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DNA sequencing technologies are transforming how environments are monitored. In this article, we pose the question: is environmental DNA (eDNA) the tool that Aotearoa New Zealand needs, but does not yet realise it does? The step change with eDNA is that genetic ‘breadcrumbs’ left behind in the environment can identify every living thing, from microbes to mammals, thus providing a more nuanced and holistic lens on ecosystems. Using eDNA, we can explore the biological networks that underpin healthy environments. Here we explore whether changes in policy setting, guidance, or pathways for uptake of eDNA are needed. Can eDNA help us make better decisions, inform policy and protections, track restoration, and act as a deterrent to reduce environmental harm?

Offshore oil and gas platforms have the potential to provide complex refugia for fish and benthic colonisers. We compare two methods of biodiversity assessment for fish and elasmobranchs at seven decommissioned oil and gas platforms as well as five sediment sites, located 5 km from platforms, in the Gulf of Thailand. Using surveys from stereo-video ROV transects, and data from Environmental DNA (eDNA) water-column samples, we detected fish and elasmobranch taxa from 39 families and 66 genera across both platform and sediment sites with eDNA, compared with 18 families and 29 genera by stereo-ROV with platforms yielding significantly greater species richness. This study demonstrates that the combination of stereo-video ROV and eDNA provide effective, non-extractive and complementary methods to enhance data capture. This approach sets new benchmarks for evaluating fish assemblages surrounding platforms and will enhance measurements of biota to inform decisions on the fate of oil/gas infrastructure.

Traditional herbal products are widely used in Australia to treat a broad range of conditions and diseases. It is popularly believed that these products are safer than prescribed drugs. While many may be safe, it is worrying that the specific effects and harmful interactions of a number of their components with prescription medications is not well understood. Some traditional herbal preparations contain heavy metals and toxic chemicals, as well as naturally occurring organic toxins. The effects of these substances can be dire, including acute hepatic and renal failure, exacerbation of pre-existing conditions and diseases, and even death. The content and quality of herbal preparations are not tightly controlled, with some ingredients either not listed or their concentrations recorded inaccurately on websites or labels. Herbal products may also include illegal ingredients, such as ephedra, Asarum europaeum (European wild ginger) and endangered animal species (eg, snow leopard). An additional problem is augmentation with prescription medications to enhance the apparent effectiveness of a preparation. Toxic substances may also be deliberately or inadvertently added: less expensive, more harmful plants may be substituted for more expensive ingredients, and processing may not be adequate. The lack of regulation and monitoring of traditional herbal preparations in Australia and other Western countries means that their contribution to illness and death is unknown. We need to raise awareness of these problems with health care practitioners and with the general public.

Outbreaks of zoonotic diseases in humans and livestock are not uncommon, and an important component in containment of such emerging viral diseases is rapid and reliable diagnostics. Such methods are often PCR-based and hence require the availability of sequence data from the pathogen. Rattus norvegicus (R. norvegicus) is a known reservoir for important zoonotic pathogens. Transmission may be direct via contact with the animal, for example, through exposure to its faecal matter, or indirectly mediated by arthropod vectors. Here we investigated the viral content in rat faecal matter (n=29) collected from two continents by analyzing 2.2 billion next-generation sequencing reads derived from both DNA and RNA. Among other virus families, we found sequences from members of the Picornaviridae to be abundant in the microbiome of all the samples. Here we describe the diversity of the picornavirus-like contigs including near-full-length genomes closely related to the Boone cardiovirus and Theiler's encephalomyelitis virus. From this study, we conclude that picornaviruses within R. norvegicus are more diverse than previously recognized. The virome of R. norvegicus should be investigated further to assess the full potential for zoonotic virus transmission.

Background Effective biodiversity monitoring is fundamental in tracking changes in ecosystems as it relates to commercial, recreational, and conservation interests. Current approaches to survey coral reef ecosystems center on the use of indicator species and repeat surveying at specific sites. However, such approaches are often limited by the narrow snapshot of total marine biodiversity that they describe and are thus hindered in their ability to contribute to holistic ecosystem-based monitoring. In tandem, environmental DNA (eDNA) and next-generation sequencing metabarcoding methods provide a new opportunity to rapidly assess the presence of a broad spectrum of eukaryotic organisms within our oceans, ranging from microbes to macrofauna. Methods We here investigate the potential for rapid universal metabarcoding surveys (RUMS) of eDNA in sediment samples to provide snapshots of eukaryotic subtropical biodiversity along a depth gradient at two coral reefs in Okinawa, Japan based on 18S rRNA. Results Using 18S rRNA metabarcoding, we found that there were significant separations in eukaryotic community assemblages (at the family level) detected in sediments when compared across different depths ranging from 10 to 40 m (p = 0.001). Significant depth zonation was observed across operational taxonomic units assigned to the class Demospongiae (sponges), the most diverse class (contributing 81% of species) within the phylum Porifera; the oldest metazoan phylum on the planet. However, zonation was not observed across the class Anthozoa (i.e., anemones, stony corals, soft corals, and octocorals), suggesting that the former may serve as a better source of indicator species based on sampling over fine spatial scales and using this universal assay. Furthermore, despite their abundance on the examined coral reefs, we did not detect any octocoral DNA, which may be due to low cellular shedding rates, assay sensitivities, or primer biases. Discussion Overall, our pilot study demonstrates the importance of exploring depth effects in eDNA and suggest that RUMS may be applied to provide a baseline of information on eukaryotic marine taxa at coastal sites of economic and conservation importance.

Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-basedgenotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the “true” composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are controlled.

BACKGROUND: Despite strong evidence implicating immune dysfunction and genetic predisposition in the pathogenesis of the chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis, the importance of the genes of the major histocompatibility complex remains uncertain. We have investigated the contribution of HLA DRB1 and DQB genes by the strategies of non-parametric linkage analysis (affected sibling pair method) as well as association study. The relation between genotype and phenotype was examined in detail. METHODS: For linkage analysis 74 families in whom two or more siblings had inflammatory bowel disease were studied. A total of 83 affected sibling pairs were involved: in 42 pairs both siblings had Crohn's disease; in 29 both had ulcerative colitis; in 12 one sibling had Crohn's disease, the other ulcerative colitis. For the association study there were 175 patients with ulcerative colitis, 173 with Crohn's disease, and 472 controls. Details of sex, age of onset, disease extent, and family history were analysed. 24 patients with ulcerative colitis and 92 with Crohn's disease required surgery for refractory disease. HLA DRB1 and DQB1 gene-typing was performed by polymerase chain reaction with sequence-specific primers. FINDINGS: In ulcerative colitis, the sharing of alleles among affected sibling pairs provided evidence for linkage with DRB1 locus (p = 0.017, chi2 = 5.32). Of 29 affected sibling pairs studied, only one pair shared no DRB1 DQB haplotypes. 15 shared two DRB DQB haplotypes. In contrast, no linkage was noted for Crohn's disease (42 sibling pairs; p = 0.30, chi2 = 0.16) or for inflammatory bowel disease overall (83 sibling pairs, p = 0.16, chi2 = 2.28). In the association study the rare DRB1*103 (8.3% vs 3.2% in controls) and DRB1*12 (8.6% vs 2.1% in controls) alleles were associated with ulcerative colitis (p = 0.0074, chi2 = 7.22, odds ratio OR = 2.9 [95% CI 1.3-6.4] and p = 0.0056, chi2 = 12.63, OR = 4.33 [1.8-11.0] respectively). No association with alleles representing DR2 (p = 0.55, chi2 = 0.34) was noted. No overall association was seen in Crohn's disease. In ulcerative colitis, the frequency of DRB1*0301 DQB*0201 (DR3 DQ2) was reduced in females (9.8% vs 26.3% in controls, p = 0.037, chi2 = 8.39 OR = 0.34 [0.15-0.71]), particularly in those with distal disease (2.3%, p = 0.001 vs controls, chi2 = 11.35, OR = 0.07 [0.00-0.39]). In both males and females, the DR3 DQ2 haplotype was predictive of extensive ulcerative colitis (32.9% vs 10.7% in distal disease, p < 0.01, chi2 = 10.94, OR 4.09 [1.70-10.6]) but not of need for surgery (p = 0.93, chi2 = 0.01). INTERPRETATION: These data provide strong evidence for genetic heterogeneity in inflammatory bowel disease. Genes of the major histocompatibility complex are implicated as important inherited determinants of susceptibility to ulcerative colitis and may also influence the pattern of disease. In Crohn's disease, important susceptibility genes are likely to exist outside the HLA region.

Linkage and association studies implicate the human leucocyte antigen (HLA) region in genetic susceptibility to ulcerative colitis (UC). However, associations with specific variants have been inconsistent, even within defined ethnic groups. A genetic basis for the disease heterogeneity of UC may account for these discrepant findings from studies in unselected populations. Here, we examine the contribution of the HLA region to the clinical phenotype of UC. We studied 321 accurately phenotyped patients recruited from a single UK centre, with a median follow-up time of 15 years. Individuals were genotyped for 340 polymorphisms constructed into 25 gene-specific allelic haplotypes between HLA-A and Tapasin. Data were analysed with respect to age of onset, disease extent and severity. Strongest association with overall susceptibility was identified with HLA-DRB1 alleles replicating previous studies (DRB1*0103, DRB1*1502 and DRB1*0401). We report a novel association with homozygosity of a tumour necrosis factor (TNF) promoter haplotype (TNF-1031T, -863C, -857C, -380G, -308G and -238G) and distal disease extent that does not extend with time (distal vs total 40.9 vs 25.7%; RR = 2.0; 95% CI 1.23-3.24). We confirm the association of DRB1*0103 with total disease and/or disease requiring colectomy and further demonstrate that DRB1*0103 is associated with shorter time to surgery. Genes in the HLA play a role in modifying disease phenotype. Further studies are required to dissect how these genes functionally interact with each other and with environmental factors to determine clinical patterns of disease

Detailed knowledge of linkage disequilibrium (LD) is regarded as a prerequisite for population-based disease gene mapping. Variable patterns across the human genome are now recognized, both between regions and populations. Here, we demonstrate that LD may also vary within a genomic region in a haplotype-specific manner. In 864 Caucasian unrelated individuals, we describe haplotype-specific LD patterns across the human MHC by the construction of gene-specific allelic haplotypes at 25 loci between HLA-A and Tapasin. Strong and extensive LD is found across both common and rare haplotypes, suggesting that haplotype structure is influenced by factors other than genetic drift, including both selection and differential haplotype recombination. Knowledge of haplotype-specific LD in the HLA may explain the apparent discrepant data from previous studies of global LD, help delineate key areas in mapping HLA-associated diseases and, together with recombination data, provide valuable information about a population's demographic history and the selective pressures operating on it.

