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Objective Determine whether variation in the HLA region is associated with the development of post-traumatic sepsis and septic shock. Background Sepsis-related deaths remain a major source of mortality after traumatic injury. Genetic characteristics may contribute to susceptibility to adverse outcomes including sepsis and septic shock. Recent advances in next-generation sequencing technology now allow comprehensive genotyping of the HLA region. Methods White adult trauma patients requiring more than 2 days of mechanical ventilation underwent HLA genotyping, and were followed for the development of sepsis and septic shock. Odds ratios (OR) for the associations between our outcomes and HLA variants were estimated, a correction for multiple comparisons was applied, and significant variants were included in regression models adjusting for potential confounders. Results A total of 1184 patients were included. Patients were severely injured (median injury severity score 33); 33% developed sepsis, 6% septic shock, and in-hospital mortality was 14%. An amino acid variant (156Q) within the HLA-A peptide-binding groove was associated with greater odds of sepsis [OR 1.50, (1.18-1.89)]. HLA-A02:01 was associated with lower odds of septic shock [OR 0.52, (0.32-0.82)]. These associations remained significant after adjusting for potential confounders. Conclusions This is the first study to apply next-generation sequencing techniques to evaluate associations between immunogenetic factors and post-traumatic sepsis and septic shock. Associations with class I HLA variants are novel as they implicate adaptive immunity in post-traumatic sepsis. These findings are a step towards developing a panel of genetic markers assessing risk of infection-related complications as we move towards more personalized medicine.

The development of next-generation sequencing (NGS) methods for HLA genotyping has already had an impact on the scope and precision of HLA research. In this study, allelic resolution HLA typing was obtained for 402 individuals from Cape Town, South Africa. The data were produced by high-throughput NGS sequencing as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with the University of Cape Town and Stanford University. All samples were genotyped for 11 HLA loci, namely HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, and -DRB5. NGS HLA typing of samples from Cape Town inhabitants revealed a unique cohort, including unusual haplotypes, and 22 novel alleles not previously reported in the IPD-IMGT/HLA Database. Eight novel alleles were in Class I loci and 14 were in Class II. There were 62 different alleles of HLA-A, 72 of HLA-B, and 47 of HLA-C. Alleles A∗23:17, A∗43:01, A∗29:11, A∗68:27:01, A∗01:23, B∗14:01:01, B∗15:10:01, B∗39:10:01, B∗45:07, B∗82:02:01 and C∗08:04:01 were notably more frequent in Cape Town compared to other populations reported in the literature. Class II loci had 21 different alleles of DPA1, 46 of DPB1, 27 of DQA1, 26 of DQB1, 41 of DRB1, 5 of DRB3, 4 of DRB4 and 6 of DRB5. The Cape Town cohort exhibited high degrees of HLA diversity and relatively high heterozygosity at most loci. Genetic distances between Cape Town and five other sub-Saharan African populations were also calculated and compared to European Americans.

The highly polymorphic human leukocyte antigen (HLA) class I molecules present peptide antigens to CD8+ T cells, inducing immunity against infections and cancers. Quality control mediated by peptide loading complex (PLC) components is expected to ensure the cell surface expression of stable peptide-HLA class I complexes. This is exemplified by HLA-B*08:01 in primary human lymphocytes, with both expression level and half-life at the high end of the measured HLA-B expression and stability hierarchies. Conversely, low expression on lymphocytes is measured for three HLA-B allotypes that bind peptides with proline at position 2, which are disfavored by the transporter associated with antigen processing. Surprisingly, these lymphocyte-specific expression and stability differences become reversed or altered in monocytes, which display larger intracellular pools of HLA class I than lymphocytes. Together, the findings indicate that allele and cell-dependent variations in antigen acquisition pathways influence HLA-B surface expression levels, half-lives and receptivity to exogenous antigens., eLife digest Most cells in the body make proteins called human leukocyte antigen class I (or HLA-I). These proteins sit on the cell surface, where they help the immune system distinguish between healthy and diseased cells. A groove in each HLA-I protein holds a fragment of a protein chain, called a peptide, from inside the cell. In healthy cells, all the peptides come from normal proteins. Yet in diseased or infected cells, the peptides may come from abnormal or foreign proteins – those encoded by viruses, for example. When the immune system sees these abnormal peptides, it responds by killing the cell. Across the human population, there are thousands of types of HLA-I, each able to carry a different set of peptides. Any individual person can only make a maximum of six types of the HLA-I, meaning we each show a different combination of peptides to our immune cells. This difference will change the way different people respond to the same disease. Before a peptide can be assembled into HLA-I, it must be moved to the correct part of the cell by a transporter known as TAP. This transport favors peptides with certain characteristics, but these characteristics do not always match the preferences of the individual's HLA-I proteins. For example, TAP is less likely to transport peptides where the second building block in the chain is a proline, but these peptides will still fit into the binding grooves of some HLA-I variants. Here, Yarzabek, Zaitouna, Olson et al. obtained blood from healthy human donors to answer questions about what happens when TAP and HLA-I have different preferences. Specifically, how many HLA-I molecules reach the surface, how long do they last, and which peptides do they carry? This analysis revealed that, when there was a mismatch between HLA-I and TAP, the amount of some HLA-I types on the surface of white blood cells called lymphocytes dropped. These HLA-I types were also able to pick up new peptides from their environment, indicating that some HLA-I were at the surface of the cell without a peptide. The role of these empty HLA-I remains to be fully defined. The reverse was true for other white blood cells called monocytes; HLA-I variants that were mismatched with TAP became more abundant on the cell surface. Monocytes also had more HLA-I molecules inside and did not pick up peptides from the environment. This suggests that monocytes may load peptides via new pathways, filling grooves left empty in lymphocytes, although other mechanisms might also explain the differences between the two types of white blood cells. Taken together, the findings reveal that HLA-I on the surface of cells depends on both the type of HLA-I and the type of immune cell. HLA-I proteins play a key role in the immune system’s ability to recognize and kill diseased cells. A better knowledge of how HLA-I variants differ could help us to understand why people respond differently to the same disease. A better grasp of HLA-I could in the future lead to improved drug and vaccine design.

