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. Author manuscript; available in PMC: 2009 Jun 30.
Published in final edited form as: J Am Coll Cardiol. 2009 Mar 24;53(12):1065–1071. doi: 10.1016/j.jacc.2008.12.023

Hypoplastic Left Heart Syndrome Links to Chromosomes 10q and 6q and Is Genetically Related to Bicuspid Aortic Valve

Robert B Hinton *, Lisa J Martin †,‡, Smitha Rame-Gowda *, Meredith E Tabangin ‡, Linda H Cripe *, D Woodrow Benson *
PMCID: PMC2703749  NIHMSID: NIHMS101168  PMID: 19298921

Abstract

Objectives

This study was designed to identify disease loci for hypoplastic left heart syndrome (HLHS) and evaluate the genetic relationship between HLHS and bicuspid aortic valve (BAV).

Background

Previously, we identified that HLHS and BAV exhibit complex inheritance, and both HLHS and BAV kindreds are enriched for BAV. However, the genetic basis of HLHS and its relationship to BAV remains unclear.

Methods

Family-based nonparametric genome-wide linkage analysis was performed in kindreds ascertained by either an HLHS or BAV proband. Echocardiograms were performed on 1,013 participants using a sequential sampling strategy (33 HLHS kindreds, 102 BAV kindreds).

Results

The recurrence risk ratio of BAV in HLHS families (8.05) was nearly identical to that in BAV families (8.77). Linkage to chromosomal regions 10q22 and 6q23 with maximum logarithm of odds scores of 3.2 and 3.1, respectively, was identified in HLHS kindreds. In addition, 5 suggestive loci were identified (7q31, 11q22, 12q13, 14q23, and 20q11). We previously identified loci at chromosomes 18q22, 13q34, and 5q21 in BAV kindreds. The relationship between these loci was examined in the combined HLHS and BAV cohort and associations between loci were demonstrated (5q21, 13q34, and 14q23; 6q23 and 10q22; 7q31 and 20q11). Subsequent subsets linkage analysis showed a significant improvement in the logarithm of odds score at 14q23 only (4.1, p < 0.0001).

Conclusions

These studies demonstrate linkage to multiple loci identifying HLHS as genetically heterogeneous. Subsets linkage analyses and recurrence risk ratios in a combined cohort provide evidence that some HLHS and BAV are genetically related.

Keywords: genetics, heart valves, cardiovascular malformation

Hypoplastic left heart syndrome (HLHS) (MIM [Mendelian Inheritance in Man] database #241550) is a severe form of cardiovascular malformation (CVM) and continues to be a significant cause of infant mortality and childhood morbidity (1). The frequent occurrence of left- and right-sided valve dysplasia in HLHS probands and the presence of bicuspid aortic valve (BAV) (MIM #109730) in family members suggests that HLHS is a severe form of valve malformation (2). This conclusion is supported by the observation that HLHS is part of the in utero natural history of aortic stenosis (3). Hypoplastic left heart syndrome has been linked with several cytogenetic abnormalities including Turner and Jacobsen syndromes, and heterozygous mutations in NKX2.5 and GJA1 have been reported in a small number of cases (4,5). However, the genetic basis of HLHS remains largely unknown. Several studies have implicated a primary genetic basis and autosomal recessive inheritance has been suggested (6,7). Heritability analysis of HLHS and associated CVM suggests that HLHS is determined largely by genetic effects but exhibits a complex inheritance (2).

Bicuspid aortic valve is the most common CVM and occurs in approximately 1% (0.4% to 2.25%) of the general population (8). Bicuspid aortic valve is a risk factor for aortic valve disease and underlies the diseased valve in the majority of the 100,000 aortic valve replacement procedures performed annually in the U.S. (9). Bicuspid aortic valve is heritable (10) and genetically heterogeneous (11,12) and exhibits complex inheritance (12). Based on studies showing that kindreds ascertained by a proband with either HLHS or BAV are enriched for BAV (2,7,10,13), a genetic relationship between HLHS and BAV has been speculated.