The woylie or brush-tailed bettong (Bettongia penicillata) is a medium-sized native Australian marsupial that has undergone a dramatic decline in numbers in recent years. Trypanosome parasites have been identified in the woylie but little is known about the prevalence and clinical impact of other haemoprotozoan parasites in these marsupials. In the present study, the occurrence and molecular phylogeny of a piroplasm was studied in woylies from six different sites in Western Australia (WA). Blood samples were screened by PCR at the 18S rRNA locus and 80.4% (123/153) of the blood samples were positive for piroplasm DNA. Sequence and phylogenetic analysis of 12 of these positives identified them as Theileria penicillata, and sequencing of cloned PCR products indicated that no other species of Theileria were present. Infected woylies had a lower body weight but microscopic evaluation of the blood films indicated that T. penicillata did not appear to cause red cell injury or anaemia. Further studies are required to determine the clinical significance of T. penicillata in woylies.

Hereditary hemochromatosis (HH) is an iron-overload disease common in populations of Northern European origin. Patients display increased iron absorption leading to excessive iron deposition and potential multiorgan failure. Using polymerase chain reaction sequence-specific primer (PCR-SSP) technology, we have developed an HH diagnosis assay capable of detecting 19 non-synonymous HFE mutations (including a previously unreported mutation, V295A) and several TFR2, SLC11A3 and H ferritin alleles implicated in HH. As part of the validation process, 159 UK renal donors were genotyped to determine HH allele frequencies in the UK population. The alleles nominally identified as HFE*01 (C282Y), HFE*02 (H63D) and HFE*03 (S65C) were found at frequencies of 0.085, 0.173 and 0.009, respectively. All other potential HH-associated alleles were absent, confirming their low prevalence in this population. This assay enables comprehensive routine HH genotyping, producing rapid, accurate and reproducible results at low cost.

The highly polymorphic nonclassical MHC class I chain-related (MIC) genes MICA and MICB encode stress inducible glycoproteins expressed on a variety of epithelial cells including intestinal cells. Interaction with the receptor NKG2D is likely to provide an important costimulatory signal for activation and proliferation of NK cells, activated macrophages and CD8 alphabeta and gammadelta T cells. Fifty-four MICA and 17 MICB alleles have been described to date. Although the functional significance of this polymorphism is not known, the high degree of nonconservative substitution, concentration to the putative ligand-binding site and recent observation that different MICA alleles bind to NKG2D with varying affinity has generated much interest. The MIC genes are attractive functional and positional candidate genes for inflammatory bowel disease susceptibility as a consequence of their position in the HLA region and expression on the gastrointestinal epithelium. We developed a robust, high-resolution PCR-SSP genotyping method that can be incorporated into the standard 'Phototyping' system and which effectively identifies 46 of 54 MICA alleles, and all 17 MICB alleles. We applied this system in combination with microsatellite genotyping of the exon 5 variable number of tandem repeats (VNTR) to the investigation of genetic susceptibility to the inflammatory bowel diseases, ulcerative colitis and Crohn's disease. We studied 248 patients with Crohn's disease, 329 with ulcerative colitis and 354 ethnically matched controls. Linkage disequilibrium patterns between HLA-B, MICA and MICB are presented. Analysis by individual allele or by multilocus haplotype failed to identify any significant disease associations.

Although the taphonomic (post-mortem) degradation processes relevant to teeth and bones have been well described, those taking place with regards to mammalian hairs have not been characterized to the same extent. This present article describes, in detail, microscopic changes resulting from the actions of biological agents that digest and degrade hairs. The most noteworthy and prevalent agents responsible for the destruction of hair structure are fungi, which use a range of strategies to invade and digest hairs. One of the most important finds to emerge from this study is that taphonomic structures and processes can easily be interpreted by the unwary as ‘real’, or as class characteristics for a particular animal taxon. Moreover, under certain conditions, ‘taphonomic’ processes normally associated with the dead are also present on the hairs of the living. This work will improve the reliability of hair examinations in forensic, archaeological and palaeontological applications—in addition, the finding has relevance in the protection of mammalian collections susceptible to infestation. This article also addresses the popular myth that ancient peoples were often red-haired and discusses phenomena responsible for this observation. Insights gained from detailed characterization of taphonomic processes in 95 hairs from a variety of species demonstrate the range and breadth of degradative effects on hair structure and colour. Lastly, the study demonstrates that hairs often tell a story and that there is value of extracting as much morphological data as possible from hairs, prior to destructive sampling for biomolecules.