HLA class I molecules are highly polymorphic cell surface proteins that trigger immune responses by CD8(+) T cells and natural killer (NK) cells. Most humans express six different HLA class I proteins encoded by the HLA-A, HLA-B and HLA-C genes. HLA class I molecules bind to peptide antigens and present these antigens to T cell receptors (TCR) of CD8(+) T cells. HLA class I expression levels also regulate NK cell activation. The presence of individual HLA class I genes is linked to many different disease, transplantation and therapy outcomes. An understanding of HLA class I expression and stability patterns is fundamentally important towards a better understanding of the associations of HLA class I genes with disease and treatment outcomes, and towards HLA class I targeting for vaccine development. Quantitative flow cytometry allows for assessments of variations in expression levels of HLA class I molecules in cells from a single blood donor over time, as well as averaged measurements across donors for the same allotype. Since all HLA class I molecules are structurally-related, cellular measurements of the HLA class I expression levels and stabilities of individual variants in human cells require careful choices of donors and antibodies, which are discussed here.

The vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species.

Background Genome-Wide Association Studies (GWAS) have produced strong evidence for association of Schizophrenia (SCZ) in the extended Major Histocompatibility Complex (chromosome 6q), including the Human Leukocyte Antigen (HLA) region. It has been reported that structural polymorphisms in the C4 gene can explain the signal in this region, but it has not been determined by direct high-resolution genotyping whether HLA alleles have independent effects. We have used a novel next-generation sequencing method to achieve HLA typing with 8-digit (4-field) resolution over classical HLA genes (A, B, C, DRB1, DRB3-5, DQA1, DQB1, DPA1 and DPB1) in cohorts of three ethnicities; and assayed C4 structural polymorphisms in a subset of the sample to permit imputation in the entire cohorts. These data permit analysis of the association of SCZ with HLA alleles while accounting for C4 variants. Methods Subjects included 21,592 individuals: SCZ cases/controls from the MGS and GPC cohorts (European-ancestry: 5,762 cases, 5,558 controls; African-American: 2,671 cases, 2,373 controls), and 1,670 Taiwan families (3,340 parents, 1,728 affected offspring). HLA genotyping was carried out using the Mia Fora NGS Flex HLA Typing Kit (Immucor), which uses long-range PCR to capture 11 HLA loci (the entire gene in most cases) followed by high-depth, multiplexed Illumina sequencing. C4 polymorphisms were assayed using ddPCR using published primers and calling algorithms, in 940 individuals representing all ancestries and genotyping platforms. Analyses are almost completed, including imputation of C4 structural alleles into all samples (HIBAG), followed by frequentist association tests of HLA alleles with and without conditioning on C4 alleles, taking ancestry covariates into account (PLINK). It is expected that imputation of HLA genotypes can also be improved for each ancestry based on the high-resolution genotyping of large cohorts. Results All analyses are in progress and will be completed prior to the Congress. Missing data rates across loci are ~1%, and Mendelian error rate in trios is low. C4 alleles can be imputed with high accuracy in all combinations of ancestry and genotyping platform. Imputation accuracy was over 90% for all HLA genes. Discussion The results of this study will provide important new information about whether HLA alleles have effects on SCZ risk, independent of their linkage disequilibrium with C4 structural polymorphisms. The study will also provide improved resolution of imputation of both C4 polymorphisms and of HLA alleles.

Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.

We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.

Here, we characterize the Arabidopsis thaliana embryo-defective mutant increased size exclusion limit2 (ise2). In contrast with wild-type embryos, ise2 mutants continue to traffic 10-kD fluorescent dextran in the mid-torpedo stage of development. ise2 embryos contain branched as well as simple plasmodesmata (PD) compared with wild-type embryos, which only contain simple PD. Positional cloning reveals that the ISE2 gene encodes a putative DEVH box RNA helicase that shares sequence homology with RNA helicases involved in RNA degradation pathways in other organisms. ISE2 localizes to granule-like structures in the cytoplasm. These granules increase in number when plant cells are stressed. These features are characteristic of stress granules (SGs) in mammalian cells, suggesting that ISE2 granules represent plant-specific SGs. Genetic data demonstrate that the ISE2 helicase is involved in posttranscriptional gene silencing and the determination of cell fate. These data together suggest that ISE2 function affects PD structure and function through the regulation of RNA metabolism and consequent gene expression.

The proper control of cell expansion is vital to plant development. It is responsible for shaping individual cells and, together with cell division, it plays a lead role in shaping plant organs. Much of the underlying mechanism by which plant cells expand anisotropically is not understood. We are taking a genetic approach to cell expansion by isolating mutants that affect the branching pattern of Arabidopsis trichomes. Here we report the identification of four new loci that control trichome morphogenesis. These loci were named the IRREGULAR TRICHOME BRANCH (ITB) loci because of the deleterious effects on branch position and length in the mutants. Our analysis of branch expansion in itb mutants shows that the ITB genes act as positive regulators of branch elongation, and that the branch position defects are caused by altered expansion of the trichome stalk. The itb mutations display synergistic effects in double mutant combinations with certain branch number mutations, suggesting that the ITB genes also play key roles in branch initiation. These results demonstrate that the ITB genes are key regulators of anisotropic cell expansion in trichomes.