Pedigree analyses have been interpreted as indicating simple Mendelian inheritance of HLHS (6) and BAV (14). Although cardiovascular genetics is steeped in examples of genetic discovery using model-based approaches, such as atrial septal defect (15), the phenomena of genetic heterogeneity, reduced penetrance, and variable expressivity underscore that even what seems to be simple inheritance is complex because genotype does not predict phenotype, that is, a single gene does not explain inheritance. Our results (2,12) and those of others (16,17) have questioned simple Mendelian inheritance of HLHS and BAV, implicating them as complex traits.

The purpose of this study was to identify loci for HLHS and define the genetic relationship between HLHS and BAV. Given the complex inheritance of HLHS and BAV, we performed nonparametric family-based genome-wide linkage analysis. Hypoplastic left heart syndrome linked to human chromosomal regions 10q22 and 6q23 with maximum logarithm of odds (LOD) scores of 3.2 and 3.1, respectively, identifying HLHS as genetically heterogeneous. In the combined HLHS and BAV cohort, a locus on 14q23 exhibited significant linkage (LOD 4.1). Taken together, these findings suggest that some HLHS and BAV are genetically related.

Methods

Study population

Two family-based cohorts were ascertained independently by identifying probands with either HLHS or BAV and were recruited from the Cardiology Clinic at Cincinnati Children's Hospital Medical Center (2,10). A complete medical history and blood sample was obtained from each participant. Cross-sectional 2-dimensional and Doppler transthoracic echocardiography were used in a sequential sampling strategy as previously described (2,10). This protocol was approved by the Institutional Review Board of Cincinnati Children's Hospital Medical Center. Informed consent was obtained from all participants.

Proband inclusion and exclusion criteria

Probands had HLHS or BAV as previously defined (2,10). We used a strict (BAV only or HLHS only) and broad (BAV or HLHS and/or associated CVM) definition of affected status; these phenotypes were defined before the genetic analysis. Patients with complex CVM and left ventricular hypoplasia (e.g., unbalanced atrioventricular septal defect) or a known genetic syndrome (e.g., Turner syndrome or Jacobsen syndrome) were excluded (2).

Genotyping

Methods for whole blood sample collection and deoxyribonucleic acid extraction as well as genotyping (using the ABI Linkage Mapping set version 2.5 MD10 [Applied Biosystems, Carlsbad, California]), allele calling (using GeneMapper software version 3.1 [Applied Biosystems]), and checking for Mendelian inconsistencies (using Infer procedure in PEDSYS [Southwest Foundation for Biomedical Research, San Antonio, Texas]) have been previously described (12). After data cleaning, >95% of genotypes were used in analyses. Marshfield sex-averaged genetic maps were used for marker map positions (18). Allele frequencies were calculated based on participant's genotype data using SOLAR version 4.1 (Sequential Oligogenic Linkage Analysis Routines, San Antonio, Texas).

Linkage analysis

Because HLHS and BAV exhibit complex inheritance and model misspecification limits the power to detect linkage, we used nonparametric methods, which rely on allele sharing rather than inheritance mode (19,20). Sample prevalence was constrained to be equal to the general population prevalence to correct for ascertainment bias. The HLHS prevalence is ~0.02%, and BAV and other CVM are each ~1% (8). Linkage analyses were run using a prevalence of 2% for HLHS, BAV, and associated CVM.

The null hypothesis (no linkage) was tested by comparing the likelihood of this model with that of a linkage model (21). The difference between the log10 likelihoods of these models produces a LOD score, a measure of linkage strength. The SOLAR software was used to adjust LOD scores to ensure they appropriately reflected the p value (22,23). This procedure simulates a fully informative unlinked marker and tests trait linkage at that marker to determine the null LOD score distribution. Empirical p values were estimated as: (number of replicates with LOD equaling or exceeding a specified LOD + 1)/(number of replicates simulated + 1) (24). Ten thousand simulations were performed; the LOD score was adjusted to reflect the underlying null distribution (i.e., LOD 3.0 is equivalent to p = 0.001). Significant linkage was considered present when LOD ≥3.0 and suggestive linkage when LOD ≥2.0.

Confirmation of loci

To confirm loci, we searched for regions that exhibited at least suggestive linkage (LOD ≥2.0) in 1 cohort (HLHS or BAV) and reached a p value of at least 0.05 (LOD ≥1.3) in the other cohort (25,26). Based on current and previous results, we tested for confirmation to loci on chromosomes 5q21, 6q23, 7q31, 10q22, 11q22, 12q13, 13q34, 14q23, 18q22, and 20q11.

