A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia

Abstract

Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn mortality. Isolated or non-syndromic CDH is considered a multifactorial disease, with strong evidence implicating genetic factors. As low heritability has been reported in isolated CDH, family-based genetic methods have yet to identify the genetic factors associated with the defect. Using the Utah Population Database, we identified distantly related patients from several extended families with a high incidence of isolated CDH. Using high-density genotyping, seven patients were analyzed by homozygosity exclusion rare allele mapping (HERAM) and phased haplotype sharing (HapShare), two methods we developed to map shared chromosome regions. Our patient cohort shared three regions not previously associated with CDH, that is, 2q11.2-q12.1, 4p13 and 7q11.2, and two regions previously involved in CDH, that is, 8p23.1 and 15q26.2. The latter regions contain GATA4 and NR2F2, two genes implicated in diaphragm formation in mice. Interestingly, three patients shared the 8p23.1 locus and one of them also harbored the 15q26.2 segment. No coding variants were identified in GATA4 or NR2F2, but a rare shared variant was found in intron 1 of GATA4. This work shows the role of heritability in isolated CDH. Our family-based strategy uncovers new chromosomal regions possibly associated with disease, and suggests that non-coding variants of GATA4 and NR2F2 may contribute to the development of isolated CDH. This approach could speed up the discovery of the genes and regulatory elements causing multifactorial diseases, such as isolated CDH.

Figure 1. Heritability plays a role in isolated CDH

Pedigrees obtained from the UPDB showing seven interrelated isolated CDH patients that were genotyped and subjected to shared segment analysis. Note that some patients are related to each other through multiple founders. By enriching our study cohort with distantly related CDH patients, we hoped to increase the probability of finding shared, disease-associated segments. The disease burden in these pedigrees (number of affecteds/number of descendants) is significantly higher that the incidence of isolated CDH in the general population, suggesting the presence of a heritable component.

Figure 2. Rationale for using HERAM to identify heritable candidate regions for a disease

HERAM is based on two ideas. First, within a shared genomic segment (red segment), alleles will never be reciprocally homozygous (that is, AA in one individual and BB in another) at the same SNP. In the upper panel, reciprocally homozygous alleles are boxed. Note that there are no boxes in the shared segment and that boxes define the boundaries of the shared segment. Second, rare-shared alleles are enriched in shared genomic segments. These are shown in the lower diagram with boxed AB genotypes, where the MAF of the B allele is <0.1. Regions that are both devoid of exclusionary reciprocally homozygous SNPs and enriched for rare shared alleles are identical-by-state, and likely identical-by-descent when the segment is large (≥ 6 cM) and individuals are distantly related. MAF= minor allele frequency.

Figure 3. HERAM analysis in isolated CDH kindreds reveals large shared chromosomal regions

a. 8p23.1 (22.2–28.4 cM) and b. 15q26.2 (111.7–126.7 cM). HERAM plots were generated by dividing each chromosome into bins of 1 cM and counting the number of reciprocally-homozygous and rare-shared alleles within each bin (reciprocally-homozygous alleles are shown in blue and rare shared alleles in red). Shared regions are identified by the co-occurrence of gaps in the homozygous exclusion map ≥ 6 cM and enrichment of rare shared alleles within the gaps. The vertical grey bar represents the location of the centromere. The names and approximate location of genes contained within each shared region are shown below the HERAM plots. Shared regions on chromosomes 8 and 15 coincide with recurrently deleted and duplicated segments found in patients with non-isolated CDH and contain the candidate genes GATA4 and NR2F2, respectively.

Figure 4. HapShare confirms that 8p23.1 and 15q26.2 are shared and unique to isolated CDH kindreds

a and c: a total of 91 pair-wise combinations were evaluated for shared segments across the entire genome (7 isolated CDH patients, total of 14 haplotypes). Results for chromosome 8 (a) and 15 (c) are plotted along HapMap centimorgan (cM) recombination maps. The maximum number of consecutive inclusion markers across each chromosome is shown at the bottom of the plot and depicted as the height of a segment along the Y axis. b and d: the shared haplotypes were compared against paternal and maternal haplotypes from 24 children who did not have CDH (total of 48 haplotypes) to eliminate the possibility of common haplotypes. Neither the chromosome 8 (b) or 15 (d) shared segment was found to be a common haplotype.

Figure 4. HapShare confirms that 8p23.1 and 15q26.2 are shared and unique to isolated CDH kindreds

a and c: a total of 91 pair-wise combinations were evaluated for shared segments across the entire genome (7 isolated CDH patients, total of 14 haplotypes). Results for chromosome 8 (a) and 15 (c) are plotted along HapMap centimorgan (cM) recombination maps. The maximum number of consecutive inclusion markers across each chromosome is shown at the bottom of the plot and depicted as the height of a segment along the Y axis. b and d: the shared haplotypes were compared against paternal and maternal haplotypes from 24 children who did not have CDH (total of 48 haplotypes) to eliminate the possibility of common haplotypes. Neither the chromosome 8 (b) or 15 (d) shared segment was found to be a common haplotype.

Figure 4. HapShare confirms that 8p23.1 and 15q26.2 are shared and unique to isolated CDH kindreds

a and c: a total of 91 pair-wise combinations were evaluated for shared segments across the entire genome (7 isolated CDH patients, total of 14 haplotypes). Results for chromosome 8 (a) and 15 (c) are plotted along HapMap centimorgan (cM) recombination maps. The maximum number of consecutive inclusion markers across each chromosome is shown at the bottom of the plot and depicted as the height of a segment along the Y axis. b and d: the shared haplotypes were compared against paternal and maternal haplotypes from 24 children who did not have CDH (total of 48 haplotypes) to eliminate the possibility of common haplotypes. Neither the chromosome 8 (b) or 15 (d) shared segment was found to be a common haplotype.

Figure 4. HapShare confirms that 8p23.1 and 15q26.2 are shared and unique to isolated CDH kindreds

a and c: a total of 91 pair-wise combinations were evaluated for shared segments across the entire genome (7 isolated CDH patients, total of 14 haplotypes). Results for chromosome 8 (a) and 15 (c) are plotted along HapMap centimorgan (cM) recombination maps. The maximum number of consecutive inclusion markers across each chromosome is shown at the bottom of the plot and depicted as the height of a segment along the Y axis. b and d: the shared haplotypes were compared against paternal and maternal haplotypes from 24 children who did not have CDH (total of 48 haplotypes) to eliminate the possibility of common haplotypes. Neither the chromosome 8 (b) or 15 (d) shared segment was found to be a common haplotype.

Figure 5. Pedigree structure of the four patients sharing chromosomal regions 8p23.1 and 15q26.2

Most relationships coincide with those identified by the UPDB, but one involves unknown founders that are likely very remote. Disease susceptibilities within shared segments were inherited though multiple unaffected ‘carrier’ individuals suggesting that they are not sufficient by themselves to cause CDH. Patient 93848 shares more than one segment which raises the possibility that she inherited two genetic susceptibilities, supporting a multigenic model for CDH. There is no clear phenotype-genotype relationship among patients with shared segments.