Mannose-binding lectin gene polymorphisms as a susceptibility factor for chronic necrotizing pulmonary aspergillosis.

It was investigated whether a deficiency of mannose-binding lectin (MBL), which binds Aspergillus species avidly in vitro, could account for chronic necrotizing pulmonary aspergillosis (CNPA), which is seen most commonly in nonimmunocompromised patients. Blood samples were obtained from 11 patients (10 white) with CNPA and were compared with blood samples from 82 white control subjects. MBL haplotype profiles were determined by polymerase chain reaction, using sequence-specific primers and sequence-specific oligonucleotide probing techniques. Seven of the 10 white patients with CNPA had MBL haplotypes that encode for low levels of the protein, compared with 25.6% of the white control subjects (P=.004). Presence of the codon 52 mutation was particularly common in patients with CNPA (P=.015), which suggests a greater involvement of this mutation.

The influence of transforming growth factor-beta1 gene polymorphisms on the severity of gingival overgrowth associated with concomitant use of cyclosporin A and a calcium channel blocker.

BACKGROUND: The purpose of this study was to determine whether the prevalence and severity of gingival overgrowth in renal transplant recipients concomitantly treated with cyclosporin and a calcium channel blocker was associated with functional polymorphisms within the signal sequence of the transforming growth factor-(TGF)beta1 gene. METHODS: The extent and severity of gingival overgrowth for 164 renal transplant recipients immunosuppressed with cyclosporin A and concomitantly taking a calcium channel blocker since transplant were entered into the study (86 in Manchester, 78 in Belfast). Two biallelic polymorphisms of the TGF-beta1 gene were studied at position +869, codon 10 (leucine to proline substitution), and position +915, codon 25 (arginine to proline substitution). RESULTS: Subjects who were homozygous for proline at codon 10 had significantly higher overgrowth scores than those who were heterozygous (P= 0.03) or homozygous for leucine (P= 0.01). Subjects who were heterozygous (arginine/proline) at codon 25 had a significantly higher (P= 0.04) gingival overgrowth score than those who were homozygous for arginine. Logistic regression analysis indicated that for codon 25 independent predictors of severe gingival overgrowth were the heterozygous arginine/proline genotype (P= 0.009) and whether the individual was young (P= 0.05). CONCLUSIONS: Polymorphisms in the TGF-beta1 gene influence the expression of gingival overgrowth in renal transplant recipients concomitantly treated with cyclosporin and a calcium channel blocker. The polymorphism in the TGF-beta1 gene at codon 25 represented an independent genetic determinant of severe gingival overgrowth in the susceptible subjects studied.

Independent association of rheumatoid factor and the HLA-DRB1 shared epitope with radiographic outcome in rheumatoid arthritis.

OBJECTIVE: Findings of a recent study suggested that HLA-DRB1 alleles encoding the rheumatoid arthritis (RA) "shared epitope" (SE) were not predictive of erosive damage at 2 years in patients with early inflammatory arthritis who were rheumatoid factor (RF) positive, but were predictive in those who were RF negative. The present study was undertaken to determine whether RF status was also important in the association between the SE and radiographic outcome in patients with longstanding RA. METHODS: The association between radiographic outcome, HLA-DRBI, and RF status was examined in 299 RA patients with established disease (5-30 years). Radiographic outcome was measured by scoring radiographs of the hands and feet using the standard radiographs of Larsen. HLA-DRB1 typing was performed using polymerase chain reaction methodology. Results were stratified by RF status and analyzed by multiple regression. RESULTS: An association between radiographic severity and the SE was found in RF-, but not RF+, patients. RF- patients carrying an SE allele had higher Larsen scores than RF- patients lacking the SE, although there was no association with SE dosage. The mean Larsen score was significantly higher in RF+ patients than in RF- patients, but there were no differences between RF+ patients with 0, 1, or 2 SE alleles. Multiple regression analysis confirmed independent associations of RF and SE positivity with radiographic outcome. No significant associations were found between RF and the SE, or RF and individual SE alleles. CONCLUSION: Our data indicate that RF and the SE are independently associated with radiographic outcome in RA. In RF+ patients with longstanding RA, there is no apparent association between the presence of the SE and radiographic damage. However, in RF-patients, although radiographic outcome is generally less severe, there is an association between severity and presence of the SE.