Subsets linkage analysis

As multiple loci were identified in both cohorts, we were concerned about locus heterogeneity; therefore, we used subsets linkage analysis to determine whether reducing locus heterogeneity would improve linkage in the combined cohort. To determine regions likely to benefit from subsets analysis, we performed regression analyses (27) on the family-specific LOD scores for all 10 loci that demonstrated suggestive linkage in either cohort. To minimize false positives, we used Bonferroni correction to adjust for the 45 tests performed (p = 0.05/45 = 0.001). Regions with family-specific LODs that exhibited significant associations (p ≤ 0.001) with other regions were selected for subsets analysis.

Subsets linkage analysis was performed in 1 of 2 ways. First, ordered subsets analysis was performed using FLOSS (flexible ordered subset analysis) (28). Ordered subsets analysis uses covariate information to identify a homogenous subset of families that yields increased linkage evidence. Second, subsets were selected based on an a priori weighting scheme. When there was positive association, families exhibiting a family-specific LOD ≥0.1 at the second (predictor) region were given a weight of 1 and all others a weight of 0. When there was a negative association, families exhibiting a family specific LOD ≤0.1 at the second region were given a weight of 1 and all others a weight of 0. This weighting scheme is more conservative than the weighting scheme proposed by Cox et al. (27), which used a cutoff of 0 instead of 0.1. For both methods, significance is assessed using permutation.

Recurrence risk ratio

The BAV recurrence risk ratios (λR) were calculated for first-degree relatives in both HLHS and BAV cohorts. Recurrence risk ratio was defined as: λR = fr/fp, where fr is the number of affected first-degree relatives over the number of first-degree relatives, and fp is the general population BAV prevalence (1%) (29).

Results

Phenotype characterization: HLHS cohort

We evaluated 208 individuals in 33 families identified by an HLHS proband (Table 1). Phenotypes in some families were previously reported (2). In 52% of families, there was more than 1 affected individual. In addition to the 33 probands (24 male, 9 female), 38 other family members had 1 or more CVMs, including 4 with HLHS. Valve abnormalities were common (30%), including 24 with aortic valve abnormalities (13 BAV) and 1 with myxomatous changes and mitral valve prolapse. Of the 3 individuals with a dilated aortic root, 1 also had an aortic valve malformation. Other CVMs were observed in 14 family members, including aortic coarctation (n = 5), ventricular septal defect (n = 3), atrial septal defect (n = 2), fatal unspecified cyanotic CVM as newborn (n = 2), atrioventricular septal defect (n = 1), persistent truncus arteriosus (n = 1), and left superior vena cava (n = 1).

Table 1.

Distribution of HLHS and Associated CVM in 33 Families (208 Individuals)

Male Female Total
HLHS 25 12 37
Valve disease 19 6 25
Aortic valve 18 6 24
BAV 11 2 13
Other abnormality 7 4 11
Mitral valve 1 0 1
Valve surgery 2 1 3
Aortic 1 1 2
Mitral 1 0 1
Aortic root dilation 0 3 3
Other CVM 8 6 14
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In the HLHS cohort, there were 71 affected individuals. The number of phenotypes exceeds the number of individuals because some individuals had more than 1 CVM.

BAV = bicuspid aortic valve; CVM = cardiovascular malformation; HLHS = hypoplastic left heart syndrome.

Phenotype characterization: BAV cohort

In a separate cohort of 102 families identified by a proband with BAV, 805 individuals were evaluated (Table 2). Phenotypes in some families were previously reported (10,12). In 56% of families, there was more than 1 affected individual. Among the 102 probands (73 male, 29 female), BAV was either isolated (n = 69) or combined with aortic coarctation (n = 12), dilated aortic root (n = 10), ventricular septal defect (n = 5), abnormal mitral valve (n = 5), atrial septal defect (n = 3), subaortic stenosis (n = 2), left superior vena cava (n = 2), abnormal tricuspid valve (n = 1), or endocardial fibroelastosis (n = 1). Eighteen probands had a history of aortic valve surgery or balloon valvuloplasty. Among 805 participants, 231 had 1 or more CVMs, including 3 with HLHS. Valve abnormalities were common (63%), including 156 with BAV. An abnormal (e.g., valve thickening, cusp redundancy, or eccentric valve closure [10]) but tricuspid aortic valve was present in 32 individuals. Dilated aortic root was present in 27 individuals, including 13 who also had an abnormal aortic valve. Mitral valve abnormalities were observed in 18 individuals, including 8 who also had an abnormal aortic valve. In some participants, aortic (n = 35) or mitral (n = 5) surgery was performed.