Aim There is very limited information on the importance of DRB1 exon 1 variations in transplantation. Routinely, clinical laboratories do not test for exon 1 assuming it codes for the leader peptide which is cleaved off of the mature protein, and therefore not clinically important. Currently there are two CWD DRB1 alleles that differ in exon 1, DRB1∗12:01 and DRB1∗12:10, which can only be resolved by sequencing exon 1. We sought to interrogate exon 1 of DRB1 for the presence of the CWD allele, DRB1∗12:10, and to determine the HLA class II haplotypes associated with it in our local population. Methods The Immucor Mia Fora Flex 11 HLA Typing kit was used to genotype eight HSCT recipients and their unrelated donors, as well as seven additional patients. All 23 of these individuals were previously genotyped as DRB1∗12:01 using SSOP. Results All 23 individuals typed as DRB1∗12:01:01. No DRB1∗12:10 was identified in this limited cohort. Two haplotypes were identified for DRB1∗12:01:01: sixteen individuals (76%) were DRB1∗12:01:01, DRB3∗02:02:01, DQA1∗05:05:01, DQB1∗03:01:01, and five individuals (24%) were DRB1∗12:01:01, DRB3∗01:01:02, DQA1∗05:05:01, DQB1∗03:01:01. Two individuals could not have their DRB3∗01:01:01 and DRB3∗02:02:01 haplotypes identified. Conclusions The Immucor Mia Fora Flex 11 HLA Typing kit is unique among several commercial NGS kits in that it contains primers capable of sequencing exon 1 of DRB1. Traditionally, this region has been avoided because of the difficulty in primer design due to the very large 8000–10,000 bp intron 1. The CWD allele, DRB1∗12:10 originally identified in Korean population, differs from DRB1∗12:01 by one bp in exon 1, but since exon 1 is not routinely typed, the individual frequencies of these alleles are not known. Retrospective studies in Asian populations indicated that 12% of DRB1 alleles previously typed as 12:01 were in Fact 12:10. In our study, 100% of our cohort of 23 patients typed as DRB1∗12:01, which was found to be predominantly associated with DRB3∗02:02:01 (76%) and less frequently with DRB3∗01:01:02 (23%). This is in contrast to the reported preferential association of DRB1∗12:01 with DRB3∗01:01:02 (97%), while association with DRB3∗02:02:01 was very rare (2.1%). Retrospective studies may be helpful to determine the importance of exon 1 mismatches in HSCT.

Aim Next Generation Sequencing (NGS) is rapidly becoming the method of choice for HLA typing for Hematopoietic Stem Cell Transplantation. Jeevan Stem Cell Foundation, Chennai, India validated the full gene HLA typing using Immucor’s MIA FORA FLEX kits from conceptualization to implementation in ninety days. This implementation, in line with the Indian Government’s policy of “Make in India”, enabled Jeevan to establish a cost effective high resolution HLA typing to international standards in India to support the transplant physicians and registries in the region and prevent Indian DNA being shipped to other countries. Methods Reference DNA used for validation were previously typed by SBT included samples from UK NEQAS, FRED HUTCHINSON and Jeevan’s registry samples. DNA was extracted using the Qiacube. HLA typing for A, B, C, DRB1 and DQB1 were performed. All workflows were performed according to the supplier’s instructions. NGS sequencing was done using the mid output flow cell kit from Illumina and sequenced on the Illumina MiniSeq platform. Immucor’s proprietary software was used to analyse the data. Results Results were evaluated per allele on the 2nd field consensus compared to the reference, the number of allelic ambiguities, genotype ambiguities and allelic drop outs were measured. The second field resolution was used because higher resolution typing was not available for all samples. MIA FORA NGS software provided high resolution typing for all samples. There results from NGS typing compared to SBT typing are shown in Table 1. Conclusions The implementation of the MIA FORA FLEX HLA typing kit at Jeevan is the first outside USA. With the MIA FORA kits, the turnaround time will depend upon the number of samples. In our experience reports for 48 samples can be delivered within 72 h. There were no ambiguous results and no allelic dropout. The proprietary software of Immucor was easy to use and significantly reduced the analysis time. Download high-res image (213KB) Download full-size image

Aim Jeevan Stem Cell Foundation, Chennai, India, is a not for profit, operating a public cord blood bank since 2008. Jeevan currently has 5972 stem cell units and is the largest active public cord blood bank in South Asia. Jeevan decided to migrate from SBT to NGS whole gene sequencing for HLA typing to improve the accuracy and reliability. Methods DNA from 47 cord blood donations were typed for HLA A, B, C, DRB1 and DQB1 using whole gene sequencing with MIA FORA FLEX kits from Immucor. NGS sequencing was done in MiniSeq from Illumina using the mid output flow cell kit. Sixteen of these samples were typed earlier by SBT using kits from Life technologies (Secore IVD). All workflows were performed according to the supplier’s instructions. Results were analyzed using Immucor’s proprietary software. Results We were able to report the results within 72 h. Because of full gene sequencing, NO ambiguity was seen in NGS typing. However, of the 16 samples done by both techniques, all 16 had ambiguous results by SBT (A-14, B-12, C-16, DRB1-11, DQB1-14) Conclusions Whole gene HLA typing using MIA FORA FLEX kits on Illumina MiniSeq for a cord blood registry is highly dependable with no ambiguous results. The proprietary software of Immucor was easy to use and significantly reduced the analysis time.