Short-form HLA-DP typing with 48 primer mixes using the polymerase chain reaction and sequence-specific primers.

We have developed a short-form SSP-based HLA-DP typing system for routine use adapted from a comprehensive HLA-DP typing method described by Gilchrist et at. (1998). Our short-form system detects 93 alleles, including the 18 most common HLA-DPB1 alleles and eight HLA-DPA1 alleles. The primer mixes described were tested using the PCR-SSP Manager (Bunce et al., 1998) database to confirm the specificity of selected primers, and to detect potentially ambiguous amplifications. This short-form HLA-DP typing system was validated using 50 fully typed samples obtained through the UCLA International DNA Exchange. All samples gave 100% concordance with the consensus type. Our laboratory routinely uses a PCR-SSP based system of 48 primer mixes for HLA-DRB and HLA-DQB typing. The advantage of the short-form HLA-DP typing system described here is that the additional 48 HLA-DP primer mixes required can be included on the second half of a 96-well format tray. This method now enables a full HLA class II typing at the level of allele group resolution in 2 1/2 h.

Mannose binding lectin (MBL) genotype distributions with relation to serum levels in UK Caucasoids.

Mannose binding lectin (MBL) gene and promoter-region polymorphisms contribute to a reduction in the levels of circulating MBL in a number of ways. Promoter polymorphisms affect the levels of MBL produced, whilst structurally encoding mutations cause non-functional protein to be assembled and subsequently degraded. MBL is important as a protein of the innate immune system in both the clearance of potential pathogens and the activation of the complement cascade. Using variations of SSP-PCR amplifications and SSO probing techniques, we have produced MBL-polymorphism haplotype and genotype profiles of a series of high-level MBL-producing, low-level MBL-producing and random individuals taken from a population of 800 UK Caucasoid controls. Structurally encoding mutant alleles were more frequent within the low-level producing cohort when compared to both high-level producers and the randomly selected sample. However, not all low-level producers could be accounted for by the possession of low-level encoding haplotypes. This may be due to the presence of additional, undetected polymorphisms governing MBL production, or another external factor that may influence the transcriptional regulation of the gene.

High serum levels of pro-matrix metalloproteinase-3 are associated with greater radiographic damage and the presence of the shared epitope in patients with rheumatoid arthritis.

OBJECTIVE: To determine if there is a relationship between serum pro-matrix metalloproteinase-3 (proMMP-3) levels and radiographic damage in rheumatoid arthritis (RA), and to investigate whether high levels are associated with presence of the HLA-DRB1 shared epitope (SE). METHODS: Serum proMMP-3 levels were measured by ELISA on 45 RA patients with early disease and 292 with established disease. Early RA was arbitrarily defined as disease duration <3 years. Clinical and laboratory measures of disease activity and severity were obtained. Radiographic damage was assessed by scoring radiographs of the hands and feet using the method of Larsen. HLA-DRB1 typing was performed by sequence-specific oligonucleotide probing. Data were analyzed by multiple regression analysis. RESULTS: In all patients, there was a correlation (r = 0.318, p<0.0001) between serum proMMP-3 levels and Larsen scores. Other correlations were found with Health Assessment Questionnaire score (r = 0.261, p<0.0001) and C-reactive protein (CRP) (r = 0.357, p<0.0001) levels. ProMMP-3 levels were significantly higher in SE+/+ patients than in those completely lacking the SE, with the highest levels in patients carrying an HLA-DRI+/DR4+ phenotype. The greatest difference in proMMP-3 levels between SE+/+ and SE-/- patients was in those with a disease duration <3 years (381.6 vs. 71.7 ng/ml; p = 0.02). CONCLUSION: Our data indicate that there is a significant relationship between radiographic damage and serum levels of proMMP-3. As well, higher circulating levels of proMMP-3 are found in patients positive for the SE, particularly in early RA, and this may partly explain the association between the SE and more erosive disease.

