroid autoimmune antibodies were also increased. There was markedly increased radiotracer uptake in the bilateral thyroid glands in Tc-99 thyroid scan. We suggest that Down's syndrome children with insulin dependent diabetes mellitus should be evaluated carefully for thyroid function and autoimmune disease.

Comparison of serum copper, magnesium, zinc and calcium levels between G6PD deficient and normal Chinese adults.

Minerals are important for normal hematopoiesis and may play a role in acute hemolytic anemia induced by G6PD deficiency. To compare serum magnesium, copper, zinc and calcium levels between G6PD deficiency and normal control adults, we investigated 69 G6PD deficient (28 male, 41 female) and 61 age- matched G6PD normal adults (26 male, 35 female). Serum magnesium, copper, zinc and calcium levels were determined by atomic absorbance spectrometry. Our results revealed that male adults with G6PD deficiency had significantly higher serum copper and magnesium levels than those of the control group (P < 0.01, < 0.05, respectively). In G6PD normal adults, serum copper levels were significantly lower in males than in females (P < 0.01). In the group of G6PD deficiency adults, serum copper levels in males (103.0 +/- 10.4 ug/dL) were significantly lower than those in females (139.0 +/- 34.3 ug/dL) (P < 0.01). Serum magnesium values and zinc values in males (2.42 +/- 0.38 mEq/L and 102.2 +/- 26.5 ug/dL) were significantly higher than those in females (2.07 +/- 0.20 mEq/L and 87.0 +/- 14.9 ug/dL) (P all < 0.01). Female adults with G6PD deficiency had significantly higher serum calcium levels and lower magnesium levels than those of the control group (P all < 0.01). The significantly higher levels of serum copper and magnesium in G6PD deficient male adults may play some role concerning red blood cells in resistance to plasmodium falciparum.

Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We have analyzed CYP21 gene sequences in 65 CAH families in Taiwan. All ten exons of the CYP21 gene were analyzed by differential polymerase chain reaction followed by single-strand conformation polymorphism electrophoresis and the amplification-created restriction site method. About 95% (123 chromosomes) contain mutations due to conversion of DNA sequences into its neighboring homologous pseudogene, CYP21P. Four novel mutations representing 5% of the total chromosomes have also been identified. The mutations were confirmed by sequencing an aberrant DNA fragment. These four mutations included a base change of the splicing donor site at intron 2 from GT to AT, a base substitution of C to T at codon 316, deletion of ten bases (TCCAGCTCCC) at codons 330-333 of exon 8, and duplication of 16 bases (CCTGGATGACACGGTC) at codons 393-397 of exon 9. The loss of the splicing donor site at intron 2 and the premature stop at codon 316 may result in aberrant splicing to reduce enzyme activity and a truncated protein with no enzyme activity, respectively. Likewise, both the duplication and the deletion forms create a frameshift and premature stop during translation. The resulting proteins lack the heme-binding domain and hence are expected to lose enzymatic activity. Since these mutations are not found in the neighboring CYP21P pseudogene, gene conversion should not be the cause of these novel mutations.

Influence of referral source on return compliance of adolescents.

PURPOSE: To determine the effect of the source of referral on appointment compliance of adolescents to referring doctors or to consultants at tertiary medical centers. METHODS: This analysis included 421 adolescent patients aged 10-18 years who were referred to the tertiary teaching hospital in Taiwan by 75 primary care doctors, including 56 pediatricians and 19 general practitioners in 1987-1996. Medical records were abstracted to determine clinical severity and demography. Data of compliance with return appointments were collected by an independently trained interviewer. RESULTS: A total of 18.07% of referrals were initiated by patients or families and 81.93% were initiated by primary care doctors. More severe illness was found in patient-initiated referrals than in doctor-initiated referrals. After controlling for demographic, family features, medical payment, and doctor specialty, this study showed that if the referral were initiated by patients or families, adolescent patients were less likely to return to either the primary care doctors or tertiary teaching hospitals. CONCLUSIONS: Although in a competitive environment, follow-up care is more likely to occur for both primary care doctors and consultants at tertiary teaching hospitals when the referral was initiated by primary care doctors.

Molecular characterization of Chinese G6PD deficiency by using polymerase chain reaction/single strand conformation polymorphism.