The role and frequency that herbal therapeutic agents play in unexpected deaths remains unclear. Herbal agents may be directly toxic in their own right, or they may act synergistically with other herbal materials, or with prescribed pharmaceutical agents. Herbal therapies are also known to sometimes contain toxic agents such as herbicides or heavy metals, or to have been adulterated with standard drugs to enhance their efficacy [1]. In the medicolegal arena the consumption of drugs that may be contraindicated in certain conditions may not be apparent at the time of autopsy, such as when steroids or non-steroidal antiinflammatory agents have been unknowingly taken by an individual with active peptic ulcer disease. Substitution of one plant for another, whether because of financial considerations or from a genuine mistake in plant identification, compounds the complexities associated with attempting to determine whether a herbal preparation has contributed to, or caused, illness or death. Thus, when compared to the assessment of prescribed medications in a standard forensic autopsy, the evaluation of the role of herbal preparations in a death is problematic [2, 3]. Death scene examiners may not document herbal preparations, as they may be considered to be ‘‘natural’’ products and therefore not likely to cause any health problems. Even if herbal medications are recorded at a death scene, there is no guarantee that the ingredients and dosages listed on the container (if this information is available) are a true reflection of the composition of the contained preparation. As standard preparations come as powders, pill or liquids, standard taxonomic classification of herbs by morphology is not possible. Finally, due to the complex organic molecules present and the blending of a number of plants, searching for active organic ingredients in forensic toxicology laboratories may resemble the time honored search for the ‘‘needle in haystack’’ [4]. Fortunately technology is now available which may assist in focusing the search for these elusive active organic agents. Two recent papers [5, 6] show how DNA sequencing can be used to identify materials in herbal medicines that would otherwise be potentially difficult to analyze. In particular, Coghlan et al.’s [5] use of next generation DNA sequencing (NGS) demonstrates how sophisticated analyses of samples, including complex mixtures unable to be evaluated effectively by previous techniques, can be rapidly screened. NGS analysis of herbal medications confiscated at a death scene may provide valuable data that can then be compared to either in-house databases or to larger collections such as the NIH genetic sequence database, GenBank (http://www.ncbi.nlm.nih. gov/genbank/). Although a potential difficulty with external databases involves the accuracy with which entries have been checked prior to uploading, they may, along with smaller databases, be extremely useful in matching the pattern of a DNA sequence to that of known plants. Thus, modern DNA sequencing can rapidly provide a genetic audit of large numbers of cases that can be matched against established standards to focus the search for ingredients [5] such as toxic plants and/or potential allergens. R. W. Byard (&) I. Musgrave C. Hoban School of Medical Sciences, Level 3 Medical School North Building, The University of Adelaide, Frome Road, Adelaide, SA 5005, Australia e-mail: roger.byard@sa.gov.au

Mycobacterium malmoense is an opportunistic mycobacterium that occasionally causes disease in non-immunosuppressed individuals. As only a few individuals exposed to these organisms actually develop clinical disease, it is possible there is a genetic component to susceptibility. CD1 molecules are capable of presenting antigens from more virulent mycobacteria to T cells; therefore, we were interested in discovering whether recently described polymorphisms in CD1 molecules modulated susceptibility to M. malmoense pulmonary disease. The CD1 system comprises five genes (CD1A, -B, -C, -D, and -E) located on chromosome 1 (1q22–23). CD1 molecules are structurally and functionally related to major histocompatibility complex (MHC) class I molecules and are expressed on dedicated antigen-presenting cells. The primary function of CD1 molecules is to present lipid and glycolipid antigens to T cells. We have developed an allele-specific polymerase chain reaction-sequence-specific primer (PCR-SSP) method of CD1 genotyping. Using this method, we compared the allele and haplotype frequencies of CD1 in 49 HIV-negative patients with M. malmoense pulmonary disease with those in 342 normal controls. The CD1A and CD1E alleles were nominally identified as CD1A*01, CD1A*02, CD1E*01 and CD1E*02, and the control gene frequencies were found to be 5%, 95%, 67% and 33%, respectively. No significant difference was observed between the patient and control cohorts. Positive linkage disequilibrium values of 0.73 were observed between CD1A*02 and CD1E*01 (P

HLA class I molecules present endogenously processed peptide ligands for surveillance by the T-cell receptor. This potentially immunogenic surface of HLA and peptide is a consequence of the polymorphism found within the HLA molecule and its preference for ligand binding together with peptide conformation within the binding groove. To investigate the relation between the polymorphic differences between some closely related HLA alleles and their effect on peptide preference, transfectants were established, each containing one of four allelic variants of HLA-A*30. Peptides from all four transfectants were eluted, and both individual ligands and peptide pools were sequenced. The data shows two distinct peptide motifs which distinguish A*3001 from the other three known A*30 variants. Differences in preferences at minor positions within the peptide sequence were noted between A*3002, A*3003 and A*3004, providing additional evidence of the implications of sequence polymorphism to HLA function.

We have identified a null HLA-A*02 allele, HLA-A*0232N, by using a combination of serology, flow cytometry, polymerase chain reaction using sequence-specific primers (PCR-SSP) and full-length sequencing. The null HLA-A2 allele was identified in an Asian individual originally typed by serology as an apparently homozygous HLA-A3, B51. Subsequent genotyping by PCR-SSP identified the genotype as HLA-A*0201, *0301, B*51, Cw*1402. The serological type and lack of detectable HLA-A2 was confirmed using monoclonal antibody typing reagents. Flow cytometry studies failed to identify any cell surface HLA-A2 expression on the patient's peripheral blood lymphocytes. Genotyping using a PCR-SSP set designed to detect null alleles revealed the mutation had not been previously described. Full-length sequencing of the allele identified an allele which was subsequently named HLA-A*0232N. This allele is identical to HLA-A*0201 except for a novel point mutation (T for C) at position 493 which creates a premature stop codon. The sequencing enabled the development of a monospecific A*0232N PCR-SSP reaction which was used to screen 973 DNA samples: no further examples of A*0232N were identified.