Aim Among HLA class I (HLA-I) molecules, HLA-B allotypes are the most polymorphic and have dominant influences upon disease outcomes. HLA-B allotypes are known to differ markedly in their intracellular folding and peptidome characteristics. The Aim of this study was to assess whether intracellular assembly or peptidome variations influence the cell surface expression levels or stabilities of HLA-B allotypes. Methods Commercial HLA-Bw4 and HLA-Bw6 antibodies were assessed for HLA-I specificities using Luminex beads, and antibody binding differences were quantified. 244 healthy donors were recruited and HLA genotyping was performed using next generation sequencing (NGS). Donors were selected for further measurements based on heterozygosity for the Bw4 and Bw6 epitopes within their HLA-B antigens, and based on the absence of cross-reactive HLA-A antigens. Using quantitative flow cytometry, antibody binding capacity (ABC) values were measured on freshly isolated lymphocyte subsets using HLA-Bw4, HLA-Bw6 and total class I-specific antibodies. For each selected donor, HLA-I ABC measurements were undertaken at multiple time points. For HLA-B alleles with measurements from at least three donors, averaged ABC values for HLA-Bw4 and HLA-Bw6 were analyzed directly or as a ratio relative to total HLA-I, after taking into account relevant antibody recognition differences. For some HLA-Bw4 and HLA-Bw6 allotypes, cell surface stabilities were also assessed on freshly isolated lymphocytes in the presence of brefeldin A. Results Statistically significant allele-dependent differences are measured in cell surface HLA-B expression levels. For some HLA-B allotypes, lower expression could be related to reduced cell surface stability. Cell surface expression and stability differences appear to be related to the individual peptide-binding characteristics of HLA-B, rather than diversities (promiscuities) of their peptide repertoires. Conclusions Overall, these studies indicate allele-dependent variations in cell surface expression and stability of HLA-B, which in some cases correlate with previously known disease progression outcomes in human immunodeficiency virus (HIV) infections.

Human leukocyte antigen class I (HLA-I) molecules comprise a highly polymorphic heavy chain complexed to a conserved light chain, b2-microglobulin (b2m), and peptide. When bound to antigenic peptides, HLA-I molecules mediate CD8+ T cell responses against cancers and intracellular pathogens. The transporter associated with antigen processing (TAP) is the major source of cellular peptides for the assembly of HLA-I molecules in the endoplasmic reticulum (ER). Consistent with previous findings of varying stabilities of peptide-deficient forms of HLA-B, we show allotype-dependent variations in the cell surface expression of HLA-B molecules in TAP-deficient and TAP-inhibited cells. HLA-B allotypes that are resistant to inhibition of TAP (RIT-HLA-B) exist on the cell surface in empty or suboptimally loaded forms under TAP-deficiency conditions, even at physiological temperatures. Furthermore peptide-deficient forms of RIT-HLA-B allotypes bind to primary CD8+ T cells via a mode of binding that is CD8-dependent. Together, these findings indicate allotype-dependent variations in the prevalence of peptide-deficient HLA-B molecules and demonstrate previously unrecognized functions for “empty” HLA-I.

Background Therapeutic decisions in cancer are generally guided by molecular biomarkers or, for some newer therapeutics, primary tumor genotype. However, because biomarkers or genotypes may change as new metastases emerge, circulating tumor cells (CTCs) from blood are being investigated for a role in guiding real-time drug selection during disease progression, expecting that CTCs will comprehensively represent the full spectrum of genomic changes in metastases. However, information is limited regarding mutational heterogeneity among CTCs and metastases in breast cancer as discerned by single cell analysis. The presence of disseminated tumor cells (DTCs) in bone marrow also carry prognostic significance in breast cancer, but with variability between CTC and DTC detection. Here we analyze a series of single tumor cells, CTCs, and DTCs for PIK3CA mutations and report CTC and corresponding metastatic genotypes. Methods We used the MagSweeper, an immunomagnetic separation device, to capture live single tumor cells from breast cancer patients’ primary and metastatic tissues, blood, and bone marrow. Single cells were screened for mutations in exons 9 and 20 of the PIK3CA gene. Captured DTCs grown in cell culture were also sequenced for PIK3CA mutations. Results Among 242 individual tumor cells isolated from 17 patients and tested for mutations, 48 mutated tumor cells were identified in three patients. Single cell analyses revealed mutational heterogeneity among CTCs and tumor cells in tissues. In a patient followed serially, there was mutational discordance between CTCs, DTCs, and metastases, and among CTCs isolated at different time points. DTCs from this patient propagated in vitro contained a PIK3CA mutation, which was maintained despite morphological changes during 21 days of cell culture. Conclusions Single cell analysis of CTCs can demonstrate genotypic heterogeneity, changes over time, and discordance from DTCs and distant metastases. We present a cautionary case showing that CTCs from any single blood draw do not always reflect metastatic genotype, and that CTC and DTC analyses may provide independent clinical information. Isolated DTCs remain viable and can be propagated in culture while maintaining their original mutational status, potentially serving as a future resource for investigating new drug therapies.

We report the genetic, phenotypic, and biochemical analyses of Catecholamines up (Catsup), a gene that encodes a negative regulator of tyrosine hydroxylase (TH) activity. Mutations within this locus are semidominant lethals of variable penetrance that result in three broad, overlapping effective lethal phases (ELPs), indicating that the Catsup gene product is essential throughout development. Mutants from each ELP exhibit either cuticle defects or catecholamine-related abnormalities, such as melanotic salivary glands or pseudotumors. Additionally, Catsup mutants have significantly elevated TH activity that may arise from a post-translational modification of the enzyme. The hyperactivation of TH in Catsup mutants results in abnormally high levels of catecholamines, which can account for the lethality, visible phenotypes, and female sterility observed in these mutants. We propose that Catsup is a component of a novel system that downregulates TH activity, making Catsup the fourth locus found within the Dopa decarboxylase (Ddc) gene cluster that functions in catecholamine metabolism.

