Rapid genotyping of MBL2 gene mutations using real-time PCR with fluorescent hybridisation probes

In this study, we describe a real-time polymerase chain reaction (PCR) for genotyping all known polymorphisms of the human mannose-binding lectin 2 (MBL2) gene. These comprised two variations in the 5' regulatory region at positions -550 (H/L) and -221 (X/Y), one in the 5' untranslated sequence at position +4 (P/Q) and three structural mutations within exon 1 at codons 52, 54, and 57, also known as the D, B and C variants, respectively. Three reactions with two different conditions were sufficient to genotype one individual unambiguously. The three mutations in exon 1 were detected in one capillary using a sensor probe covering the three mutations, whereas amplification of the variants located upstream of the coding sequence was performed in only two reactions. Single colour detection was used for detection of the (H/L) polymorphism and multiplexing by dual colour probes was used for simultaneous genotyping of (X/Y) and (P/Q). The reliability of the system was evaluated by comparison with a conventional PCR method with sequence-specific primers (PCR-SSP). For this study, 100 individuals of Danish and 30 of African descent were analysed, and the genotypes obtained were concordant in all cases. This new method is rapid and provides reliable results without ambiguities.     

Incidence and diagnosis of acute rheumatic fever in Denmark, 1980 and 1983. A retrospective analysis of the fulfillment of the revised Jones criteria in hospitalized patients

A review of 1547 official hospital record summaries concerning discharges during the period 1980 through 1983 of patients whose diagnoses had been coded as acute rheumatic fever revealed that in only 61% of the cases had this illness been diagnosed or suspected. A substantial proportion of the remaining patients had had acute non-rheumatic pericarditis diagnosed. The medical records were analyzed for 141 patients diagnosed in 1980 or 1983 by hospital departments as having acute rheumatic fever with regard to the revised Jones criteria. They were fulfilled in 47 patients, 23 of whom were considered unlikely cases of rheumatic fever. Eight patients were considered possible cases, although they did not fulfill the revised Jones criteria. The current annual incidence of acute rheumatic fever was estimated to be at most 0.3 per 100,000 inhabitants.     

Clonal chromosome abnormalities in human breast carcinomas I. Twenty-eight cases with primary disease

Cytogenetic analysis was performed on a selected series of short-term cultures of primary breast carcinomas from 28 patients. All patients had histopathologically confirmed malignancies, with the majority (25/28 cases) demonstrating infiltrating ductal carcinoma. All 28 cases evidenced clonal chromosome abnormalities, with 10/28 displaying only numeric aberrations, whereas 18/28 displayed clonal structural alterations. In near-diploid tumors the most common numeric changes were — 17 and — 19. However, trisomy 7 was the only numeric change in two near-diploid tumors. Structural chromosome alterations were primarily isochromosomes, apparent terminal deletions, and unbalanced non-reciprocal translocations. Chromosomes 1 (10/18–56%) and 6 (8/18–44%) were most frequently altered in this series. Breakpoints of clonal structural abnormalities were shown to cluster to several chromosome segments, including 1p22-q11, 3p11, 6p11–13, 7p11-q11, 8p11-q11, and 19q13. Analysis of the gain or loss of specific chromosome segments revealed that the most consistent tendency was over-representation of 1q, 3q, and 6p. © 1993 Wiley-Liss, Inc.     

Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila

Polyglutamine (polyQ) diseases are a growing class of inherited neurodegenerative diseases including Huntington's disease, which are caused by abnormal expansions of the polyQ stretch in each unrelated disease protein. The expanded polyQ stretch is thought to confer toxic properties on the disease proteins through alteration of their conformation leading to pathogenic protein-protein interactions including oligomerization and/or aggregation. Hypothesizing that molecules with selective binding affinity to the expanded polyQ stretch may interfere with the pathogenic properties, we previously identified Polyglutamine Binding Peptide 1 (QBP1) from combinatorial peptide phage display libraries. We show here that a tandem repeat of the inhibitor peptide QBP1, (QBP1)(2), significantly suppresses polyQ aggregation and polyQ-induced neurodegeneration in the compound eye of Drosophila polyQ disease models, which express the expanded polyQ protein under the eye specific promoter. Most importantly, (QBP1)(2) expression dramatically rescues premature death of flies expressing the expanded polyQ protein in the nervous system, resulting in the dramatic increase of the median life span from 5.5 to 52 days. These results suggest that QBP1 can prevent polyQ-induced neurodegeneration in vivo. We propose that QBP1 prevents polyQ oligomerization and/or aggregation either by altering the toxic conformation of the expanded polyQ stretch, or by simply competing with the expanded polyQ stretches for binding to other expanded polyQ proteins. The peptide inhibitor QBP1 is a promising candidate with great potential as a therapeutic molecule against the currently untreatable polyQ diseases.     