We have analyzed a series of HLA region markers in 207 UK Caucasoids with early-onset myasthenia gravis (EOMG, onset before age 40), where there is a strong female bias. The well known associations with HLA-DR3 and -B8 have now proved to be significantly stronger in the 165 females than in the 42 males. In patients (of either sex) lacking -DR3, there was also a significant increase in HLA-DR2. Although the muscle weakness in EOMG is clearly mediated by autoantibodies, the associations are consistently stronger with HLA-B8 (in class I) than with HLA-DR3 (in class II), as confirmed here. We therefore typed 87–137 cases for polymorphisms at four loci in the intervening class III region, and also at three in the adjacent stretch of class I. At each locus, one allele tended to co-occur with HLA-B8 and showed strong and highly significant associations in the patients. There appeared to be a region of maximal susceptibility extending from HSP70 (in class III) past HLA-B and HLA-C at least 600kb telomerically into the class I region, which is now being mapped in detail. Any candidate genes here that act shortly after puberty may allow more precise localization of susceptibility.

HLA-A*2418 is described for the first time. It segregates in a Mendelian fashion. Serological analyses indicate that the new allele encodes epitopes from both HLA-A9 and -A3 specificities. Results from nucleotide sequencing analyses of polymerase chain reaction (PCR) amplification products derived from genomic and cDNA are compatible with those findings.

Molecular methods are now commonplace for HLA typing and they have replaced traditional serological methods in many histocompatibility laboratories. A consequence of reliance on molecular methods using primers or probes based on existing sequence information is that unsequenced or partially-sequenced null, or low expressed variants are not discriminated from expressed alleles. Failure to identify null alleles might have deleterious implications for allogeneic transplants. Expression variants may be classified into two categories: unique mutations and repeat mutations. For example, the alleles A*0303N, A*2409N, and B*1526N have apparently unique mutations. In contrast, repeat mutations may occur frequently at points where unusual nucleotide sequences make accurate DNA replication by DNA polymerases difficult. One example is between nucleotide positions 621–627, where HLA class I alleles may exhibit between three and seven consecutive cytosine residues. Incorrect insertion of an extra cytosine in this region is the cause of expression failure in A*2411N and A*0104N alleles. We hypothesise that insertion of an extra cytosine into the cytosine island between nucleotide positions 621–627 is likely to recur not only in other HLA-A alleles but also in HLA-B and even HLA-C alleles. We describe here a polymerase chain reaction using sequence-specific primers (PCR-SSP) system that can not only detect all previously-sequenced HLA class I expression variants but can also screen for mutations between positions 621–627 in HLA-A, B or C alleles which may give rise to potentially null alleles. Overall, in this study HLA class I expression variants were identified in 5 of 931 tested samples (0.53%).

Molecular typing for HLA class I was introduced in the 12th International Histocompatibility Workshop. Following a pilot study using three methods, sequence specific oligotyping (SSO), reverse dot blot and amplification refractory mutation system (ARMS)-PCR, the ARMS-PCR method was selected for use. A great advantage of an ARMS-PCR method is that, unlike the other two methods, it can determine whether sequence motifs are in cis or in trans, as ARMS-PCR detects two cis located motifs per reaction using forward and reverse sequence specific primers. Resolution was designed to be low to medium level for HLA-A, -B and -C alleles. Two hundred and fifty class I kits and 83 HLA-A2 subtyping kits were distributed. The A2 subtyping kit used a two round nested PCR system to identify all of the A2 alleles known at the time. Typing results on control DNA samples distributed with both the kits showed a very satisfactory performance. Since the 12th Workshop, the kits have been developed with the addition of new primers and primer mixes to increase the resolution of the test.

Exon 3 of DRB1 is known to be polymorphic, but thought to be conserved within allelic groups. This implies that exon 3 polymorphisms would not need to be considered in evolutionary studies or clinical settings when assessing immunogenicity of allelic mismatches in stem cell transplantation. To further assess this, we determined the sequences of DRB1 exon 3 by hemizygote amplification and direct sequencing on 55 selected DNA samples containing 42 DRB1 alleles for which no exon 3 sequence data were previously available. The data confirmed the high degree of overall sequence conservation. The DRB4- and DRB5-associated alleles were completely conserved within their DRB1 groups. However, it could be shown that exon 3 is more diverse than previously expected. Multiple allelic differences within each group of DRB3-associated DRB1 alleles were found, without identifying unique group-related sequence motifs differentiating between these groups. For DRB1*1402 and DRB1*1406, it could be shown that they originated from DRB1*0302. In several samples previously typed as DRB1*1401, a novel DRB1 allele was identified: DRB1*1454. Thus, from a clinical viewpoint, the availability of exon 3 sequence information may be useful for optimizing typing as well as matching strategies. Additionally, it will allow for more detailed evolutionary studies, further elucidating the origin of alleles and the mechanisms driving sequence diversification.

An almost complete and bidirectional association exists between HLA-Cw*17 and the HLA-B antigens B41 and B42. Serological and molecular analysis of an individual in which HLA-B*4101 was identified in the absence of Cw*17 provides experimental evidence to prove a previously proposed hypothesis predicting that alloantisera classified as "B41+B42" are instead specific reagents for HLA-Cw*17.

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed.