Table 2.

Distribution of BAV and Associated CVM in 102 Families (805 Individuals)

Male Female Total
Valve disease 138 68 206
Aortic valve 130 58 188
BAV 111 45 156
Other abnormality 19 13 32
Mitral valve 8 10 18
Valve surgery 28 11 39
Aortic 27 8 35
Mitral 1 3 4
Aortic root dilation 21 6 27
HLHS 3 0 3
Other CVM 33 17 50
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In the BAV cohort, there were 231 affected individuals. The number of phenotypes exceeds the number of individuals because some individuals had more than 1 CVM.

Abbreviations as in Table 1

Linkage analysis: HLHS cohort

Linkage analyses using the broad (HLHS and associated CVM) phenotype are presented. Linkage analyses using the strict (HLHS only) phenotype demonstrated the same patterns of linkage; however, the identified linkage signals were attenuated and did not reach statistical significance (data not shown). Among 33 HLHS families, 2 significant loci (LOD ≥3) were identified (Fig. 1). The pedigrees of the 10 families contributing to the 2 significant HLHS loci are shown in Figure 2. The maximum LOD scores for significant loci were 3.2 on chromosome 10q22.1 near marker D10S537 and 3.1 on chromosome 6q23.3 near marker D6S292 (Fig. 3). In addition, 5 suggestive linkage peaks (LOD ≥2) were identified on chromosomes 7q31.2 (D7S486; LOD 2.7), 11q22.1 (D11S898; LOD 2.4), 12q13.1 (D12S368; LOD 2.2), 14q23.2 (D14S63; LOD 2.1), and 20q12 (D20S107; LOD 2.0). Two additional suggestive loci were identified on chromosomes 16q24.1 (D16S520; LOD 2.6) and 20q13.33 (D20S173; LOD 2.6); however, enthusiasm for these loci was diminished because the peaks were identified in intervals without flanking markers (e.g., telomeric regions), which are prone to false-positive linkage (30).

Figure 1. Multipoint LOD Scores From Nonparametric Linkage Analyses Across the Genome for HLHS and Associated CVM.

Figure 1

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The x axis designates the chromosomes using proportional spacing. The y axis indicates the nonparametric LOD score. The solid line at LOD 3.0 indicates evidence of significant linkage, and the dotted line at LOD 2.0 indicates evidence of suggestive linkage. Two significant linkage peaks were identified (6q23 and 10q22) (*), and 5 suggestive linkage peaks were identified (7q31, 11q22, 12q13, 14q23, and 20q11) (+). There were 2 additional suggestive linkage peaks identified on telomeric regions (16q24 and 20q13) (arrows). CVM = cardiovascular malformation; HLHS = hypoplastic left heart syndrome; LOD = logarithm of odds.

Figure 2. Pedigrees Depicting 10 Kindreds Contributing to Linkage at Chromosomes 6q23 and 10q22.

Figure 2

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Darkened quadrants indicate phenotype including HLHS (left upper), abnormal aortic valve including bicuspid aortic valve (right upper), aortopathy including dilated aorta (AOD) and coarctation of the aorta (COA) (left lower), and other CVM (right lower). An arrow indicates the proband. An open symbol indicates normal cardiac anatomy and a gray symbol indicates phenotype status unknown. ASD = atrial septal defect; AVSD = atrioventricular septal defect; CCVM = fatal unspecified cyanotic cardiovascular malformation as newborn; LSVC = left superior vena cava; MVA = mitral valve abnormality; VSD = ventricular septal defect; other abbreviations as in Figure 1.

Figure 3. Multipoint LOD Scores From Nonparametric Linkage Analyses in HLHS Kindreds.

