Exome Sequencing of Only Seven Qataris Identifies Potentially Deleterious Variants in the Qatari Population

<div><p>The Qatari population, located at the Arabian migration crossroads of African and Eurasia, is comprised of Bedouin, Persian and African genetic subgroups. By deep exome sequencing of only 7 Qataris, including individuals in each subgroup, we identified 2,750 nonsynonymous SNPs predicted to be deleterious, many of which are linked to human health, or are in genes linked to human health. Many of these SNPs were at significantly elevated deleterious allele frequency in Qataris compared to other populations worldwide. Despite the small sample size, SNP allele frequency was highly correlated with a larger Qatari sample. Together, the data demonstrate that exome sequencing of only a small number of individuals can reveal genetic variations with potential health consequences in understudied populations.</p> </div

Affymetrix Microarray Validation of Qatari Exome Potentially Deleterious SNPs Where the SNP and Gene Have Been Previously Linked to Human Health¹.

1<p>Analysis of the exomes in the QE7 14 alleles identified 131 missense coding SNPs where the SNP and gene have been previously identified as linked to human health (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone-0047614-t001" target="_blank">Table 1</a>, 4^th row). To validate this observation in a larger group of Qataris, the Affymetrix Genome-Wide SNP Array 5.0 was used to assess an independent group of 149 Qataris (QA149, 298 alleles). Of the 2,750 missense potentially deleterious SNPs identified in at least 1 of the QE7 14 alleles, 131 were on the microarray. Of these, 49 were in genes linked to human health, including 16 where both the gene and the SNP are linked to human health. Of these 16, listed are 10 chosen as examples of missense SNPs linked to human health.</p>2<p>Gene symbol and name obtained from the Consensus Coding Sequence (CCDS) NCBI database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Pruitt1" target="_blank">[32]</a>, amino acid substitution position and residues obtained from dbSNP when available, otherwise SIFT online webserver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Kumar1" target="_blank">[34]</a>. Transcript position and amino acid substitution were verified to be consistent with the literature.</p>3<p>SNP information includes chromosome amino acid substitution, dbSNP build 134 rsID if available, chromosome, position in GRCh37 human reference genome assembly, reference and alternate allele in QE7. Ref = references; alt = alternative.</p>4<p>Phenotype information from OMIM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Online1" target="_blank">[12]</a>, HGMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Stenson1" target="_blank">[37]</a>, PharmGKB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Klein1" target="_blank">[38]</a> or HUGE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Yu1" target="_blank">[39]</a> database.</p>5<p>For more details and references, see Details S1.</p>6<p>Shown is the alternate allele frequency determined by exome sequencing in QE7 individuals.</p>7<p>Shown is the risk allele frequency in the validation set of QA149 individuals (n = 149 Qatari, 298 alleles). Failed genotypes are accounted for in the allele frequency. For statistical comparisons of the QE7 and QA149 allele frequencies, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone-0047614-g002" target="_blank">Figure 2</a>.</p

Functional classification of single nucleotide polymorphism (SNP) sites in seven Qatari exomes.

<p>Genotypes for 126,924 SNPs in target exons ±500 bp were confidently called as ref/ref, ref/alt or alt/alt (where ref = GRCh37 reference allele and alt = non-reference allele) using GATK <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-DePristo1" target="_blank">[33]</a> and classified using databases of SNP function (NCBI dbSNP build 134, SIFT online webserver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Kumar1" target="_blank">[34]</a>, and GATK VariantAnnotator function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-DePristo1" target="_blank">[33]</a>. Shown are bar plots of: <b>A.</b> SNPs observed in ≥1 of 1,099 exomes [QE7 and 1000 G]; <b>B.</b> SNPs identified in ≥1 of 14 QE7 alleles; <b>C.</b> SNPs significantly higher or lower in QE7 <i>vs</i> at least one population; and <b>D.</b> Subset of significantly higher or lower SNPs in genes with a health-related role (OMIM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Online1" target="_blank">[12]</a>, HGMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Stenson1" target="_blank">[37]</a>, PharmGKB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Klein1" target="_blank">[38]</a> or HUGE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Yu1" target="_blank">[39]</a>). In the four plots, the x-axis lists the functional categories (noncoding, coding, silent, missense, splice, nonsense) and the y-axis the number of SNPs. There were 20,857 (52%) missense SNPs predicted deleterious by SIFT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Kumar1" target="_blank">[34]</a> or PolyPhen2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Adzhubei1" target="_blank">[35]</a> polymorphic in 1,099 exomes (QE7 and 1000 G), a subset 2,750 polymorphic in QE7 with ≥1 of 14 alleles (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone-0047614-t001" target="_blank">Table 1</a>). There were 1,853 significantly higher or lower missense SNPs predicted deleterious by SIFT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Kumar1" target="_blank">[34]</a> or PolyPhen2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Adzhubei1" target="_blank">[35]</a> polymorphic in 1,099 exomes (QE7+1000 G), and a subset of 510 relevant to health; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone-0047614-t002" target="_blank">Table 2</a>). Red = predicted deleterious SNPs.</p

Potentially Deleterious Missense Coding SNPs in the Qatari Genome Identified by Exome Sequencing¹.

