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Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories.

AimCoeliac disease(CD) is a highly prevalent, gluten-dependent, autoimmune enteropathy. While the diagnosis is based on serological and histological criteria, genotyping of the human leucocyte antigens (HLA) DQ2 and DQ8 has been shown to have substantial clinical utility, especially in excluding the diagnosis in patients who do not carry either antigen. As a result, HLA genotyping is now being performed by more laboratories and has recently become one of the most frequently requested genetic tests in Australia. To date, there has been little scrutiny on the accuracy and reporting of results by laboratories new to HLA typing. In response to clinician feedback that identified potentially clinically significant discrepancies in HLA typing results, the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) undertook a pilot study to assess laboratory performance in the detection of HLA-DQ2/DQ8 and their associated HLA-DQA1and HLA-DQB1alleles.MethodsDNA was extracted from 5 patients and sent to 10 laboratories for external quality assurance (EQA) testing. Laboratories were assessed for reporting in genotyping, interpretation and methodology.ResultsOur findings showed that at least 80% of laboratories underperform with respect to recommended guidelines for HLA typing and reporting for CD, with 40% of laboratories failing to provide any clinical interpretation or full genotyping data. This suboptimal level of reporting may lead to ambiguities for downstream clinical interpretation that may compromise patient management.ConclusionsThese findings highlight the importance of adherence to standardised guidelines for optimal performance and reporting of HLA results and substantiate the need for EQA and proficiency testing for laboratories providing this service.

The +1169A allele of the A/T single nucleotide polymorphism (SNP; rs2665802), located within intron 4 of the human growth hormone I (GHI) gene, has been associated with reduced levels of circulating GH and insulin-like growth factor I, a reduced risk of colorectal cancer and a predisposition to osteoporosis. Whether this intronic SNP is itself the functional polymorphism responsible for exerting a direct effect on GHIgene expression, however, or whether it is instead in linkage disequilibrium with the functional SNP, has been an open question. The evolutionary conservation of the +1169T allele (and the surrounding intronic sequence) in the bovine genome, as well as in primate genomes, is, however, suggestive of its functionality. Although a potential alternative splice site spans the location of the +1169 SNP, polymerase chain reaction-based assays failed to yield any evidence for alternative splicing associated with either allele. To determine whether the +1169 SNP, in different allelic combinations with SNPs at -278 (G/T), -57 (T/G) and +2103 (C/T), exerts a direct effect on gene expression and/or GH secretion, we performed a series of transfections of various GHIhaplotype-expressing constructs into rat GC (somatotroph) cells. The results obtained provided evidence to support the contention that the +1169A allele contributes directly to the observed reduction in both GHIgene expression and GH secretion. Part of the apparent influence of the +1169A-bearing allele on GHIgene expression and GH secretion may still, however, be attributable to alleles of additional SNPs in cisto +1169A and located within either the promoter or the 3'-flanking region.

The regulatory regions of the neurofibromatosis type 1 (NF1) gene have scarcely been screened either for mutations of potential pathological importance or for functional polymorphisms. To address this question, a 987 bp sequence spanning the promoter and 5′ flanking sequence of the human NF1 gene was screened for sequence variants in 570 unrelated NF1 patients and 105 controls. Five novel sequence variants were identified, comprising a 14 bp deletion at −142 within the promoter region, three single nucleotide substitutions in the 5′UTR (C + 247T, C + 261G, G + 462C), and a substitution (C + 514T) at the 5′ end of the coding region that served to generate a Stop codon. The latter is likely to be of pathological significance since it is predicted to lead to the synthesis of a truncated protein. The functional significance of three of the other variants (14 bp del, C + 261G, G + 462C) was explored by luciferase reporter gene expression and electrophoretic mobility shift assays. The del14 variant demonstrated allele‐specific protein binding without altered reporter gene expression and the G + 462C allele showed slightly decreased reporter gene expression. © 2004 Wiley‐Liss, Inc.

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