Aim Africans represent the most genetically diverse population in the world, but have not been as well studied with respect to their HLA types compared to the inhabitants of western countries. Thus it was of interest to apply our high-throughput HLA sequencing technology to a cohort of 403 healthy adolescents from Cape Town, South Africa, as part of a study of T-cell responses to Mycobacterium tuberculosis in collaboration with Stanford University. Methods Cryopreserved PBMC samples from 403 adolescents were collected and shipped to Stanford University from Cape Town. High resolution HLA typing was performed using MIA FORA NGS kit and analysis software. The whole gene coverage included HLA-A, -B, -C, -DPA1, -DQA1, and -DQB1; all exons and introns for -DRB1 and -DRB3/4/5 except partial coverage for exon 6 and intron 1; and all exons and introns between exons 2 and 4 for HLA-DPB1. NGS sequencing libraries were prepared using a semi-automated protocol and sequenced in high-throughput format on the Illumina NextSeq platform. HLA allele candidates were computed and final HLA typing was confirmed using MIA FORA NGS analysis software. Results HLA typing revealed a unique population, including unusual haplotypes, and novel alleles not previously reported in the IMGT/HLA database. The frequency of HLA-A, -B, -DRB1 haplotypes in Cape Town were compared to those represented in the US National Marrow Donor Program. As might be expected, 14 of the top 16 haplotypes in the Cape Town cohort were observed in the African American cohort. However there were important exceptions, including a frequent haplotype found in African American but not observed in Cape Town and two frequent Cape Town haplotypes not observed in African American samples. Conclusion High resolution HLA typing is a powerful method to characterize HLA allele and haplotype diversity in population studies. C. Wang: Consultant; Company/Organization; Immucor. M. Li: Employee; Company/Organization; Immucor. M. Fukushima: Employee; Company/Organization; Immucor. R. Kuehn: Employee; Company/Organization; Immucor. S. Krishnakumar: Employee; Company/Organization; Immucor. M. Mindrinos: Employee; Company/Organization; Immucor.

Aim Next generation sequencing of extended long-range HLA gene products provides better molecular characterization of HLA genes and improves the assessment of HLA population diversity than traditional HLA genotyping methods. In addition, accurate comprehensive analyses of HLA diversity would be expected to increase the efficiency of unrelated donor selection. In this study we assessed the genomic and haplotype diversity in European Americans (EA) and African Americans (AFA). Methods We used a high-throughput NGS HLA typing method developed by Sirona Genomics to genotype class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA, -DQB, -DRB1, -DRB3, -DRB4, -DRB5) loci in >2400 EAs and >840 AFAs unrelated healthy individuals. Sample libraries were sequenced using Illumina HiSeq and NextSeq instruments. Sequence data was analyzed and genotypes were assigned using the MIA FORA NGS HLA software. Statistical analyses was performed using the GENE[RATE] computer pipeline (hla-net.eu). Results The distribution of allele frequencies in both EA and AFA populations is similar across all loci with an extreme right skew in each case. Rare alleles and novel DPA and DQB alleles were detected in both populations. The AFA population contained higher levels of mean heterozygosity at class I loci and most class II loci compared to the EA population. Tests of selective neutrality revealed a significant excess of homozygosity in B, C, and DPB1 in EAs and significant excess of heterozygosity in A and DQB1 in the AFA group. Haplotype analyses of neighbouring loci pairs in positive LD, such as B ∼ C and B ∼ DRB1, revealed novel associations at the 4th field level of resolution. Conclusions Our findings show unique 4-field associations that were not apparent in results obtained using testing methodologies with limited sequence coverage and phasing. In addition, our results provide new 4-field allele and haplotype frequencies. Our data provides a useful reference source for future population studies that in turn may allow for precise fine mapping of coding and non-coding sequences determining disease susceptibility and allo-immunogenicity. S. Krishnakumar: Employee; Company/Organization; Immucor. C. Wang: Employee; Company/Organization; Immucor. M. Li: Employee; Company/Organization; Immucor. M.N. Mindrinos: Employee; Company/Organization; Immucor.

Introduction Unambiguous, high resolution HLA typing plays a central role in hematopoietic stem cell transplantation. Current methodologies, primarily Sanger SBT, often fails to resolve all ambiguities creating the need for additional testing and longer turnaround times. Next generation sequencing (NGS) has the potential to address these shortcomings by providing full length, phase resolved sequences of HLA alleles with a single test. The Immucor Mia Fora NGS TM is an HLA typing kit and analysis software package that allows for typing of multiple samples per run in multiplex using the Illumina platform. In this study, we evaluated the NGS system against Sanger SBT methods. Methods NGS was performed on 11 HLA genes (HLA -A, B, C, DQA1/B1, DPA1/B1, and DRB1/3/4/5) for 69 subjects (1221 alleles) in alpha test, 69 (1223 alleles) in beta, and 35 (618 alleles) in further test. Automated liquid handling instruments were used for long range PCR and library preparation. Size selection was performed using the Pippin Prep followed by real-time PCR quantification on the ABI 7500. The final library was sequenced on the Illumina using the 300-cycle v2 kit. FASTQ files were analyzed with the Mia Fora software. NGS alleles to the 3rd fields were compared to the typing obtained by SBT, SSO, and SSP. Results A panel of 173 subjects carrying 3062 HLA class I and II alleles (total of 369 unique HLA alleles including novel alleles) was selected and typed by NGS. The overall concordance between NGS (automatic allele assigned by the software without editing) and Sanger SBT typing ranged from 98.5–99.7% in class I and 96.7–100.0% in class II. After manual review using the software’s tools for flagged alleles, the overall concordance will reach even higher. Conclusions The current NGS HLA typing product is able to produce robust and reliable allelic level HLA typing at all 11 HLA class I and II loci with no or very few ambiguities. Discordance was primarily observed in class II, most notably certain DRB1 ∗ 04 and DQB1 ∗ 03 alleles which often suffered from low read coverage in the presence of other alleles, and the ability to assign novel alleles or ambiguous results (e.g. DPB1) by the software. Improvements on test condition to prevent potential preferential amplification of certain HLA alleles would improve even more the accurate assignment of alleles at all HLA loci.