Blue genes: genetic variation in our immune genes may be a mixed blessing.

Today we know that almost all aspects of disease are affected in some way by our genes. The size of the genetic contribution towards each disease shows tremendous variation, but ultimately, the key to our survival of onslaught from environmental agents lies within our gene pool.

The influence of HLA-DRB1 alleles encoding the DERAA amino acid motif on radiological outcome in rheumatoid arthritis.

OBJECTIVES: To investigate the influence of HLA-DRB1 alleles encoding the QK/RRAA shared epitope (SE) on radiological outcome in rheumatoid arthritis (RA), and to determine whether it is modulated by alleles carrying the putative rheumatoid arthritis-protective (RAP) sequence DERAA. Patients and methods. The association between erosive damage and HLA-DRB1 status was examined in 315 RA patients with a disease duration of 5-30 yr. Radiological outcome was measured by scoring X-rays of the hands and feet using the standard radiographs of Larsen (Larsen score). HLA-DRB1 typing was carried out using polymerase chain reaction methodology. RESULTS: Patients with two alleles encoding the QK/RRAA SE had significantly higher Larsen scores than SE-negative patients (96.9 vs 83.3; P=0.04, after correction for multiple testing), with DRB1*0401/*0401 homozygotes demonstrating the greatest radiological damage (99.9). The lowest Larsen score (65.6) was observed in patients carrying the DERAA motif without an accompanying SE allele (RAP+/SE-). This was significantly lower than in patients with RAP+/SE+ (105.6; P=0.04), RAP-/SE- (88.2; P=0.05) and RAP-/SE+ (95.8; P=0.009), after correction for multiple testing. There was no evidence that the RAP sequence was modulating the effect of the SE since radiological outcome in RAP+/SE+ patients was not significantly different to that in RAP-/SE+ individuals. CONCLUSIONS: Our data support a possible role for DRB1 alleles encoding the DERAA motif in protection against severe erosive damage in patients lacking the QK/RRAA SE, but not in patients heterozygous for the SE. This suggests that DRB1 alleles encoding the SE have a dominant influence over 'protective alleles' and are not merely 'non-protective'.

A study of HLA-DPB1 phenotypes reveals DPB1*6301 in a rural population from Cameroon.

Several recently reported HLA-DPB1 alleles have only been identified in a single family or individuals and are of unknown distribution world-wide. Many new DPB1 alleles appear to arise as a result of gene conversion-like events, which may localize variant DPB1 alleles to the population in which they were first identified. Using two SSOP-based typing methods in parallel, we have identified HLA-DPB1*6301 in an individual from rural Cameroon which has previously only been reported in a family of Mexican-American origin. The presence of DPB1*6301 was confirmed by sequence-based typing of exon 2.

Definition of HLA-C alleles using sequence-specific oligonucleotide probes (PCR-SSOP).

Many new HLA-C locus alleles have recently been identified by DNA sequencing, and a molecular based method for their detection using PCR with sequence specific primers has been reported. However, other methods may be more appropriate for the identification of C locus alleles in larger studies. Here we describe one such system, based on PCR sequence specific oligonucleotide probes, (SSOP) for C locus typing. Advantages of SSOP typing compared to SSP are that it is easier to detect new alleles, more cost effective and less time consuming. We have developed a DNA typing method to identify the broad C locus antigens (including those not yet defined serologically) using a minimum of probes with one amplification. We use a C locus specific sense primer in exon 2 and a consensus antisense primer in exon 3, in a two-step PCR, giving a product of 710 bp. Probes were designed with similar melting temperatures (54-56 degrees C) that would identify as many alleles as possible. The method was established using DNA from B lymphoid cell lines of known C locus type, mostly 10th workshop homozygous cell lines, plus as many other sequenced cell lines as possible. The system was able to correctly identify their C locus types using only 26 probes. DNA was tested from a panel of serologically typed individuals which included many different heterozygous combinations. We found a high concordance of results, with all discrepancies being additional antigens identified by molecular typing, filling in serological blanks. We can identify all common heterozygote combinations using this method.