ESwab as an optional collection device for use with the Affirm VPIII microbial test system.

The ESwab collection device was compared to the collection swab provided as part of the Affirm VPIII microbial identification test kit for testing vaginal specimens with the Affirm test system. There was excellent agreement between the two sampling devices for Candida spp., Gardnerella vaginalis, and Trichomonas vaginalis.

Viability of Trichomonas vaginalis in Copan universal transport medium and eSwab transport medium.

We compared the use of universal transport medium and eSwab transport medium held at room temperature or 37 degrees C to bedside inoculation and immediate incubation of culture media for the detection of Trichomonas vaginalis. There were no significant culturable differences in the sensitivity of either of the transport media to that of bedside inoculation.

The human growth hormone receptor gene - characterisation of the liver-specific promoter.

Several variants (V1-V8) have been described for the 5' untranslated region of human growth hormone receptor cDNA (Pekhletsky, R.I., Chernov B.K., Rubtsov, P.M., 1992. Variants of the 5'-untranslated sequence of human growth hormone receptor mRNA. Mol. Cell. Endocrinol. 90, 103-109). Transcription of one of these, variant V1, is under the control of a liver-specific promoter (Zou, L., Burmeister L.A., Sperling, M.A., 1997. Isolation of a liver-specific promoter for human growth hormone receptor gene. Endocrinology 138, 1771-1774). Further characterisation of this promoter shows that: (1) a cluster of exon 1 variants, which includes that coding for V1, is located about 13 kb upstream of the first coding exon; (2) the human V1 promoter (and not V7, as previously suggested) is homologous to liver-specific regulatory regions of rat and mouse GH receptor genes; (3) the transcription start site for V1 is 27 bp further upstream than previously reported; (4) a 158-bp sequence is sufficient for basal promoter activity, while larger constructs provide evidence for negative elements further upstream; and (5) nuclear proteins from HepG2 hepatoma cells protect regions of the V1 promoter from DNase digestion, revealing putative regulatory sites.

A rapid PCR-SSP assay for the hemochromatosis-associated Tyr250Stop mutation in the TFR2 gene.

Several genes associated with hemochromatosis and primary iron overload have been identified. Mutations in the HFE gene have been detected in 60-100% of hemochromatosis patients of northern, central, and western European descent, although the frequencies of these mutations vary among racial and ethnic groups. Recently, a mutation in the gene encoding transferrin receptor-2 (exon 6, nucleotide 750 C --> G; Y250X) was detected by a PCR-restriction fragment length polymorphism (RFLP) method in Sicilians with hemochromatosis. We describe a modification of the original assay in which the sequence-specific priming PCR assay does not require the use of restriction endonuclease. The modified assay is robust and cost-efficient, and may be more useful for large-scale population studies because it can be performed rapidly on DNA extracted from buccal swabs.

DMPK-associated myotonic dystrophy and CTG repeats in Alabama African Americans.

Myotonic dystrophy type 1 (DM1) is a result of a CTG expansion in the 3'-untranslated region of the DMPK gene. DM1 is rare among African blacks who have fewer large CTG repeats in the normal range than other racial/ethnic groups. Neither the prevalence of DM1 nor the relationship of CTG expansion to clinical status in African Americans (AAs) is well documented. We describe two AA brothers with DM1, each of whom had CTG repeats of 5/639; their father was reported to have DM1 and had CTG repeats of 5/60. Other family members had CTG repeats of 5-14. An unrelated AA patient from a second kinship also had DM1; an analysis revealed CTG repeats of 27/191. In 161 Alabama AA control subjects, we observed 18 CTG alleles from 5 to 28 repeats; the most common allele had five CTG repeats. The frequency of CTG repeats >or=15 were greater (p < 0.0003) in Pygmy, Amhara Ethiopian, Ashkenazi Jewish, North African Jewish, Israeli Muslim Arab, European white, and Japanese populations than in the Alabama AA population. These data suggest that the risk for DM1 in AAs is intermediate between that of African blacks and whites of European descent.

Racial differences in the prevalence of Factor V Leiden mutation among patients on chronic warfarin therapy.

