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author:("gorgan, Neda")
1.  Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry 
Science translational medicine  2013;5(208):208ra145.
Natural Killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 35 parameters, including 28 NK cell receptors, on peripheral blood NK cells from five sets of monozygotic twins and twelve unrelated donors of defined HLA and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6,000-30,000 phenotypic populations within an individual and >100,000 phenotypes in this population. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors, while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.
PMCID: PMC3918221  PMID: 24154599
2.  Exome capture from saliva produces high quality genomic and metagenomic data 
BMC Genomics  2014;15:262.
Targeted capture of genomic regions reduces sequencing cost while generating higher coverage by allowing biomedical researchers to focus on specific loci of interest, such as exons. Targeted capture also has the potential to facilitate the generation of genomic data from DNA collected via saliva or buccal cells. DNA samples derived from these cell types tend to have a lower human DNA yield, may be degraded from age and/or have contamination from bacteria or other ambient oral microbiota. However, thousands of samples have been previously collected from these cell types, and saliva collection has the advantage that it is a non-invasive and appropriate for a wide variety of research.
We demonstrate successful enrichment and sequencing of 15 South African KhoeSan exomes and 2 full genomes with samples initially derived from saliva. The expanded exome dataset enables us to characterize genetic diversity free from ascertainment bias for multiple KhoeSan populations, including new exome data from six HGDP Namibian San, revealing substantial population structure across the Kalahari Desert region. Additionally, we discover and independently verify thirty-one previously unknown KIR alleles using methods we developed to accurately map and call the highly polymorphic HLA and KIR loci from exome capture data. Finally, we show that exome capture of saliva-derived DNA yields sufficient non-human sequences to characterize oral microbial communities, including detection of bacteria linked to oral disease (e.g. Prevotella melaninogenica). For comparison, two samples were sequenced using standard full genome library preparation without exome capture and we found no systematic bias of metagenomic information between exome-captured and non-captured data.
DNA from human saliva samples, collected and extracted using standard procedures, can be used to successfully sequence high quality human exomes, and metagenomic data can be derived from non-human reads. We find that individuals from the Kalahari carry a higher oral pathogenic microbial load than samples surveyed in the Human Microbiome Project. Additionally, rare variants present in the exomes suggest strong population structure across different KhoeSan populations.
PMCID: PMC4051168  PMID: 24708091
Exomes; KhoeSan; Genetic diversity; Metagenomics; Microbiome
3.  Co-evolution of Human Leukocyte Antigen (HLA) Class I Ligands with Killer-Cell Immunoglobulin-Like Receptors (KIR) in a Genetically Diverse Population of Sub-Saharan Africans 
PLoS Genetics  2013;9(10):e1003938.
Interactions between HLA class I molecules and killer-cell immunoglobulin-like receptors (KIR) control natural killer cell (NK) functions in immunity and reproduction. Encoded by genes on different chromosomes, these polymorphic ligands and receptors correlate highly with disease resistance and susceptibility. Although studied at low-resolution in many populations, high-resolution analysis of combinatorial diversity of HLA class I and KIR is limited to Asian and Amerindian populations with low genetic diversity. At the other end of the spectrum is the West African population investigated here: we studied 235 individuals, including 104 mother-child pairs, from the Ga-Adangbe of Ghana. This population has a rich diversity of 175 KIR variants forming 208 KIR haplotypes, and 81 HLA-A, -B and -C variants forming 190 HLA class I haplotypes. Each individual we studied has a unique compound genotype of HLA class I and KIR, forming 1–14 functional ligand-receptor interactions. Maintaining this exceptionally high polymorphism is balancing selection. The centromeric region of the KIR locus, encoding HLA-C receptors, is highly diverse whereas the telomeric region encoding Bw4-specific KIR3DL1, lacks diversity in Africans. Present in the Ga-Adangbe are high frequencies of Bw4-bearing HLA-B*53:01 and Bw4-lacking HLA-B*35:01, which otherwise are identical. Balancing selection at key residues maintains numerous HLA-B allotypes having and lacking Bw4, and also those of stronger and weaker interaction with LILRB1, a KIR-related receptor. Correspondingly, there is a balance at key residues of KIR3DL1 that modulate its level of cell-surface expression. Thus, capacity to interact with NK cells synergizes with peptide binding diversity to drive HLA-B allele frequency distribution. These features of KIR and HLA are consistent with ongoing co-evolution and selection imposed by a pathogen endemic to West Africa. Because of the prevalence of malaria in the Ga-Adangbe and previous associations of cerebral malaria with HLA-B*53:01 and KIR, Plasmodium falciparum is a candidate pathogen.
Author Summary
Natural killer cells are white blood cells with critical roles in human health that deliver front-line immunity against pathogens and nurture placentation in early pregnancy. Controlling these functions are cell-surface receptors called KIR that interact with HLA class I ligands expressed on most cells of the body. KIR and HLA are both products of complex families of variable genes, but present on separate chromosomes. Many HLA and KIR variants and their combinations associate with resistance to specific infections and pregnancy syndromes. Previously we identified basic components of the system necessary for individual and population survival. Here, we explore the system at its most genetically diverse by studying the Ga-Adangbe population from Ghana in West Africa. Co-evolution of KIR receptors with their HLA targets is ongoing in the Ga-Adangbe, with every one of 235 individuals studied having a unique set of KIR receptors and HLA class I ligands. In addition, one critical combination of receptor and ligand maintains alternative forms that either can or cannot interact with their ‘partner.’ This balance resembles that induced by malfunctioning variants of hemoglobin that confer resistance to malaria, a candidate disease for driving diversity and co-evolution of KIR and HLA class I in the Ga-Adangbe.
PMCID: PMC3814319  PMID: 24204327
4.  Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa 
Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing™ for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing™ as a substitute for traditional methods as it provides a rapid and reliable technique for determinating the antibiotic resistance pattern of a given bacterial strain in <1 h.
PMCID: PMC2744841  PMID: 19656662
Pyrosequencing; Pseudomonas aeruginosa; Antibiotic resistance

Results 1-4 (4)