Insights on the relationship between complement component C4 serum concentrations and C4 gene copy numbers in a Western Australian systemic lupus erythematosus cohort.

The relationship between serum concentration of complement C4 ([C4]) and C4 gene copy number (GCN) was investigated in 56 systemic lupus erythematosus (SLE) patients and 33 age and sex-matched controls in a Western Australian population. C4A and C4B gene copy numbers (C4A & B GCN) together with the presence or absence of the ≈6.4-kb human endogenous retroviral element type K (hereafter HERV-K) in intron 9 were estimated by two TaqMan™ real-time PCR (RT-PCR) assays that measured total C4 and HERV-K GCNs, respectively. There was good correlation between the two methods; however, the HERV-K GCN method showed a positive bias (≈6%) relative to the C4A & B total GCN. Despite individual variation, excellent correlation between total C4 GCN and mean [C4] per GCN was observed for both the SLE and control cohorts ( R2 = 88% and R2 = 99%, respectively). It was noted that serum [C4] was significantly lower in the SLE patients than the controls ( p = 0.006) despite there being no difference between C4A and C4B GCN in both cohorts. The data therefore confirm previous reports that the C4A genes are preferentially associated with the presence of the HERV-K insertion relative to C4B genes and does not support the hypothesis that low [C4] in SLE is explained by low C4A GCNs. There was no evidence also that the presence of the HERV-K insertion in C4 genes influenced [C4]. This study supports the view that low [C4] in SLE patients is due to consumption rather than deficient synthesis related to lower C4A & B GCN.

Sequencing-based typing identifies novel alleles due to single nucleotide polymorphisms in 'conserved' regions.

The Royal Perth Hospital laboratory has been using sequencing-based typing for all HLA loci since 2002. In the period to October 2005, approximately 12,000 HLA A and HLA B, 5000 HLA C and DQB1, and 17,000 DRB1 requests have been processed. Twenty nine novel alleles have been identified in that time. These comprise 10 HLA-A (including one null allele), five HLA-B, six HLA-C, six DRB1 (including a null allele), and one DQB1 novel allele. (At the time of identifying the DRB1 null allele, there were no other reported examples.) In addition, we have seen one example of a blast-specific HLA-A null allele. One HLA-A allele (HLA-A*0264) and one HLA-B allele (HLA-B*400104) were subsequently identified in other laboratories and submitted to the international ImMunoGeneTics project (IMGT) database.

A multicenter international evaluation of single-tube amplification protocols for sequencing-based typing of HLA-DRB1 and HLA-DRB3,4,5.

We have described previously a novel single-tube polymerase chain reaction (PCR) amplification (STAmp) protocol for the efficient sequencing-based typing (SBT) of human leukocyte antigen (HLA)-DRB1. The PCR amplification mix includes primers to each of seven allele group-sequence motifs. We have applied this principle to the simultaneous SBT of HLA-DRB3, -DRB4, and -DRB5 using locus specific primers. We report here a multicenter international evaluation of the STAmp protocols performed as a component of the 13th International Histocompatibility Workshop. Identical amplification primer mixes, sequencing primers, and DNA were sent to participating laboratories. The primer mixes contained the amplification primers and the PCR buffer. Each laboratory was requested to amplify the DNA with the primer mixes and perform SBT on the resulting PCR protocols, using their own protocols, and return the typing results for analysis. The reported results indicated that the expected sequence could be obtained with a variety of PCR amplification and sequencing platforms and protocols. There were difficulties but these seemed unrelated to STAmp reagents and suggest that optimal SBT results can be obtained if bi-directional sequencing is performed and software is used for sequence verification and editing. This indicates that SBT by STAmp can be applied in many laboratories for high-throughput HLA-DRB1 and HLA-DRB3,4,5 SBT.

HLA-DRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors.

The HLA-DRB1 sequencing based typing strategies reported to date require separate amplifications of each sample with a series of group-specific primers followed by sequencing of any resulting polymerase chain reaction (PCR) products. Whilst this results in high resolution typing in the majority of cases, a number of unnecessary amplifications are performed. We report here a novel approach where amplification of the second exon of HLA-DRB1 is performed in a single tube for all alleles. Retrospective analysis of 642 consecutive Western Australian unrelated bone marrow registry donors has shown that this approach results in unambiguous typings in 71.1% of cases. Ambiguities can be readily resolved if necessary with a single additional sequencing reaction on the original PCR product.