1<p>In order to identify potentially deleterious missense health-linked SNPs in Qatar, genotypes of the 2,750 predicted to be potentially deleterious alternate alleles observed in QE7 were subdivided by frequency [≥1/14 or ≥6/14 alternate allele frequency] and by functional category.</p>2<p>In order to identify potentially deleterious SNPs of medical interest in Qatar, the 2,750 predicted to be potentially deleterious SNPs were subclassified into 3 groups based on prior link of the gene or SNP to a health-related phenotype using four major databases of disease and metabolism SNPs (OMIM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Online1" target="_blank">[12]</a>, HGMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Stenson1" target="_blank">[37]</a>, PharmGKB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Klein1" target="_blank">[38]</a> and HUGE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Yu1" target="_blank">[39]</a>). 1^st row - total number of potentially deleterious SNPs; 2^nd row - number of potentially deleterious of SNPs where no SNP in the gene has been previously associated with a phenotype relevant to human health; 3^rd row - SNPs in genes linked to human health, but the SNP has not been previously tested for phenotypic effect; and 4^th - number of potentially deleterious SNPs where the specific SNP and gene has been reported to be health-linked. SNPs in the fourth row (previously identified) are not counted in the third row (in a gene, but not SNP, previously linked).</p>3<p>1^st column - the 2,750 SNPs where the potentially deleterious alternate allele was observed at least once in QE7 (≥1 of 14), representing 2% of the sites confidently genotyped in QE7, subdivided by the health-linked classification described above. 2^nd column - the 339 potentially deleterious alleles observed at least 6 times in QE7 (12% of the 2,750), subdivided by the health-linked classification described above. For each column, percentages are based on the total in the first row for that column.</p

Principal component analysis (PCA) validation of exome genotypes for the QE7 individuals.

<p>In order to verify the overall quality of the genotyping call set, the seven Qatari exomes were compared to 1,092 individuals from four continents (1000 Genomes Project October 2011 Integrated Phase 1 Variant Set Release) at 18,865 SNPs segregating in both QE7 and 1000 Genomes that are present in dbSNP build 134 using SMARTPCA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-Price1" target="_blank">[14]</a>. Plotted is PCA 1 (x-axis) <i>vs</i> PCA 2 (y-axis). Individuals are color-coded by continent of origin (European = red, Asian = green, African = blue, American = grey, Qatar = orange). Clustering of the Qatari individuals was verified to be consistent with our prior report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone.0047614-HunterZinck1" target="_blank">[9]</a>, where Q1 cluster near Europeans, Q2 in between Q1 and Asians, and Q3 between Q1 and Africans.</p

Validation of allele frequency for potentially deleterious nonsynonymous missense SNPs observed in n = 7 Qatari exomes using Affymetrix 5.0 array genotyping of n = 149 Qataris or TaqMan genotyping of n = 86 Qataris (n = 82 overlapping).

<p>A. To confirm the allele frequency estimates for the Qatari population based on the number of alleles observed in QE7 (n = 14 alleles) for potentially deleterious SNPs, the QE7 allele frequency observed in at least 1 of 14 (7%) QE7 alleles was compared to the allele frequency in QA149 (n = 298 alleles) generated using Affymetrix 5.0 SNP microarrays. Of the 2,750 potentially deleterious nonsynonymous SNPs identified in QE7, 149 probes were on the Affymetrix 5.0 array. Shown is the QE7 allele frequency along the x-axis and the QA149 allele frequency along the y-axis for 131 SNPs, excluding 18 Affymetrix 5.0 SNPs where the QE7 allele frequency could not be validated due to partial missing genotypes. B. Validation of allele frequency for potentially deleterious nonsynonymous missense SNPs significantly higher or lower in Qatari exomes using TaqMan genotyping of n = 86 Qataris. To confirm the allele frequency estimates for the Qatari population based on the number of alleles observed in QE7 (n = 14 alleles) for deleterious SNPs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047614#pone-0047614-t004" target="_blank">Table 4</a>, the QE7 allele frequency observed in at least 1 of 14 (7%) QE7 alleles was compared to the allele frequency in QT86 (n = 172 alleles) generated using TaqMan. Shown is the QE7 allele frequency along the x-axis and the QT86 allele frequency along the y-axis.</p