A locus for posterior polymorphous corneal dystrophy (PPCD3) maps to chromosome 10

Posterior polymorphous corneal dystrophy (PPCD) is an autosomal dominant disorder characterized by corneal endothelial abnormalities, which can lead to blindness due to loss of corneal transparency and sometimes glaucoma. We mapped a new locus responsible for PPCD in a family in which we excluded the previously reported PPCD locus on 20q11, and the region containing COL8A2 on chromosome 1. Results of a 317-marker genome scan provided significant evidence of linkage of PPCD to markers on chromosome 10, with single-point LOD scores of 2.63, 1.63, and 3.19 for markers D10S208 (at (circumflex)theta = 0.03), D10S1780 (at (circumflex)theta = 0.00), and D10S578 (at (circumflex)theta = 0.06). A maximum multi-point LOD score of 4.35 was found at marker D10S1780. Affected family members shared a haplotype in an 8.55 cM critical interval that was bounded by markers D10S213 and D10S578. Our finding of another PPCD locus, PPCD3, on chromosome 10 indicates that PPCD is genetically heterogeneous. Guttae, a common corneal finding sometimes observed along with PPCD, were found among both affected and unaffected members of the proband's sib ship, but were absent in the younger generations of the family. Evaluation of phenotypic differences between family members sharing the same affected haplotype raises questions about whether differences in disease severity, including differences in response to surgical interventions, could be due to genetic background or other factors independent of the PPCD3 locus.     

Identifying undiagnosed diabetes: cross-sectional survey of 3.6 million patients' electronic records.

BACKGROUND: Around 1% of the UK population has diabetes that is either undiagnosed or unrecorded on practice disease registers. AIM: To estimate the number of people in UK primary care databases with biochemical evidence of undiagnosed diabetes. To develop simple practice-based search techniques to support early recognition of diabetes. DESIGN OF STUDY: Cross-sectional survey of 3 630 296 electronic records. SETTING: Four hundred and eighty UK practices contributing to the QRESEARCH database. METHOD: Electronic searches to identify people with no diabetes diagnosis in one of two categories (A and B), using the most recently recorded blood glucose measurement: random blood glucose level >or=11.1 mmol/l or fasting blood glucose level >or=7.0 mmol/l (A); either a random or a fasting blood glucose level >or=7.0 mmol/l (B). An additional outcome measure was the proportion of the population with at least one blood glucose measurement in the record. RESULTS: The number (percentage) identified in category A was 3758 (0.10% of the total population); the number in category B was 32 785 (0.90%). Projected to a practice of 7000 patients, around eight patients have biochemical evidence of undiagnosed diabetes, and 68 have results suggesting the need for further follow-up. One-third of people aged over 40 years without diabetes have a blood glucose measurement in the past 2 years in their record. CONCLUSION: People with possible undiagnosed diabetes are readily identifiable in UK primary care databases through electronic searches using blood glucose data. People with borderline levels, who may benefit from interventions to reduce their risk of progression to diabetes, can also be identified using practice-based software.     

Chronic progressive multiple sclerosis: double-blind controlled study of plasmapheresis in patients taking immunosuppressive drugs

Fifty-four patients with chronic progressive multiple sclerosis received prednisone plus oral low-dose cyclophosphamide and either true plasmapheresis (PP) or "sham" PP weekly for 20 weeks in a double-blind controlled study. Immunosuppressive drug therapy alone (sham PP group, n = 29) was associated with improvement (greater than or equal to one step in Kurtzke Disability Status Scale [DSS]; mean change of 1.5) in 8 and stabilization of MS in 18 patients, with this status sustained in 23 patients at follow-up, 11 months after entry. In contrast, 14 of 26 patients who received "true" PP improved (greater than or equal to one step in DSS; mean change of 2.6), and 11 more were stable, with these changes sustained in 23 of 26 patients at follow-up. These differences, overall, between the PP and sham PP groups were significant at p less than 0.007.     

Effects of the co-carcinogen catechol on benzo[a]pyrene metabolism and DNA adduct formation in mouse skin

We have studied the effects of the co-carcinogen catechol (1,2-dihydroxybenzene)on the metabolic activation of [³H] benzo[a]pyrene (BaP) inmouse skin, in vivo and on the binding of BaP metabolites toDNA and protein at intervals from 0.5–24 h. Upon topicalapplication of 0.015 mg [³H]BaP and 0.25 or 0.5 mg catecholper mouse, catechol had little effect on the total amount of[³H]BaP metabolized in mouse skin, but it affected the relativeproportions of [³H]BaP metabolites. Catechol (0.5 mg/mouse)decreased the proportion of watersoluble [³H]BaP metabolites,ethyl acetate-soluble polar metabolites and quinones, but doubledthe levels of unconjugated 3-hydroxy-BaP at all measured intervalsafter treatment. Catechol also caused a small increase in thelevels of trans-7,8-dihydroxy-7,8-dihydroBaP and trans-9,10-dihydroxy-9,10-dihydroBaP0.5 h after treatment. Two hours after treatment, the levelsof these metabolites subsided to those of the controls. Catecholdid not affect the levels of glutathione conjugates of BaP.However, it caused a decrease in glucuronide and sulphate conjugateformation from BaP. Catechol caused an 2-fold increase in theformation of anti-7, 8-dihydroxy-9, 10-epoxy-7, 8, 9, 10-tetrahydroBaP(BPDE) DNA adducts and elevated the ratio of anti-syn-BPDE-DNAadducts 1.6 to 2.9-fold. Catechol treatment increased the radioactivityassociated with epidermal proteins after [³H]BaP application.Because catechol increased levels of 3-hydroxyBaP, we consideredthe possibility that 3-hy-droxyBaP might enhance the tumor initiatingactivities of BaP or BPDE in mouse skin; a bioassay demonstratedthat this was not the case. The results of this study indicatethat one important effect of catechol related to its co-carcinogenicityis its ability to enhance formation of anti-BPDE-DNA adductsin mouse skin.