Comparative Genomic Analyses of the Human <i>NPHP1</i> Locus Reveal Complex Genomic Architecture and Its Regional Evolution in Primates

<div><p>Many loci in the human genome harbor complex genomic structures that can result in susceptibility to genomic rearrangements leading to various genomic disorders. Nephronophthisis 1 (NPHP1, MIM# 256100) is an autosomal recessive disorder that can be caused by defects of <i>NPHP1</i>; the gene maps within the human 2q13 region where low copy repeats (LCRs) are abundant. Loss of function of <i>NPHP1</i> is responsible for approximately 85% of the NPHP1 cases—about 80% of such individuals carry a large recurrent homozygous <i>NPHP1</i> deletion that occurs via nonallelic homologous recombination (NAHR) between two flanking directly oriented ~45 kb LCRs. Published data revealed a non-pathogenic inversion polymorphism involving the <i>NPHP1</i> gene flanked by two inverted ~358 kb LCRs. Using optical mapping and array-comparative genomic hybridization, we identified three potential novel structural variant (SV) haplotypes at the <i>NPHP1</i> locus that may protect a haploid genome from the <i>NPHP1</i> deletion. Inter-species comparative genomic analyses among primate genomes revealed massive genomic changes during evolution. The aggregated data suggest that dynamic genomic rearrangements occurred historically within the <i>NPHP1</i> locus and generated SV haplotypes observed in the human population today, which may confer differential susceptibility to genomic instability and the <i>NPHP1</i> deletion within a personal genome. Our study documents diverse SV haplotypes at a complex LCR-laden human genomic region. Comparative analyses provide a model for how this complex region arose during primate evolution, and studies among humans suggest that intra-species polymorphism may potentially modulate an individual’s susceptibility to acquiring disease-associated alleles.</p></div

Features of the LCRs at the human <i>NPHP1</i> locus.

<p>Features of the LCRs at the human <i>NPHP1</i> locus.</p

The standard evolutionary phylogeny and a hypothetical model of structural changes from baboon to human.

<p>The meaning of each colored boxes or lines are indicated underneath the diagram. The information from <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005686#pgen.1005686.t003" target="_blank">Table 3</a></b>, including the BLAT results of human 45MID and 5936Ins in different nonhuman primates, are annotated in each diagram.</p

Deletion of the 45 kb LCRs is accompanied with a 5936 bp insertion (5936Ins).

<p><b>A.</b> UCSC Genome Browser custom track of the five fosmids (WIBR2-2033B13, ABC12-49282900B12, ABC12-47010700M12, ABC24-1014G11 and ABC24-2481A19) with fully sequenced inserts. <b>B.</b> Diagram on the top shows the SV haplotype in the haploid reference. A hypothetical SV haplotype with 45DIST deleted from the reference is shown at the bottom. This hypothetical SV haplotype is shown only for the purpose of illustrating the deletion/insertion haplotype. In the middle, the breakpoints of the 45 kb LCR deletion and 5936Ins are shown at base pair resolution. The full insert sequence of the fosmid from individual ABC12 can be found in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005686#pgen.1005686.s003" target="_blank">S3 Fig</a>.</b></p

Structural haplotype delineation by combined OM and aCGH approach.

<p>Structural haplotype delineation by combined OM and aCGH approach.</p

Copy number estimation of the human 45 kb LCRs and their orthologs in humans and non-human primates using aCGH.

<p>Copy number estimation of the human 45 kb LCRs and their orthologs in humans and non-human primates using aCGH.</p

Copy number polymorphisms of the 45 kb LCRs.

<p><b>A.</b> Discordant fosmids selected from individual fosmid libraries shown as custom track in UCSC Genome Browser. Diagram of SV haplotype of human reference is shown underneath the custom track. Green segments, discordant fosmids representing deletions; yellow segments, discordant fosmids representing inversions with one ESP mapped to the 358PROX; red segments, discordant fosmids representing inversions with one ESP mapped to the 358DIST. <b>B.</b> Pie chart showing copy number distribution of the 45 kb LCRs in each population. Percentage of each copy number (CN) is plotted. CHB and JPT are combined. YRI, Yoruba in Ibadan, Nigeria; CHB, Han Chinese in Beijing, China; JPT, Japanese in Tokyo, Japan; CEU, CEPH (Utah residents with ancestry from northern and western Europe); all populations, an aggregate of all the populations investigated in Conrad <i>et al</i>. 2010; AJHG 2000, population data in Saunier <i>et al</i>. 2000 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005686#pgen.1005686.ref039" target="_blank">39</a>]; Red, CN2 in 2% of YRI, 1% of CHB/JPT and 1% of CEU; orange, CN3 in 18% of YRI, 12% of CHB/JPT and 8% of CEU; yellow, CN4 in 38% of YRI, 52% of CHB/JPT and 74% of CEU; green, CN5 in 31% of YRI, 31% of CHB/JPT and 16% of CEU; blue, CN6 in 11% of YRI, 3% of CHB/JPT and 1% of CEU; grey, CN2 plus CN4 (the frequencies of which were not specifically indicated by Saunier <i>et al</i>. 2000). <b>C.</b> Bar plot showing copy number distribution of the 45 kb LCRs in each population. Percentage of each copy number is plotted.</p

