Sujatha Krishnakumar

High-throughput, high-fidelity HLA genotyping with deep sequencing

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.

INCREASED SIZE EXCLUSION LIMIT2 Encodes a Putative DEVH Box RNA Helicase Involved in Plasmodesmata Function during Arabidopsis Embryogenesis

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.

Sequential Elimination of Major-Effect Contributors Identifies Additional Quantitative Trait Loci Conditioning High-Temperature Growth in Yeast

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.

A comprehensive assay for targeted multiplex amplification of human DNA sequences

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.

High-quality DNA sequence capture of 524 disease candidate genes

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 <100–500 bp in length and only a single-tube capture reaction and one microarray was required per sample. Our approach was highly reproducible and quick (<8 h) and detected DNA variants at high accuracy (false discovery rate 1%, false negative rate 3%) on the basis of known sample SNPs and Sanger sequence verification. In a patient with clinical and biochemical presentation of ornithine transcarbamylase (OTC) deficiency, we identified copy-number differences in the OTC gene at exon-level resolution. This shows the ability of LPPs to accurately preserve a samples genome information and provides a cost-effective strategy to identify both single nucleotide changes and structural variants in targeted resequencing.

SNP discovery and molecular evolution in Anopheles gambiae, with special emphasis on innate immune system

BackgroundAnopheles innate immunity affects Plasmodium development and is a potential target of innovative malaria control strategies. The extent and distribution of nucleotide diversity in immunity genes might provide insights into the evolutionary forces that condition pathogen-vector interactions. The discovery of polymorphisms is an essential step towards association studies of susceptibility to infection.ResultsWe sequenced coding fragments of 72 immune related genes in natural populations of Anopheles gambiae and of 37 randomly chosen genes to provide a background measure of genetic diversity across the genome. Mean nucleotide diversity (π) was 0.0092 in the A. gambiae S form, 0.0076 in the M form and 0.0064 in A. arabiensis. Within each species, no statistically significant differences in mean nucleotide diversity were detected between immune related and non immune related genes. Strong purifying selection was detected in genes of both categories, presumably reflecting strong functional constraints.ConclusionOur results suggest similar patterns and rates of molecular evolution in immune and non-immune genes in A. gambiae. The 3,214 Single Nucleotide Polymorphisms (SNPs) that we identified are the first large set of Anopheles SNPs from fresh, field-collected material and are relevant markers for future phenotype-association studies.

Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition

IntroductionTriple-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).MethodsWe 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.ResultsPatient-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.ConclusionsA 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.

Targeted and highly multiplexed detection of microorganisms by employing an ensemble of molecular probes.

ABSTRACT 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.

Allelic resolution NGS HLA typing of Class I and Class II loci and haplotypes in Cape Town, South Africa

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.

Abstract 3528: Genotype discordance between circulating tumor cells in blood and disseminated tumor cells in bone marrow at single cell level in breast cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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