The accurate and complete selection of candidate genomic regions from a DNA sample before sequencing is critical in molecular diagnostics. Several recently developed technologies await substantial improvements in performance, cost, and multiplex sample processing. Here we present the utility of long padlock probes (LPPs) for targeted exon capture followed by array-based sequencing. We found that on average 92% of 5,471 exons from 524 nuclear-encoded mitochondrial genes were successfully amplified from genomic DNA from 63 individuals. Only 144 exons did not amplify in any sample due to high GC content. One LPP was sufficient to capture sequences from

The GTP cyclohydrolase I gene of Drosophila melanogaster, the Punch locus, encodes alternative transcripts of 1.7 and 1.75 kilobases (kb). These transcripts are differentially expressed throughout Drosophila development. cDNA clones representing these transcripts, corresponding genomic regions and polymerase chain reaction-amplified primer extensions of the 5' ends of the RNAs were sequenced. Both RNAs contain five exons and derive from primary transcripts that extend over approximately 8 and 4 kb for the 1.7- and 1.75-kb RNAs, respectively. Their 5'-most exons are unique and spliced onto common 3' exons. The cDNAs each contain a single long open reading frame, which can be translated into a polypeptide of 273 amino acids for the 1.7-kb mRNA and 308 amino acids for the 1.75-kb mRNA. The unique exons confer distinct N-terminal domains to each predicted protein. Sequence comparisons reveal that the Drosophila GTP cyclohydrolase isoforms encoded by the multiple transcripts are highly similar to GTP cyclohydrolases from humans, rodents, and bacteria, with one significant exception. The N-terminal domains encoded by the transcript-specific 5' exons cannot be aligned with the N termini of any other GTP cyclohydrolases. These domains are predicted to confer distinct physical characteristics to the alternate isoforms, and it is hypothesized that they aid in regulating the expression of the enzyme in diverse cellular environments.

Several quantitative trait loci (QTL) mapping strategies can successfully identify major-effect loci, but often have poor success detecting loci with minor effects, potentially due to the confounding effects of major loci, epistasis, and limited sample sizes. To overcome such difficulties, we used a targeted backcross mapping strategy that genetically eliminated the effect of a previously identified major QTL underlying high-temperature growth (Htg) in yeast. This strategy facilitated the mapping of three novel QTL contributing to Htg of a clinically derived yeast strain. One QTL, which is linked to the previously identified major-effect QTL, was dissected, and NCS2 was identified as the causative gene. The interaction of the NCS2 QTL with the first major-effect QTL was background dependent, revealing a complex QTL architecture spanning these two linked loci. Such complex architecture suggests that more genes than can be predicted are likely to contribute to quantitative traits. The targeted backcrossing approach overcomes the difficulties posed by sample size, genetic linkage, and epistatic effects and facilitates identification of additional alleles with smaller contributions to complex traits.

The accurate identification of the true allele frequency of HLA genes in various ethnic groups may be a challenging task. The current methodology to catalog the common and well-documented (CWD) HLA alleles is based on the 3rd or 4th field made from assumptions for gene segments that were not tested by Sanger Sequencing methods. We examined the allelic frequencies in all HLA loci by leveraging the power of Next Generation Sequence (NGS) and long range PCR to interrogate the entire sequence of the class I genes and most of the extent Class II genes. In a HLA typing study of 6,000 subjects we unexpectedly found several examples of alleles with common occurrence that were thought to be rare, and represent the most common allele of the 1st-2nd fields allele group. For example, we found that the alleles B ∗ 40:01:02 and DRB1 ∗ 08:01:03 represent the common alleles of their groups while the alleles with the lowest digit number (B ∗ 40:01:01, DRB1 ∗ 08:01) were absent. Extending the sequences also failed to find alleles thought to be common, while other alleles appeared to replace them; DRB3 ∗ 02:24 that associates tightly with DRB1 ∗ 14:01:01 is common while the closely related allele DRB3 ∗ 02:01 thought to be common, was not found; these alleles differ in exon-3, that had not been tested frequently. Our study also showed that several subtypes of the allele groups A ∗ 29:02, B ∗ 44:02, B ∗ 18:01, B ∗ 52:01, C ∗ 05:01, C ∗ 06:02, C ∗ 07:01, C ∗ 07:02, DQA1 ∗ 01:01, DQA1 ∗ 01:02, DQA1 ∗ 01:03, DQA1 ∗ 05:01, DQA1 ∗ 05:05, DQB1 ∗ 03:01 and DQB1 ∗ 05:01 differing at non coding regions or by silent substitutions at areas not tested frequently, are very common. Intriguingly, the alleles differing in areas not tested routinely in previously used assays, show distinctive associations with alleles of contiguous or close loci and contrary to previous predictions indicate that the evolutionary processes in HLA are more complex and modern populations show strongly conserved common haplotype blocks. Our findings show that HLA diversity may have arisen from opposing mechanisms such as allele diversification and convergent evolution.