Figure 3

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Chromosomes 6q23 (A) and 10q22 (B) exhibit significant linkage. The vertical lines represent the upper and lower bound of the 1-LOD unit support interval. Abbreviations as in Figure 1.

Given the presence of multiple linkage peaks, we examined each family's contribution to the loci. Family-specific LOD scores ranged from -0.21 to 0.91 and -0.37 to 1.2, for chromosomes 6q23.3 and 10q22.1, respectively. Using a cutoff of 0.1 LOD units, 7 of 33 (21%) families contributed to each of the peaks on chromosomes 6 and 10 (Fig. 4). Four families contributed to both peaks, consistent with complex inheritance of HLHS. Taken together, these findings identify 2 significant HLHS loci and demonstrate that HLHS is genetically heterogeneous.

Figure 4. Family-Specific LOD Scores Exceeding 0.1 LOD at Chromosomes 6q23 and 10q22.

Figure 4

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Family identification numbers in the overlapping area of the 2 circles indicate families contributing to both linkage peaks. The table presents only those families contributing at least 0.1 logarithm of odds (LOD) units. Therefore, the family-specific LOD sum will not equal the cohort LOD score. Chr = chromosome.

Linkage analysis and confirmation: BAV cohort

In 38 BAV families, we previously identified linkage to chromosome 18q22, 13q34, and 5q21 (12). The results of the current study including an additional 64 BAV families (total 102 BAV families) did not identify new significant or suggestive loci, but continued to show evidence of linkage to 18q22 and 5q21. Only 1 HLHS locus, 14q23, was confirmed in the BAV cohort (15 BAV families in addition to the original 6 HLHS families). The 1-LOD unit support interval of the BAV cohort (LOD 1.5) overlapped the HLHS locus (Fig. 5A). The BAV loci were not identified in the HLHS cohort.

Figure 5. Linkage to Chromosome 14q23 in the HLHS and BAV Cohorts.

Figure 5

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The original suggestive linkage peak (LOD ≥2) was identified in the HLHS cohort (dashed line) and confirmed (LOD ≥1.3) in the bicuspid aortic valve (BAV) cohort (dotted line) (A). The horizontal arrows represent 1-LOD unit support intervals with the overlapping region shown in yellow. In the combined cohort, linkage at 14q23 (dotted-dashed line) was strengthened using subsets linkage analysis (solid line) (B). NPL = nonparametric logarithm of odds; other abbreviations as in Figure 1.

Association between loci using family-specific LOD scores

Regressing the family-specific LOD scores, we found several statistically associated regions including 14q23 with 13q34 (beta = 0.49 ± 0.10, p < 0.0001) and 5q21 (beta = 0.41 ± 0.09, p < 0.0001), 6q23 with 10q22 (beta = 0.22 ± 0.07, p = 0.001), and 7q31 with 20q12 (beta = 0.27 ± 0.07, p = 0.0001). Association between family-specific LOD scores identifies loci that co-segregate within families providing evidence that information at 1 locus is associated in part with another locus suggesting interaction. These related loci may identify regions in which subsets linkage analysis improves linkage evidence.

Subsets linkage analysis: combined cohort

To reduce the impact of locus heterogeneity, we performed subsets linkage analyses; we limited analysis to regions exhibiting significantly associated family-specific LOD scores to minimize false-positive associations. This approach strengthened linkage at the 14q23 locus. Ordered subsets analysis using either family-specific LOD scores at chromosomes 5 and 13 did not statistically improve the LOD score. However, when we incorporated information from both variables by taking the residuals from a regression of the family-specific LODs at 5 and 13 (n = 10 families), there was a significant improvement in the LOD (p = 0.0001), increasing the LOD from 1.6 in the combined cohort to 3.2. Using the a priori weighting scheme (n = 121 families), there was a significant improvement in the LOD (p < 0.0001) increasing to 4.1 (Fig. 5B).