Using the polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP), we studied the molecular characterization of 97 (86 male and 11 female) glucose-6-phosphate dehydrogenase (G6PD) deficient Chinese newborn babies and infants in southern Taiwan. Movement shifts were clearly seen in exon 2, 5, 6 and 12, respectively. No movement shift was seen in exon 9. Mutation confirmations were followed by direct sequencing. Using this approach we identified the molecular defect in 90 of the 97 above samples. Our results show a total of seven variants, the most common being at nucleotide (nt) 1376 mutation G-->T 42.3% (41/97) and at nt 1388 mutation G-->A 34.0% (33/97). The other mutation sites were at nucleotide 95 A--G (5.2%), nucleotide 392 G-->T (4.1%), nucleotide 493 A-->G (3.1%) and two new variants with mutation at nucleotide 371 A-->G (2.1%) and nucleotide 519 C-->G (2.1%). Still 7.2% (7/97) remained unidentified. There was no significant difference in G6PD activity among the different mutations. Simple and fast, PCR/SSCP may be suitable for molecular screening for G6PD deficiency in Chinese and other people.

Cleidocranial dysplasia: a rare case associated with congenital hypothyroidism and severe neonatal hyperbilirubinemia.

Cleidocranial dysplasia is an autosomal dominant disorder affecting skeletal ossification and tooth development. This disorder can be rarely associated with blood, intestinal and vascular anomalies. There has been no case reported in previous literature that discusses this disorder in association with congenital hypothyroidism and severe neonatal hyperbilirubinemia. Herein, we report on a 4-year-old boy who in an outpatient clinic was diagnosed as having cleidocranial dysplasia, with defective ossification over bilateral hypoplastic clavicles, narrowed thoracic cage on chest x-ray and prominent wormian bones over the lambdoid sutures on skull x-ray. Physical examination revealed a 4-fb wide open anterial fontanel, frontal bossing and malalignment of teeth. He was a primpara with birth weight of 2350 gm and gestation age of 33 weeks. He had received blood exchange transfusion on the 4th postnatal day at Ping-Tong Christian Hospital because of severe neonatal hyperbilirubinemia. Congenital hypothyroidism was diagnosed on the 10th day. He was then treated with thyroxine and transferred to our hospital. Thyroid scan revealed diffusely decreased radioactivity in bilateral lobes of the thyroid gland. According to out clinic findings at his regular follow ups, with continued use of thyroxine supplement, he now has a normal thyroid function and has a body length of about 50 percentile.

Prevalence of permanent primary congenital hypothyroidism in Taiwan.

The purpose of this study was to find the prevalence of permanent primary congenital hypothyroidism in Taiwan. From January 1988 to December 1990, there were 991,132 live births in Taiwan. Of these, 329,891 neonates were screened for primary congenital hypothyroidism. Fifty-seven cases of permanent primary congenital hypothyroidism were confirmed. Hence, the prevalence of permanent primary congenital hypothyroidism in that period in Taiwan was 1 in every 5,788 live births. There was a female preponderance with a female to male ratio of 1.7. Of 54 infants who had a thyroid scan, 36 were found to have an ectopic thyroid gland, while 11 were found to have dyshormonogenesis. Among the 57 infants, one case with a delayed rise in the serum thyrotropin level was missed on initial screening. When compared with other studies, these data suggest that the prevalence of permanent primary congenital hypothyroidism varies in different ethnic groups throughout the world.

[Lysosomal enzyme activity of cultured fetal cells in Chinese and its clinical application].

Lysosomal storage diseases (LSD) are caused by deficient activity of specific lysosomal enzymes. Early diagnosis and selective termination is still the trend of therapy. The purpose of this study was to establish an assay system and investigate the reference range of lysosomal enzyme activity of cultured fetal cells in the Chinese population. Seventy amniotic fluid and 9 chorionic villi samples were collected and cultured in this study. Enzyme activity assay was done by synthesized 4-Mu-binded substrates. The activity was expressed as nmol/mg protein/hour. In cultured amniotic cells, the results showed 14-138 of alpha-glucosidase, 8-133 of alpha-galactosidase, 32-470 of alpha-mannosidase, 101-1121 of alpha-fucosidase, 106-1321 of beta-galactosidase, 15-268 of beta-glucosidase, 11-279 of beta-glucuronidase, 101-1193 of Hexosaminidase A, and 886-6204 of N-acetyl-alpha-glucosaminidase. In cultured chorionic villi samples, it showed 22-335 of alpha-glucosidase, 31-230 of alpha-galactosidase, 47-250 of alpha-mannosidase, 35-218 of alpha-fucosidase, 49-934 of beta-galactosidase, 34-329, of beta-glucosidase, 57-379 of beta-glucuronidase, and 328-3412 of Hexosaminidase A. The enzyme activity was not correlated with the gestation age when sample was obtained. Furthermore, there was no statistical significance among the range of amniotic cells, chorionic villi samples, skin fibroblasts and peripheral leukocytes for each enzyme studied. It is suggested that the synthesis of lysosomal enzymes has been mature since the early fetal state, and the samples obtained as early as 8 weeks of gestation age can be used for early diagnosis of lysosomal storage diseases.