Background: Circulating tumor cells (CTCs) in blood and disseminated tumor cells (DTCs) in bone marrow are being studied to monitor disease and guide therapy, but the relationship between CTCs and DTCs is weak and may confound clinical decision-making. Because blood sampling is easier than sampling bone marrow, CTC analyses are used more frequently than DTC analyses, although the relationship between CTCs and DTCs and the mutational heterogeneity within both populations at the single cell level are not usually examined simultaneously. Methods: We used the MagSweeper to immunomagnetically isolate single tumor cells from blood and bone marrow samples in breast cancer patients. Isolated tumor cells were used for immunohistochemical identification, PIK3CA gene mutation analysis, and to propagate cells in culture. In one patient, CTC and DTC single cell genotypes were compared during multiple treatment courses as the disease course progressed. Results: 242 individual tumor cells were isolated from 17 breast cancer patients. All tumor cells were assayed for single nucleotide mutations on exons 9 and 20 of the PIK3CA gene, and 48 mutated cells were identified in three patients. Heterogeneity and temporal discordance were observed in and between CTCs and DTCs in the same patient. All DTCs from bone marrow overgrown by tumor cells in a metastatic breast cancer patient carried the same PIK3CA single nucleotide mutation even though CTCs isolated within the same time period were wild type or heterogeneous for the mutation, providing evidence of both concordance and discordance of single cell PIK3CA genotype between CTCs and DTCs at different blood sampling time points. DTCs isolated by the MagSweeper could be directly cultured and consistently maintained the mutant PIK3CA genotype despite morphological changes over time in cell culture. Conclusions: DTCs isolated live by the MagSweeper can be propagated in culture, and a DNA single nucleotide mutation was maintained as a stable marker during cell culture multiple passages. This same mutation was used to monitor CTCs and DTCs at the single cell level. Although others have shown variable correlation between presence of CTCs and DTCs in the same patients, we show here potential discordance at the genotype level of single CTCs isolated from the same patient at different time points and between individual CTCs and DTCs. Our data support that CTCs and DTCs can have independent clinical value and suggest that it may be necessary to independently sample both during overall treatment course. Citation Format: Glenn Deng, Sujatha Krishnakumar, Marc A. Coram, Ashley A. Powell, Haiyu Zhang, Michael N. Mindrinos, Melinda L. Telli, Katharina E. Effenberger, Michael Herrler, Klaus Pantel, Ronald W. Davis, Stefanie S. Jeffrey. Genotype discordance between circulating tumor cells in blood and disseminated tumor cells in bone marrow at single cell level in breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3528. doi:10.1158/1538-7445.AM2014-3528

The plant cytoskeleton plays a pivotal role in determining the direction of cell wall expansion, and ultimately the cell’s final shape. However, the mechanisms by which localized expansion events are initiated remain obscure. Mutational analysis of the trichome (plant hair) morphogenic pathway in Arabidopsis has identified at least eight genes that determine trichome branch number. One of these genes, ZWICHEL (ZWI), encodes a novel member of the kinesin superfamily of motor proteins. Mutations in the ZWI gene cause a reduction in the number of trichome branches. To identify additional genes involved in trichome branch initiation, we screened for extragenic suppressors of the zwi-3 mutation and isolated three suppressors that rescued the branch number defect of zwi-3. These suppressors define three genes, named suz, for suppressor of zwichel-3. All of the suppressors were shown to be allele specific. One of the suppressors, suz2, also rescued the trichome branch number defect of another branch mutant, furca1-2. Plants homozygous for suz2 have more than the wild-type number of trichome branches. This suggests that SUZ2 is a negative regulator of trichome branching and may interact with ZWI and FURCA1. The suz1 and suz3 mutants display no obvious phenotype in the absence of the zwi-3 mutation. The suz1 zwi-3 double mutants also exhibited a male-sterile phenotype due to a defect in pollen tube germination and growth, whereas both the suz1 and the zwi-3 single mutants are fertile. The synthetic male sterility of the suz1 zwi-3 double mutants suggests a role for SUZ1 and ZWI in pollen germination and pollen tube growth. DNA sequence analysis of the zwi-3 mutation indicated that only the tail domain of the zwi-3 protein would be expressed. Thus, the suz mutations show allele-specific suppression of a kinesin mutant that lacks the motor domain.

Introduction: Most cancer deaths are caused by metastatic disease. Tumor cells exit the primary tumor and travel through blood and/or lymphatic channels to distant sites. Circulating tumor cells (CTCs) from blood and disseminated tumor cells (DTCs) from bone marrow may play key roles in the metastatic process. Primary tumor heterogeneity and how the metastatic process evolves over time may confound the study of metastatic biology. To better distinguish the cells of origin in metastases, we developed new methodologies to isolate single tumor cells from tissues, blood, and bone marrow for DNA and RNA analyses. We compared these analyses in individual cells from primary and metastatic tumors, CTCs and DTCs. Materials and Methods: Single tumor cell suspensions from primary and metastatic tissues were prepared from fresh human specimens or mouse xenografts. Single cells were isolated from blood, bone marrow, or single tumor cell suspensions from tissue using EpCAM-conjugated microbeads and the MagSweeper, a device invented by our group. Unbounded microbeads were removed and the single cells were placed into individual PCR tubes for further nucleic acid analyses. Live CTCs and DTCs from some breast cancer patients were cultured for further testing. Single cell DNA mutational analyses were performed by multiplex PCR sequencing. Single cell transcriptional profiling was performed by multiplex qRT-PCR. Single tumor cells with mutations were compared among multiple compartments: primary tumor, metastatic tumor, blood and bone marrow. Array CGH analysis was used to focus single cell analysis on genes in potential areas of DNA deletion or amplification. Xenograft mouse models were generated using primary or tumor tissues and single cells were compared. Results: Single tumor cells from fresh or fixed solid tumor tissues were successfully isolated for nucleic acid analyses. CTCs and DTCs isolated by MagSweeper from breast cancer patients could be isolated live and cultured or injected for growth in xenografts. Single cell DNA mutation analysis of the PIK3CA gene was identified in 3/27 breast cancer patients, showing the mutation in only 45/178 single tumor cells. Sequential PIK3CA mutational analyses of CTCs, DTCs, and metastases showed that DTCs better reflected the mutational status of metastases at time points earlier in the metastatic process. Discussion: Our results indicate that isolating single tumor cells from tumor tissues, blood, and bone marrow provides detailed information about different tumor compartments in individual cancer patients. Single cell DNA mutational analysis can provide a biomarker for tracking CTCs and DTCs over time during the metastatic process. Based on our initial findings, we postulate that the bone marrow serves as a tumor cell reservoir for early metastatic spread in breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-03.