Genetic relationship between HLHS and BAV

In addition to the shared locus on chromosome 14, the recurrence risk ratio (lambda-R) for BAV is nearly identical in the 2 independently ascertained cohorts. The lambda-R in HLHS families was 8.05, which is consistent with previous findings (2), and was similar to the lambda-R of 8.77 found in BAV families. In addition, 1 HLHS family includes a set of monozygotic twins with discordant phenotypes; 1 twin has HLHS and the other has isolated BAV. These findings, together with the observation of individuals with BAV in the HLHS kindreds and individuals with HLHS in the BAV kindreds (Tables 1 and 2), support a genetic relationship between HLHS and BAV.

Discussion

Using family-based linkage analysis, 2 significant loci on 10q22 and 6q23 were identified providing evidence that nonsyndromic HLHS is genetically heterogeneous. A significant proportion of kindreds (21%) contributed to each locus, suggesting these loci account for a substantial number of HLHS cases. Further, a suggestive HLHS locus on 11q22 has been previously described (31) in a case of HLHS with a balanced translocation t (10;11) (q24;q23), externally validating these analyses. Loci at chromosome 10q22 and 6q23 are gene-rich, containing approximately 300 positional candidate genes. Based on the speculation that HLHS is a severe form of valve malformation (2), we anticipate disease-causing genes to be involved in valvulogenesis. However, because HLHS and BAV exhibit complex inheritance, identifying their genetic basis may require discovering a genetic variation at multiple loci.

To identify genomic regions that encode genes influencing the complex inheritance of HLHS and BAV, we used non-parametric, family-based genome-wide linkage analysis. This approach was chosen rather than population-based association analysis for several reasons. First, several multiplex families were identified, making linkage analysis feasible. Second, with association-based methods, linkage disequilibrium becomes unpredictable with rare single nucleotide polymorphisms. Furthermore, many genes implicated in CVM exhibit different mutations in different families (allelic heterogeneity), for example, NKX2.5 and TBX5 (19). Allelic heterogeneity reduces power to detect associations, further limiting the value of association for genetic studies of CVM. Therefore, linkage analysis was used to search for loci in kindreds identified by HLHS or BAV probands. Like other complex conditions, notably mitral valve prolapse (32), the finding of multiple unconfirmed significant loci may result from phenotypic imprecision or false-positive linkage peaks in addition to locus heterogeneity. Subsets linkage analysis is one strategy to deal with genetic heterogeneity.

The VEGF gene is a growth factor that regulates several endothelial cell functions including proliferation and differentiation, and the vascular endothelial growth factor (VEGF) pathway is an important signaling module in heart valve development (33). Although VEGF is not encoded at any loci identified in this study, several VEGF pathway members are. For example, HIF-1, encoded at 14q23, is a signaling molecule in the VEGF pathway; other VEGF pathway members include genes encoded at 14q23 (PGF, ACTN1, and EIF2) as well as genes encoded at other loci (e.g., VCL at 10q22). Because HLHS and BAV exhibit complex inheritance, identification of the genetic basis of these phenotypes may require a mutation analysis of coding sequence as well as regulatory sequence that may alter expression and/or interaction of pathway members. We speculate that similar consideration may be required for analysis of other genetic discovery methods used to assess these complex traits, such as comparative genomic hybridization and genome-wide association study.

Identification of shared chromosomal loci for HLHS and BAV provides the first direct evidence of a genetic relationship between HLHS and BAV. However, only a limited number of HLHS and BAV families share loci (21 of 135, 16%), and the majority of linkage peaks including the strongest HLHS and BAV loci could not be confirmed in the combined cohort. Although this may reflect power limitations in the current study, this finding suggests that the genetic relationship between all “left-sided” CVMs may not be as strong as has been presumed (17).

Acknowledgments

The authors are grateful to the participating families. The authors thank clinical coordinators Kerry Shooner, MS, CGC, and Wendi Long, as well as the Cincinnati Children's Hospital Genomics Core and Imaging Core for their assistance.

Dr. Hinton received financial support from the Cincinnati Children's Research Foundation and grants (HD43005, HL085122) from the National Institutes of Health. Dr. Benson received a grant (HL069712) from the National Institutes of Health. Drs. Martin, Cripe, and Benson received a grant (HL074728) from the National Institutes of Health.

Abbreviations and Acronyms

BAV

bicuspid aortic valve

CVM

cardiovascular malformation

HLHS

hypoplastic left heart syndrome

LOD

logarithm of odds

VEGF

vascular endothelial growth factor

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