Genetic mutations in patients with possible familial hypercholesterolaemia in South East Scotland.

Familial hypercholesterolaemia (FH) is one of the most common genetic disorders in the general population. Genetic testing of this condition is increasingly available in the UK to confirm its diagnosis, but the strategies of genetic testing vary. In this pilot study, we sought to investigate whether a strategy that focuses on the low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) genes can identify the majority of genetic variants in patients with possible FH in South East Scotland. Forty patients with a clinical diagnosis of possible FH according to the Simon Broome criteria were recruited in a lipid clinic serving South East Scotland. All 18 exons of the LDLR gene were sequenced and multiplex ligation probe amplification was performed to identify major deletions and duplications. Variants of the APOB gene at codon 3527 were investigated by direct sequencing. Genetic mutations were detected in 45% of the patients. Sixteen patients (40%) were found to have mutations in their LDLR gene, whereas two other patients (5%) were identified as heterozygous for the APOB variant commonly associated with FH (c.10580G>A; p.R3527Q). None of these genetic variants were detected in more than two patients. Multiple genetic mutations are associated with a clinical phenotype of FH in South East Scotland. A genetic testing strategy which focuses on a limited number of mutations is unlikely to confirm the diagnosis of FH in the majority of patients in this part of Scotland.

Comparison of two multilocus variable-number tandem-repeat methods and pulsed-field gel electrophoresis for differentiating highly clonal methicillin-resistant Staphylococcus aureus isolates.

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson's index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.

Burkholderia fungorum sp. nov. and Burkholderia caledonica sp. nov., two new species isolated from the environment, animals and human clinical samples.

A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analyses, whole-cell protein and fatty acid analyses and an extensive biochemical characterization was performed on 16 strains isolated from the environment, animals and human clinical samples. The isolates belonged to the genus Burkholderia, were phylogenetically closely related to Burkholderia graminis, Burkholderia caribensis and Burkholderia phenazinium and had G+C contents between 61.9 and 62.2 mol%. Seven strains isolated from the rhizosphere were assigned to Burkholderia caledonica sp. nov. [type strain LMG 19076T (= CCUG 42236T)]. Nine strains isolated from the environment, animals and human clinical samples were assigned to Burkholderia fungorum sp. nov. [type strain LMG 16225T (= CCUG 31961T)]. Differential tests for B. graminis, B. caribensis, B. phenazinium, B. caledonica and B. fungorum include the following: assimilation of trehalose, citrate, DL-norleucine, adipate and sucrose; nitrate reduction; growth in the presence of 0.5% NaCl; and beta-galactosidase activity.

Transcriptional activation of the factor VIII gene in liver cell lines by interleukin-6.

Circulating factor VIII (fVIII) levels increase during inflammation suggesting that fVIII synthesis or secretion is stimulated during acute inflammation. To examine the mechanisms underlying this increase in circulating factor VIII, we have developed a sensitive and reliable semiquantitative assay for fVIII mRNA utilising competitive reverse transcriptase-polymerase chain reaction (RT-PCR, and used this to study two human liver cell lines, Hep-G2 and Chang Liver cells. These cells were cultured under basal conditions or following treatment with interleukin-1, -2 and 6 (IL-1, -2, and -6). Following 18h culture with IL-6 (maximum concentration 40 U/ml), these levels had risen 6 and 9 fold respectively, with no concomitant rise in control RNA levels. The dose responses for both cell types were similar, with an ED50 of 11 U/ml. The time course of this response was also similar in both cell lines with the increase in fVIII mRNA first reaching significance by 3 h, and reaching maximum levels by 12 h. IL-1 and IL-2 had no effect at any of the doses studied. This study provides the first evidence for regulated expression of fVIII in human cell lines, and suggests that increased plasma fVIII levels during the acute phase response may be due to increased expression of fVIII mRNA.