Holoprosencephaly and trisomy 13 in a fetus with maternal early gestational amphetamine abuse--a case report.

We report a congenital anomalous fetus with holoprosencephaly, prenatally diagnosed by sonography and MR scan at the twenty-sixth week during the gestational period. Chromosome analysis by amniocentesis revealed trisomy 13 (47,XY,+13). The diagnosis was confirmed by autopsy. After induction, external examination showed a proboscis-like nose on the normal nose position. The right foot showed polydactylia with wide separation of the fifth and sixth toes. We found that the mother was an abuser of amphetamine at an early gestational age. The teratogenic effect of D-amphetamine on animals has been noted, but no previous clinical case of a congenital anomalous fetus and maternal abuse of amphetamine has been reported. This case report is the first to reveal holoprosencephaly and trisomy 13, with maternal early gestational abuse of amphetamine.

Leukemic manifestation in a case of alpha-chain disease with multiple polypoid intestinal lymphocytic lymphoma.

We describe a female aborigine from Taiwan with alpha-chain disease associated with multiple polypoid intestinal lymphocytic lymphoma and leukemic manifestation. Initially, the patient experienced intermittent diarrhea, abdominal pain, and leukemic manifestation. No evidence of bone marrow involvement was found. Alpha-chain protein was demonstrated in the serum. Gastroendoscopy and a series of radiographs of the small intestine revealed multiple polypoid tumors involving the entire length of the small intestine. Duodenal biopsy showed diffuse lymphocytic lymphoma. Immunohistochemical staining of tumor samples revealed features typical of alpha-chain disease. Cytogenetic analysis showed the same abnormal karyotypes of neoplastic clones in intestinal tumor cells and in circulating leukemic cells. The data suggest that alpha-chain disease can present initially with intestinal multiple polypoid lymphocytic lymphoma and leukemic manifestation without evidence of bone marrow involvement. The data also support the homing theory of lymphomas from mucosa-associated lymphoid tissue.

Chromosomal and biochemical screening on mentally retarded school children in Taiwan.

Governmental officials as well as medical scientists in Taiwan have worked hard in recent years to develop and to implement various measures, such as prenatal diagnosis and neonatal screening, to lower the incidence of hereditary diseases and mental retardation in the population. An inquiry into the possibility of devising a chromosomal and biochemical screening program and to apply it routinely to all the mentally retarded school children island-wide was the major aim of the present study. A collection of 1,614 blood samples was screened for phenylketonuria (PKU), galactosemia, homocystinuria, biotinidase deficiency, and congenital hypothyroidism. The IQ of these children ranged from 50-75 (1,397 children, moderate group) to less than 50 (217 children, severe group). Six cases of PKU (one tetrahydrobiopterin deficient and five classical) and three cases of thyroid dysfunction were found. The overall incidence of these two diseases was 0.56%. Of the 1,614 blood samples, 1,323 were cultured and karyotyped successfully. One hundred and twenty-five of them had chromosome abnormalities. The majority (64 out of 125) were trisomy 21. A remarkable difference in the percentage of mentally retarded children with chromosome abnormalities was observed between the moderate (7.87%) and severe (17.51%) retarded.

[Anthropometric study on Down syndrome in Taiwan].

Anthropometric studies of Down syndrome in foreign children had been reported. To have similar information for Chinese children with Down syndrome, data based on 1624 measurements of height and 1208 measurements of weight were done on 496 children who took part in a collaborative study done via several hospitals and institutions in Taiwan. Height and weight were measured using standard methods. Additional data about these children were also solicited from hospital and school records to obtain semilongitudinal data which were then analyzed with SAS PC software. Descriptive statistics and percentiles were estimated using flexible mathematical functions. Results showed that ethnic Chinese children with Down syndrome were significantly shorter than normal children. Mean weight, however, was not significantly different from normal children. Centile charts for assessment of stature and weight are also presented.