Background: Cancer evolves through the accumulation of multiple genetic alterations. How such alterations are related to tumor growth and metastatic dissemination is being widely investigated. Tissue level analyses, however, may be confounded by tumor cell heterogeneity and contamination by wildtype stromal and immune cells. Single cell analysis of tumor cells may contribute to a better understanding of tumorigenesis and metastasis. Single cell analyses using flow cytometry or microdissected cells from tissues require large numbers of cells, expensive equipment, laborious technical training, and the analyzed single cell information may be impacted by technical processing artifacts. We developed an efficient, reliable and simple new method to isolate fresh single tumor cells from tissues and blood for mutational analysis and to evaluate additional biomarkers. Materials and Methods: Tumor tissues and blood samples were obtained from breast cancer patients after informed consent. Primary or metastatic tissue was used for preparation of single cell suspensions for subsequent isolation. Single cells were isolated using EpCAM-conjugated microbeads and the MagSweeper, a device invented by our group. Individual microbead-labeled single cells were easy to observe and extract. Single cells were placed into individual PCR tubes for further DNA and RNA analyses. Single cell DNA mutational analysis was performed by sequencing using multiplex pre-amplification followed by PCR amplification. Multiplex RNA expression analyses were performed by qRT-PCR from single tumor cells. Enriched and unenriched single cell suspensions were used for biomarker evaluation and results were compared with normal tissues. Tumor cells with mutations were compared on a single cell level among multiple compartments: primary tumor, blood, and metastatic tumor. Results and Discussion: Single cells were isolated from suspensions prepared from primary and metastatic tumor tissues, EpCAM-enriched and unenriched single cells microscopically observed without requiring additional treatment and used for immunostaining for tumor cells identification. Some single cells not bound to EpCAM microbeads still met tumor cell criteria (cytokeratin positive, CD45 negative, and DAPI positive). The percentage of EpCAM -positive and cytokeratin-positive was determined in the single cell suspensions. Single cell DNA showing a single nucleotide mutation in the PIK3CA gene was identified in individual tumor cells from different tissues but not white blood cells. Results from our model indicate that isolating single tumor cells from tumor tissues or blood provides more detailed information about different tumor compartments in individual cancer patients. Comparison of individual tumor cells from primary and/or metastatic sites to tumor cells in blood should provide further insight into the metastatic process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1182.

Introduction Triple-negative breast cancer (TNBC) is aggressive and lacks targeted therapies. Phosphatidylinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathways are frequently activated in TNBC patient tumors at the genome, gene expression and protein levels, and mTOR inhibitors have been shown to inhibit growth in TNBC cell lines. We describe a panel of patient-derived xenografts representing multiple TNBC subtypes and use them to test preclinical drug efficacy of two mTOR inhibitors, sirolimus (rapamycin) and temsirolimus (CCI-779). Methods We generated a panel of seven patient-derived orthotopic xenografts from six primary TNBC tumors and one metastasis. Patient tumors and corresponding xenografts were compared by histology, immunohistochemistry, array comparative genomic hybridization (aCGH) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) sequencing; TNBC subtypes were determined. Using a previously published logistic regression approach, we generated a rapamycin response signature from Connectivity Map gene expression data and used it to predict rapamycin sensitivity in 1,401 human breast cancers of different intrinsic subtypes, prompting in vivo testing of mTOR inhibitors and doxorubicin in our TNBC xenografts. Results Patient-derived xenografts recapitulated histology, biomarker expression and global genomic features of patient tumors. Two primary tumors had PIK3CA coding mutations, and five of six primary tumors showed flanking intron single nucleotide polymorphisms (SNPs) with conservation of sequence variations between primary tumors and xenografts, even on subsequent xenograft passages. Gene expression profiling showed that our models represent at least four of six TNBC subtypes. The rapamycin response signature predicted sensitivity for 94% of basal-like breast cancers in a large dataset. Drug testing of mTOR inhibitors in our xenografts showed 77 to 99% growth inhibition, significantly more than doxorubicin; protein phosphorylation studies indicated constitutive activation of the mTOR pathway that decreased with treatment. However, no tumor was completely eradicated. Conclusions A panel of patient-derived xenograft models covering a spectrum of TNBC subtypes was generated that histologically and genomically matched original patient tumors. Consistent with in silico predictions, mTOR inhibitor testing in our TNBC xenografts showed significant tumor growth inhibition in all, suggesting that mTOR inhibitors can be effective in TNBC, but will require use with additional therapies, warranting investigation of optimal drug combinations.