Desiree C. Petersen

Complete Khoisan and Bantu genomes from southern Africa

The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial and small sets of nuclear markers have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans. However, until now, fully sequenced human genomes have been limited to recently diverged populations. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.

Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii (Tasmanian devil)

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction because of a contagious cancer known as Devil Facial Tumor Disease. The inability to mount an immune response and to reject these tumors might be caused by a lack of genetic diversity within a dwindling population. Here we report a whole-genome analysis of two animals originating from extreme northwest and southeast Tasmania, the maximal geographic spread, together with the genome from a tumor taken from one of them. A 3.3-Gb de novo assembly of the sequence data from two complementary next-generation sequencing platforms was used to identify 1 million polymorphic genomic positions, roughly one-quarter of the number observed between two genetically distant human genomes. Analysis of 14 complete mitochondrial genomes from current and museum specimens, as well as mitochondrial and nuclear SNP markers in 175 animals, suggests that the observed low genetic diversity in todays population preceded the Devil Facial Tumor Disease disease outbreak by at least 100 y. Using a genetically characterized breeding stock based on the genome sequence will enable preservation of the extant genetic diversity in future Tasmanian devil populations.

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments.

Mutation detection has, until recently, relied heavily on the use of gel‐based methods that can be both time consuming and difficult to design. Nongel‐based systems are therefore important to increase simplicity and improve turn around time without compromising assay sensitivity and accuracy, especially in the diagnostic/clinical setting. In this study, we assessed the latest of the nongel‐based methods, namely high‐resolution melt (HRM) curve analysis. HRM is a closed‐tube method that incorporates a saturating dye during DNA amplification followed by a monitoring of the change in fluorescence as the DNA duplex is denatured by an increasing temperature. We assessed 10 amplicons derived from eight genes, namely SERPINA1, CXCR7, MBL, VDR, NKX3A, NPY, TP53, and HRAS using two platforms, the LightScanner® System using LC Green® PLUS DNA binding dye (Idaho Technology, Salt Lake City, UT, USA) and the LightCycler® 480 using the HRM Master dye (Roche Diagnostics, Indianapolis, IN, USA). DNA variants (mutations or polymorphims) were previously identified using denaturing gradient gel electrophoresis (DGGE) a method, similarly to HRM, based upon the different melting properties of double‐stranded DNA. Fragments were selected based on variant and fragment complexity. This included the presence of multiple sequence variants, variants in alternate orientations, and single or multiple variants (constitutional or somatic) in GC‐rich fragments. We demonstrate current limitations of the HRM method for the analysis of complex DNA regions and call for caution when using HRM as the sole method to make a clinical diagnosis based on genetic analysis. Hum Mutat 30, 1–8, 2009

Risk for HIV-1 infection associated with a common CXCL12 (SDF1) polymorphism and CXCR4 variation in an African population.

CXC chemokine ligand 12 (CXCL12), or stromal cell-derived factor 1 (SDF1), is the only known natural ligand for the HIV-1 coreceptor, CXC chemokine receptor 4 (CXCR4). A single nucleotide polymorphism (SNP) in the CXCL12 gene (SDF1-3′A) has been associated with disease progression to AIDS in some studies, but not others. Mutations in the CXCR4 gene are generally rare and have not been implicated in HIV-1/AIDS pathogenesis. This study analyzed the SDF1-3′A SNP and performed mutation screening for polymorphic markers in the CXCR4 gene to determine the presence or absence of significant associations with susceptibility to HIV-1 infection. The study consisted of 257 HIV-1-seropositive patients and 113 HIV-1-seronegative controls representing a sub-Saharan African population belonging to the Xhosa ethnic group of South Africa. The SDF1-3′A SNP was associated with an increased risk for HIV-1 infection (P = 0.0319) whereas no significant association was observed between the occurrence of the SDF1-3′A SNP and increased or decreased plasma levels of CXCL12. Comprehensive mutation analysis of the CXCR4 gene confirmed a high degree of genetic conservation within the coding region of this ancient population.

Interpretation of custom designed Illumina genotype cluster plots for targeted association studies and next-generation sequence validation

BackgroundHigh-throughput custom designed genotyping arrays are a valuable resource for biologically focused research studies and increasingly for validation of variation predicted by next-generation sequencing (NGS) technologies. We investigate the Illumina GoldenGate chemistry using custom designed VeraCode and sentrix array matrix (SAM) assays for each of these applications, respectively. We highlight applications for interpretation of Illumina generated genotype cluster plots to maximise data inclusion and reduce genotyping errors.FindingsWe illustrate the dramatic effect of outliers in genotype calling and data interpretation, as well as suggest simple means to avoid genotyping errors. Furthermore we present this platform as a successful method for two-cluster rare or non-autosomal variant calling. The success of high-throughput technologies to accurately call rare variants will become an essential feature for future association studies. Finally, we highlight additional advantages of the Illumina GoldenGate chemistry in generating unusually segregated cluster plots that identify potential NGS generated sequencing error resulting from minimal coverage.ConclusionsWe demonstrate the importance of visually inspecting genotype cluster plots generated by the Illumina software and issue warnings regarding commonly accepted quality control parameters. In addition to suggesting applications to minimise data exclusion, we propose that the Illumina cluster plots may be helpful in identifying potential in-put sequence errors, particularly important for studies to validate NGS generated variation.