Known SV haplotypes at the <i>NPHP1</i> locus in the human 2q13 region.

<p><b>A.</b> Comparison between human reference builds hg18 and hg19 by Miropeats. The traces between the top and bottom track represent the aligned paralogous sequences greater than 1 kb in size between the two builds. Inverted orientations of paralogous LCRs could be inferred (e.g. 45PROX and 45DIST, 358PROX and 358DIST) using the intersection point in a cluster as the indicator. Blue traces, aligned sequences from the 358 kb LCRs; red traces, aligned sequences from the 45 kb LCRs. Top track, human reference build hg18; bottom track, human reference build hg19. The positions of gaps in build hg18 are shown above the top track. “Segmental Dups” track from UCSC Genome Browser is shown below the Miropeats plots with the diagram of the SV haplotypes of the reference build hg19 shown underneath. <b>B.</b> Known SV haplotypes at the <i>NPHP1</i> locus. Green block, <i>NPHP1</i> gene; blue arrows, the 358 kb LCRs; red arrows, the 45 kb LCRs; CEN, centromeric side (proximal); TEL, telomeric side (distal).</p

Copy number analysis of genomic sequences flanking <i>NPHP1</i> in primates.

<p><b>A.</b> UCSC Genome Browser tracks showing the diagram of 358 kb LCRs and 45 kb LCRs on the proximal side of <i>NPHP1</i> (Chr2: 110430525–110962639, hg19). Blue and red arrows, one copy of the 358 kb LCRs and 45 kb LCRs for illustration purpose (the copy number of each LCR in NA10851, the universal aCGH reference, is annotated above the arrows); green segment, <i>NPHP1</i>. <b>B-C.</b> Plot of aCGH species average log₂ ratios for humans (N = 10), baboon (N = 1), rhesus macaques (N = 2), gorillas (N = 3), chimpanzees (N = 7) and orangutan (N = 1). The probes target the genomic region defined in <b>Fig 6A</b>. The species average log₂ ratio calculated for each probe is used for the plot. <b>D.</b> Whole genome sequencing (WGS) read-depth analysis in humans (N = 10), gorillas (N = 32), chimpanzees (N = 23) and orangutans (N = 17). The copy numbers indicated by WGS read-depth data are extracted from the dataset published by Sudmant <i>et al</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005686#pgen.1005686.ref054" target="_blank">54</a>]. The species average copy number calculated for each position is used for the plot. Blue, humans; yellow, baboon; brown, rhesus macaques; red, gorillas; green, chimpanzees; orange, orangutan. Regions between red dashed lines indicate the region that corresponds to the 45 kb LCRs.</p

SV haplotypes at the <i>NPHP1</i> locus delineated by OM and aCGH.

<p><b>A.</b> UCSC Genome Browser custom track showing discordance fosmids identified in NA15510. Discordant fosmids representing <i>NPHP1</i> inversion (red) and 45 kb LCR deletion (green) are shown on the custom track. <b>B.</b> SV haplotypes delineated by OM analysis. Four OM diagrams, from top to bottom, represent the <i>in silico</i> Rmap of hg19, the SwaI Rmap of NA10860, NA18994 and NA15510, respectively. Vertical black lines, individual restriction sites; purple area, the 45 kb LCR and its deletion product; green area, <i>NPHP1</i> region and its inversion product. <b>C.</b> aCGH result of NA15510. The log₂ ratio plot is shown. No CNV can be observed from the plot. <b>D.</b> Diagram showing the H2 SV haplotype of NA15510. The diagram results from data obtained from both OM and aCGH. <b>E.</b> OM results of CHM. <b>F.</b> Diagram showing the SV haplotype of CHM. Only one consensus map is found, which supports the H5 SV haplotype. <b>G.</b> OM results of H1-ES-P208. <b>H.</b> Diagram showing the SV haplotypes of H1-ES-P208. In total, three consensus maps are identified, indicating three different SV haplotypes (H5, H6 and H7).</p