Two previously reported PCR-SSP variants of the HLA-B locus, B51GAC and B45v, were investigated by RT-PCR cloning and nucleotide sequence analysis of their complete coding regions. They have been shown to correspond to the new alleles B*5108 and B*5002, both of which differ from the common B*5101 and B*5001 subtypes, respectively, by amino acid replacements at their alpha-2 domain alpha-helices. The primary structure of B*5002, intermediate between those of B*4501 and B*5001, raises further concern about the current classification of B*45 as a B12 rather than as a B*50 subtype.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system that operates under identical conditions to HLA phototyping was devised for characterizing polymorphisms in tumor necrosis factor (TNF) and lymphotoxin alpha (LT-alpha). Mismatches at the 3' end were incorporated into the forward and reverse primers of each PCR so as to unequivocally establish the cis/trans status between the biallelic sites. Three previously described biallelic polymorphisms in TNF and three in LT-alpha were characterized in a 24-reaction PCR-SSP system. The method was used to genotype 20 cell lines and 201 HLA class I and II typed controls from the United Kingdom at the TNF and LT-alpha loci. Population frequencies of TNF haplotypes were determined as was linkage disequilibrium with HLA-A, B, Cw, DRB1 and DQB1 loci. In each gene there were 8 theoretical polymorphic combinations; 4 were observed in TNF and 4 in LT-alpha. A total of 11 TNF-LT-alpha haplotypes were determined from apparent homozygous controls and statistical analysis.

The HLA-Cw*1801 specificity, a Cw7/Cw4 hybrid allele, has recently been described in association with B*8101 (formerly B"DT"). In this study, the new Cw*1802 variant, differing from Cw*1801 at exon 5, is found associated with B*5703 in Bubi individuals from Equatorial Guinea. Confirmatory complete coding regions of B*5703 and B*3910 are also reported.

Human killer cell inhibitory receptors (KIR) are novel members of the immunoglobulin superfamily of cell surface glycoproteins, which are expressed by lymphocytes with natural killer (NK) and cytotoxic T-cell (CTL) phenotypes. These receptors have specificity for relatively conserved epitopes of HLA-A, -B, and -C class I antigens. Recent studies have identified KIR as being involved in the transmission of negative, inhibitory signaling events to the cytotoxic cell which prevent or diminish target cell lysis. KIR are thus likely to play an important role in the responses of alloreactive NK cells and CTL to allogeneic HLA antigens. In this article, we review the known structural and functional characteristics of KIR, suggest a possible mechanism for the transmission of intracellular negative signaling by these receptors, and discuss the relevance of KIR function and HLA specificity to the clinical transplantation of allogeneic tissues.

We report here the identification and characterization of a novel human leucocyte antigen (HLA)-DPB1 allele that was subsequently named HLA-DPB1*0302 by the WHO Nomenclature Committee. HLA-DPB1*0302 was identified in a single Sicilian individual by a combination of sequence-specific primers, reverse line sequence-specific oligonucleotide probing and DNA sequencing-based typing. The DPB1*0302 allele is most similar to the DPB1*3101 allele, differing by a single mismatch at nucleotide position 301 (T to G).

Nucleotide sequence analysis of the HLA-C alleles of the GB92 cell line, heterozygous for B*8101 and B*4407, revealed the existence of two new allelic variants: Cw*1801 and Cw*0706. The former allele, initially detected as a PCR-SSP variant, displays a hybrid aspect, sharing sequence motifs with Cw*07 at exons 1 and 2, and with Cw*04 at distal exons. In serological assays, Cw*1801 is only recognized by some cross-reactive sera. Cw*0706 shows a primary structure closely related to previously known Cw7 alleles, but carries new sequence motifs at its 3'-end. Preliminary data indicate that Cw*1801 is associated to B*8101 and that Cw*0706, B*4407 could account for a part of the Cw7, B44 haplotypes observed in African populations.

We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.

The novel HLA-Cw*0704 allele, previously detected as the PCR-SSP variant Cw7/8v, has been cloned and sequenced from the homozygous typing cell KR03/4 after amplification by anchored PCR. The nucleotide sequence of Cw*0704 is closely related to those of other Cw*07 alleles, but carries specific changes in exon 3 consistent with its serological behavior - a short Cw7 cross-reactive with antibodies directed against HLA-Cw8. Some of the substitutions of Cw*0704 have not been previously described for HLA-C but are found in HLA-B alleles and in published C sequences of non-human primates. The new allele carries a novel polymorphism in its 5′ untranslated region (5′ut) that could be shared by all Cw*07 alleles. By PCR-SSP, Cw*0704 is a relatively common allele in English Caucasoids at a frequency of 4.6%. It is most often observed on HLA-B44 haplotypes previously described as HLA-C “blank”, although linkage disequilibria with other HLA-B specificities have been found.

AIM--To develop polymerase chain reaction based artificial restriction fragment length polymorphism (artificial RFLP PCR) assays for DR3 and DR4 alleles of the multiallelic DRB1 locus and to apply them to paraffin wax embedded archival material. METHODS--Sixty five samples from DRB1 typed cell lines were analysed using the artificial RFLP PCR method to determine the specificity and sensitivity of the system. RESULTS--The artificial RFLP PCR method for typing the DRB1 locus showed 100% accuracy in the 65 samples previously typed using allele specific PCR and serology. The samples included 18 combinations of alleles that included DR3, 18 that included DR4, four that were DR3/DR4 heterozygotes, and 10 samples that were neither DR3 nor DR4. Typing of 10 paraffin wax embedded samples using artificial RFLP PCR was in complete agreement with previous typing at the DRB1 locus. CONCLUSION--The application of artificial RFLP PCR for the analysis of multiallelic loci, such as those of the HLA system, in archival DNA samples has been achieved. Artificial RFLP PCR is a robust, easily implemented, non-isotopic system and may be useful for large retrospective studies.