We report the prevalence of Factor V Leiden (FVL) in European American and African American patients on warfarin therapy residing in Alabama. METHODS.: Detailed history was obtained and FVL genotype was determined for 288 patients enrolled in a prospective cohort: Pharmacogenetic Optimization of Anticoagulation Therapy. Racial differences in genotype frequency were assessed by the Chi-square statistics and HWE assumptions by G-statistics. Race-specific analysis for the association between site of thromboembolism and the presence of FVL mutation was assessed using logistic regression. RESULTS.: The overall heterozygote (GA genotype) frequency was 4.9%. No patient was found to be homozygous (AA) for the variant allele. The prevalence of GA was higher in European American (8.6%) compared to African American (1.4%) patients (p=0.004). The FVL genotype frequency was significantly different across race for venous thromboembolic events (p=0.014) but not for arterial thromboembolic events (p=0.20). Multivariable race-specific analysis highlights the contribution of FVL mutation to the risk of venous thromboembolic events in European American (p=0.03) but not in African American patients (p=0.95). European American patients with the GA mutation were approximately 6.3 times more likely to have experienced a venous, rather than arterial thromboembolic event. CONCLUSION.: In Alabama, among patients on warfarin, the GA genotype is more prevalent in European Americans compared to African Americans. In European Americans, but not in African Americans, the GA genotype was more prevalent in patients with venous compared to arterial thromboembolic events.

Transferrin receptor-2 (TFR2) mutation Y250X in Alabama Caucasian and African American subjects with and without primary iron overload.

Most cases of hemochromatosis are associated with mutations of the HFE gene on Ch6p. In southern Italy and central Alabama, the percentages of patients with hemochromatosis who have "atypical" HFE genotypes (defined as lack of C282Y homozygosity, C282Y/H63D compound heterozygosity, or H63D homozygosity) are relatively great. A mutation of the transferrin receptor-2 gene (TFR2; exon 6, nt 750 C --> G, replaces TAC with stop signal TAG; Y250X) on Ch7q22 was recently identified in two Sicilian families with HFE mutation-negative hemochromatosis. We wanted to estimate the frequency of this mutation in persons from central Alabama. We evaluated Caucasian hemochromatosis probands with atypical HFE genotypes and African Americans with primary iron overload. We also studied control Caucasians, including persons of southern Italian/Sicilian heritage, and control African Americans. Analysis of genomic DNA was performed using a PCR-sequence-specific priming assay and positive control specimens from Sicilian hemochromatosis subjects heterozygous and homozygous for Y250X. Among Alabama subjects, this allele was not detected in 113 Caucasians, including 21 hemochromatosis probands with atypical HFE genotypes and 92 normal control subjects (including 27 of southern Italian/Sicilian descent). In African Americans, Y250X was not detected in 20 index cases with primary iron overload or in 274 unrelated control subjects. We conclude that Y250X is uncommon in Caucasians with hemochromatosis associated with atypical HFE genotypes, in African Americans with primary iron overload, and in the general Caucasian and African American population subgroups in central Alabama.

Characterization of 18 new mutations in COL7A1 in recessive dystrophic epidermolysis bullosa provides evidence for distinct molecular mechanisms underlying defective anchoring fibril formation.

We have characterized 21 mutations in the type VII collagen gene (COL7A1) encoding the anchoring fibrils, 18 of which were not previously reported, in patients from 15 unrelated families with recessive dystrophic epidermolysis bullosa (RDEB). COL7A1 mutations in both alleles were identified by screening the 118 exons of COL7A1 and flanking intron regions. Fourteen mutations created premature termination codons (PTCs) and consisted of nonsense mutations, small insertions, deletions, and splice-site mutations. A further seven mutations predicted glycine or arginine substitutions in the collagenous domain of the molecule. Two mutations were found in more than one family reported in this study, and six of the seven missense mutations showed clustering within exons 72-74 next to the hinge region of the protein. Patients who were homozygous or compound heterozygotes for mutations leading to PTCs displayed both absence or drastic reduction of COL7A1 transcripts and undetectable type VII collagen protein in skin. In contrast, missense mutations were associated with clearly detectable COL7A1 transcripts and with normal or reduced expression of type VII collagen protein at the dermo/epidermal junction. Our results provide evidence for at least two distinct molecular mechanisms underlying defective anchoring fibril formation in RDEB: one involving PTCs leading to mRNA instability and absence of protein synthesis, the other implicating missense mutations resulting in the synthesis of type VII collagen polypeptide with decreased stability and/or altered function. Genotype-phenotype correlations suggested that the nature and location of these mutations are important determinants of the disease phenotype and showed evidence for interfamilial phenotypic variability.