No Association between Common Chemokine and Chemokine Receptor Gene Variants and Prostate Cancer Risk

There is growing evidence that inflammation and infection play important roles in the etiology of prostate cancer. As the chemokine network is directly involved in inflammation and infectious diseases, we tested for an association between six common putative functional variants and prostate cancer risk using an Australian case-control study. We measured CCL5 −403G>A, CXCL12 +801G>A, CCR2V64I (G>A), CCR5Δ32, CX3CR1V249I (G>A), and CX3CR1T280M (C>T) for 815 cases and 738 controls. Of these, only CXCL12 +801G>A has previously been tested and found to be associated with prostate cancer risk. We found no significant associations with prostate cancer risk (all P > 0.4). All per allele odds ratios ranged from 0.96 (95% confidence intervals, 0.80-1.16) to 1.06 (95% confidence intervals, 0.90-1.23). This suggests that these common chemokine and chemokine receptor variants do not play a major, if any, role in susceptibility to prostate cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3615–7)

Functional analysis of novel SLC11A1 (NRAMP1) promoter variants in susceptibility to HIV-1

The divalent cation transporter is the natural resistance associated macrophage protein 1 (formerly NRAMP1 and now named SLC11A1) for solute carrier 11A1 (OMIM accession number 600266). The gene that codes for this transporter has been studied intensively for its role in conferring susceptibility to infectious diseases such as tuberculosis, leprosy, meningococcal meningitis, visceral leishmaniasis, and HIV infection, as well as to autoimmune diseases such as rheumatoid arthritis, diabetes, sarcoidosis, inflammatory bowel disease,1 and, more recently, Kawasaki disease.2 Most studies have investigated a functional GT repeat sequence in the promoter region of this gene3 and have identified two commonly occurring repeat alleles and four rare repeat alleles.4,5 The common alleles are T(GT)5AC(GT)5AC(GT)10 (allele 2) and T(GT)5AC(GT)5AC(GT)9 (allele 3; GenBank accession number AF229163, 5768 to 5808). Allele 2, which decreases gene expression, has been associated with susceptibility to infectious diseases; the more common allele 3 enhances gene expression to protect against infectious diseases while enhancing susceptibility to autoimmune diseases. Although HIV is classified as an infectious disease, it affects the autoimmune system, which may explain why allele 3 is associated with susceptibility to HIV-1.6,7 This study aimed to screen the promoter region of SLC11A1 for novel sequence variations in people from sub-Saharan Africa infected with HIV-1 compared with uninfected people and to determine the effect of novel variants on normal promoter function. ### Participants We studied 84 HIV-1 seropositive people (60 African and 24 of mixed African–European descent) and 133 HIV-1 seronegative people (64 African, 62 of mixed African–European descent, and seven Asian) who lived in the Western Cape of South Africa and who attended one of the HIV-1 clinics in Tygerberg or Langa or the blood transfusion services of the Western Cape. In this study, we …

Clinical presentation of prostate cancer in black South Africans.

Compared with White Americans, Black American men are at a significant increased risk of presenting with prostate cancer (PCa) and associated mortality, suggesting a link to African‐ancestry. However, PCa status within Africa is largely unknown. We address the clinical presentation of PCa within Black South African men.

African-based CCR5 single-nucleotide polymorphism associated with HIV-1 disease progression.

In this study we show a statistically significant association between the CCR5-A335V SNP and decreased disease progression from HIV infection to AIDS within individuals of African ethnic origin residing in the Western Cape of South Africa. The variable penetrance observed for this protective association together with previous findings that this polymorphic marker results in a functional response similar to that of wild-type CCR5 suggests that A335V does not contribute directly in slowing disease progression. We therefore hypothesize that this genetic marker in combination with other weaker genetic events collectively acts in slowing disease progression and may ultimately contribute to providing an individual HIV susceptibility risk profile for sub-Saharan Africans. (excerpt)

Revised timeline and distribution of the earliest diverged human maternal lineages in southern Africa

The oldest extant human maternal lineages include mitochondrial haplogroups L0d and L0k found in the southern African click-speaking forager peoples broadly classified as Khoesan. Profiling these early mitochondrial lineages allows for better understanding of modern human evolution. In this study, we profile 77 new early-diverged complete mitochondrial genomes and sub-classify another 105 L0d/L0k individuals from southern Africa. We use this data to refine basal phylogenetic divergence, coalescence times and Khoesan prehistory. Our results confirm L0d as the earliest diverged lineage (∼172 kya, 95%CI: 149–199 kya), followed by L0k (∼159 kya, 95%CI: 136–183 kya) and a new lineage we name L0g (∼94 kya, 95%CI: 72–116 kya). We identify two new L0d1 subclades we name L0d1d and L0d1c4/L0d1e, and estimate L0d2 and L0d1 divergence at ∼93 kya (95%CI:76–112 kya). We concur the earliest emerging L0d1’2 sublineage L0d1b (∼49 kya, 95%CI:37–58 kya) is widely distributed across southern Africa. Concomitantly, we find the most recent sublineage L0d2a (∼17 kya, 95%CI:10–27 kya) to be equally common. While we agree that lineages L0d1c and L0k1a are restricted to contemporary inland Khoesan populations, our observed predominance of L0d2a and L0d1a in non-Khoesan populations suggests a once independent coastal Khoesan prehistory. The distribution of early-diverged human maternal lineages within contemporary southern Africans suggests a rich history of human existence prior to any archaeological evidence of migration into the region. For the first time, we provide a genetic-based evidence for significant modern human evolution in southern Africa at the time of the Last Glacial Maximum at between ∼21–17 kya, coinciding with the emergence of major lineages L0d1a, L0d2b, L0d2d and L0d2a.