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate ( k ) of 5.50 × 10 –6 per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model ( R 2 = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.

Cw*1602, a novel HLA-C allele belonging to the newly assigned Cw*16 group, has been cloned and sequenced from a Spanish Caucasoid cell expressing a "Cw6.2" phenotype. Some of the polymorphic substitutions of the new allele, and linkage disequilibrium to B51, had been predicted on the basis of previously published studies. The primary structure of Cw*1602 is in agreement with its serologic reactivity and, in comparison with that of Cw*1601, underlines the dimorphism of HLA-C molecules at residues 77 and 80 of the alpha 1-domain alpha helix.

HLA-B15 embraces a multiplicity of antigenic specificities which vary in their distribution amongst human populations. To correlate B15 molecular structure with the serological picture we have sequenced alleles encoding the various subspecificities of the B15 antigen: B62, B63, B75, B76 and B77, and a number of "variants" of these antigens including the 8w66 split of B63. HLA-B63 (B*1517) and 8w66 (B*1516) heavy chains have sequence identity to B17 in the alpha 1 helix correlating with the antigenic crossreactivity of these molecules. HLA-B77(B*1513) and B75 (B*1502) heavy chains differ solely in segments determining the Bw4 and Bw6 public epitopes, consistent with the serological description of the B77 and B75 antigens. One allele encoding the B76 antigen (B*1512) appears to be the product of gene conversion between the HLA-A and -B loci and differs from B*1501 in codons 166 and 167. In contrast, a second allele encoding the B76 antigen (B*1514) differs from B*1501 by an unrelated substitution in codon 167 which confers similarily with B45, an antigen crossreactive with B76. A third allele encoding B76, B*1519, differs from B*1512 by a unique point substitution in exon 4. Three alleles encoding variant B15 and B62 antigens (B*1508, B*1511 and B*1515) differ from B*1501 by localized clusters of substitutions that probably result from interallelic conversion. The B15 sequences described in this paper, in combination with those previously determined, define a family of 22 alleles, including those encoding the B46 and B70 antigens. Within this family the patterns of allelic substitution are analogous to those of other HLA-A and -B families, in that pairwise differences almost always involve functional positions of the antigen recognition site and recombination is the major agent of diversification.

Detection of HLA-C antigens by complement mediated cytotoxicity using human alloantisera is often difficult. Between 20 to 40% of individuals in every race have undetectable HLA-C locus antigens and 9 out of the 29 sequenced HLA-C alleles so far published encode serologically undetected antigens. In addition, HLA-C molecules are expressed at the cell surface at about 10% of the levels of HLA-A and HLA-B. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable and rapid method for typing HLA-DR, HLA-DQA and HLA-DQB genes. PCR-SSP takes two hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers which will positively identify the HLA-C alleles corresponding to the serologically defined series HLA-Cw1, Cw2, Cw3, Cw4, Cw5, Cw6, Cw7 and Cw8. The serologically undetectable alleles have also been detected in groups according to sequence homology. In addition, three new unsequenced variants have been identified. DNA samples from 56 International Histocompatibility Workshop reference cell lines and 103 control individuals have been typed by the HLA-C PCR-SSP technique. 4/56 cell line types and 11/103 normal control individuals types were discrepant with the reported serological types. All combinations of serologically detectable and most of the serologically blank HLA-C antigens can be readily identified. DNA typing for HLA-Cw by PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

We have identified a new HLA-B*15 allele and a new HLA-DRB1*12 allele, named B*1568 and DRB1*1208, respectively. The alleles were identified using a combination of sequence specific primers, reverse line sequence specific oligonucleotide probing and sequence-based typing. Both alleles were identified in a single individual of Korean origin. HLAB*1568 appears to be an HLA-B*4801/B*1507 hybrid combining the exon 2 sequence of B*4801 and the exon 3 and 4 sequences of B*1507. Exon 2 of DRB1*1208 was most similar to DRB1*1201 or 1206, with a single mismatch at nucleotide position 165 (A to C). At the protein level, this substitution results in a phenylalanine substitution at position 26 that creates an identical amino acid sequence to DRB3*0202 between amino acid positions 17 and 36.

Molecular genotyping of HLA class II genes using group-specific DNA amplification by the PCR followed by probing with (PCR-SSO) probes is too time consuming for the typing of cadaveric organ donors. Recently, amplification of DNA using PCR-SSP has proved a reliable and rapid method for typing HLA-DRB1 genes. PCR-SSP takes 2 hours to perform and is therefore suitable for the genotyping of cadaveric donors. We have designed a set of primers that in eight PCR reactions will positively identify the HLA-DQB1 alleles corresponding to the serologically defined series HLA-DQ2, DQ4, DQ5, DQ6, DQ7, DQ8, and DQ9. Presently, 30 homozygous cell lines and 138 individuals have been typed by the DQB1 PCR-SSP technique and compared with a combination of serology and RFLP with 100% concordance. No false-negative or false-positive amplifications were recorded. All combinations of DQB1 can be readily identified. DQB1 PCR-SSP can take as little as 130 minutes from start to finish, including DNA preparation.

