Homozygosity mapping and exome sequencing reveal GATAD1 mutation in autosomal recessive dilated cardiomyopathy

Abstract

Background: Dilated cardiomyopathy (DCM) is a heritable, genetically heterogeneous disorder that typically exhibits autosomal dominant inheritance. Genomic strategies enable discovery of novel, unsuspected molecular underpinnings of familial DCM. We performed genome-wide mapping and exome sequencing in a unique family wherein DCM segregated as an autosomal recessive (AR) trait.

Methods and results: Echocardiography in 17 adult descendants of first cousins revealed DCM in 2 female siblings and idiopathic left ventricular enlargement in their brother. Genotyping and linkage analysis mapped an AR DCM locus to chromosome arm 7q21, which was validated and refined by high-density homozygosity mapping. Exome sequencing of the affected sisters was then used as a complementary strategy for mutation discovery. An iterative bioinformatics process was used to filter >40,000 genetic variants, revealing a single shared homozygous missense mutation localized to the 7q21 critical region. The mutation, absent in HapMap, 1000 Genomes, and 474 ethnically matched controls, altered a conserved residue of GATAD1, encoding GATA zinc finger domain-containing protein 1. Thirteen relatives were heterozygous mutation carriers with no evidence of myocardial disease, even at advanced ages. Immunohistochemistry demonstrated nuclear localization of GATAD1 in left ventricular myocytes, yet subcellular expression and nuclear morphology were aberrant in the proband.

Conclusions: Linkage analysis and exome sequencing were used as synergistic genomic strategies to identify GATAD1 as a gene for AR DCM. GATAD1 binds to a histone modification site that regulates gene expression. Consistent with murine DCM caused by genetic disruption of histone deacetylases, the data implicate an inherited basis for epigenetic dysregulation in human heart failure.

Figure 1

Pedigree structure and chromosome 7q21 haplotypes for family with autosomal recessive dilated cardiomyopathy. * = proband; square = male; circle = female; solid = DCM; half symbol = idiopathic left ventricular enlargement; open = unaffected; gray = uncertain; slash through the symbol = deceased with age at death indicated for generations I and II. Markers are listed from centromere to q telomere, with map locations according to the National Center for Biotechnology Information website (36.3 Build) and given in megabases (MB) and centimorgans (cM). Haplotypes for marker genotypes are shown in columns below pedigree symbols and are unambiguously inferred for individuals without DNA samples (II.1, II.2, III.7). Consistent with homozygous recessive inheritance, identical disease-associated haplotypes (boxed) are inherited by the three affected family members from their first-cousin parents. Recombination events account for homozygous inheritance of portions of the haplotype in an unaffected (III.4) and affected (III.6) individual, defining D7S669 as the upper flanking marker. The lower flanking marker, D7S515, is defined by heterozygous genotypes in all three affected individuals (III.6, III.10, and III.12). GATAD1 maps within this 24 MB DCM locus. Presence of the identified GATAD1-S102P mutation is indicated by a plus symbol and its absence indicated by a minus symbol. Clinically affected individuals are homozygous for the mutation.

Figure 2

Genome-wide linkage analysis revealed a locus for autosomal recessive DCM on chromosome 7, with a peak multipoint LOD score of 3.1 (>1,000:1 odds). The graph illustrates the peak multipoint LOD scores for individual chromosomes with the lower threshold set at zero. The inset details the LOD score plot for chromosome 7, highlighting the linked 7q21 region flanked by markers D7S669 (78 MB) and D7S515 (102 MB).

Figure 3

Genome-wide homozygosity mapping revealed a single region of homozygosity on chromosome 7q21 in the three affected siblings. Adjacent to the chromosome ideogram is a depiction of the SNVs for these individuals (III.12, III.10, and III.6) and an unaffected individual (III.4), which enabled fine mapping of the critical region. The dotted lines represent blocks of contiguous, homozygous SNVs identified in each family member. The unaffected individual (III.4) shared a portion of the homozygous block with her affected siblings, thus establishing the upper flank for the locus. The lower flank was defined by the telomeric ends of the homozygous blocks in the three affected individuals. A block of 889 contiguous, homozygous SNVs defined the 7.3 MB critical region encompassing 61 genes. Subsequent analysis of exome sequencing data within this region identified a total of 23 SNVs homozygous for the non-reference allele. All but one SNV were present in HapMap or 1000Genomes and several of our 16 other exomes.

Figure 4

To identify a pathogenic mutation that underlies autosomal recessive DCM, an iterative filtering process was applied to SNVs and INDELs identified in III.12 and III.10 by exome sequencing. The list of potential candidate genes was narrowed to three, each harboring a novel, non-silent, homozygous SNV shared by the affected sisters. Only one of these – GATAD1-S102P - mapped to the critical region on chromosome 7q21.

Figure 5

(A) The gene topology of GATAD1, including functional GATA-type Zinc Finger (black) and Glycine-Rich (diagonal lines) domains and the location of the homozygous mutation c.304 T>C in exon 2. ATG, start codon; TAG, stop codon. (B) Sanger sequencing verified the mutation, which results in an S102P substitution. (C) Conservation of this residue is illustrated whereby (•) indicates identical residues and (-) indicates a gap. Overall, the zebrafish and human homologues share only 73% protein identity and the N-terminus of the zebrafish homologue lacks the glycine-rich domain, implicated in protein-protein interactions and present in all other species.

Figure 6

Immunohistochemistry was performed with a monoclonal antibody against GATAD1 on left ventricular tissue from four individuals. Normal tissue from a male (A) and a female (B) display nuclear staining of GATAD1 with a homogeneous striated pattern in the extranuclear space, similar to what was noted in tissue from an individual with DCM due to a mutation in TPM1 (C). In contrast, GATAD1 in biopsy tissue from the proband shows an abnormal staining pattern (D). While there is still nuclear localization, extranuclear distribution of GATAD1 is perturbed whereas actin staining of an adjacent section (E) was indistinguishable from normal controls (data not shown). In addition, the globular morphology of nuclei is unique from the spindle shape observed in control tissue.