In the West African population of the Gambia the class I antigen HLA-Bw53 is found at high frequency. We used the polymerase chain reaction to amplify cDNA from an individual homozygous for this allele and determined the nucleotide sequence of the polymorphic alpha 1 and alpha 2 domains. The HLA-Bw53 sequence is identical to HLA-B35 except for a short sequence at the 3' end of exon 2 (encoding the alpha 1 domain) which specifies a Bw4 rather than a Bw6 motif. This suggests an origin for HLA-Bw53 involving a gene conversion of HLA-B35 by an allele containing this Bw4 sequence. The alpha 2 domain shared by HLA-Bw53, -B35, and -Bw58 is particularly common in sub-Saharan Africans.

HLA class-I and class-II allele frequencies and two-locus haplotypes were examined in 367 unrelated Melanesians living on the islands of Vanuatu and New Caledonia. Diversity at all HLA class-I and class-II loci was relatively limited. In class-I loci, three HLA-A allelic groups (HLA-A*24, HLA-A*34 and HLA-A*11), seven HLA-B alleles or allelic groups (HLA-B*1506, HLA-B*5602, HLA-B*13, HLA-B*5601, HLA-B*4001, HLA-B*4002 and HLA-B*2704) and four HLA-C alleles or allelic groups (HLA-Cw*04, HLA-Cw*01, HLA-Cw*0702 and HLA-Cw*15) constituted more than 90% of the alleles observed. In the class-II loci, four HLA-DRB1 alleles (HLA-DRB1*15, HLA-DRB1*11, HLA-DRB1*04 and HLA-DRB1*16), three HLA-DRB3-5 alleles (HLA-DRB3*02, HLA-DRB4*01 and HLA-DRB5*01/02) and five HLA-DQB1 alleles (HLA-DQB1*0301, HLA-DQB1*04, HLA-DQB1*05, HLA-DQB1*0601 and HLA-DQB1*0602) constituted over 93, 97 and 98% of the alleles observed, respectively. Homozygosity showed significant departures from expected levels for neutrality based on allele frequency (i.e. excess diversity) at the HLA-B, HLA-Cw, HLA-DQB1 and HLA-DRB3/5 loci on some islands. The locus with the strongest departure from neutrality was HLA-DQB1, homozygosity being significantly lower than expected on all islands except New Caledonia. No consistent pattern was demonstrated for any HLA locus in relation to malaria endemicity.

An emerging problem of molecular typing methods such as PCR amplification using sequence-specific primers (PCR-SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis-linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class Il means that, for example, new HLA-A sequences have to be compared with HLA-B and HLA-C primer mixes to rule out cross-locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class TI sequences obtained from Internet-accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR-SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR-SSP class I and class TI typing set. The SSP Manager is available on request from the authors.

HLA-DP is the third of the class II molecules. Its role is antigen presentation, and it has been suggested to play a part in the susceptibility to certain diseases such as berylliosis, sarcoidosis and juvenile chronic arthritis. The standard typing method is SSO typing, although other methods have been used. Probably the best is sequence-based typing, but this is time-consuming and requires expensive equipment. We describe a method for comprehensive HLA-DPB1 and HLA-DPA1 typing using sequence-specific primers. This method has the advantages that it is rapid - typing a single DNA sample takes under 3 hours - and does not require any special equipment or reagents. The method has been shown to be highly accurate by typing 60 cell line DNA samples in which there was 100% agreement between the types obtained and the published information. Similarly typing of 20 DNA samples previously typed by sequence-based typing gave 100% concordance. We used the method to type DNA samples from 102 UK Caucasoid kidney donors. The allele frequencies agree with previously published data. Linkage disequilibria between HLA-DPB1, HLA-DPA1 and the other class II antigens have been investigated. Strong linkage disequilibria exist between certain HLA-DPB1 and HLA-DPA1 alleles. This is unsurprising in view of their proximity on the chromosome. More unexpectedly, the data also suggest that genes further away along the chromosome are in linkage disequilibrium with HLA-DP, forming extended haplotypes.

Polymorphism at HLA-DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32-reaction PCR-SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.

We hypothesize that kidneys from non-secretor blood group A2 donors may be used for transplant into non-A recipients. In addition, we believe that organs from A2 donors may be used in non-A recipients where the anti-A titer is low. In order to reliably identify non-secretor A2 kidneys from cadaver donors, we have developed a rapid molecular method. The PCR-SSP-based method was developed to genotype ABO blood group and secretor status. Samples of known blood group ABO and Lewis phenotype determined by standard serological methods were used to appraise the method. A retrospective renal cadaver donor study was conducted to assess the potential of using A2 non-secretor organs for transplantation into non-A recipients. Phenotype frequencies of blood group A donors were 76% and 24% for A1 and A2 subgroups respectively, whereas 27% of the donor sample population were non-secretors. Three donors were identified as A2 non-secretors, and analysis was performed to theoretically place the organs by considering them as blood group O. These results coupled with a detailed analysis of HLA type and antibody status of our panel suggests that using A2 donors would be a useful adjunct to strategies for transplanting highly sensitized patients and redressing the donor-recipient imbalance in terms of blood group.

The sequence of B*5603 has been deposited with the Genome Sequence Database and has the accession number U73113. The name B*5603 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee in September 1996. This follows the agreed policy that, subject to the conditions stated in the most recent nomenclature report (1), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO nomenclature report. CJL is a Wellcome Trust prize student.