Flaquer et al. BMC Genetics 2013, 14:44 http://www.biomedcentral.com/1471-2156/14/44 RESEARCH ARTICLE Open Access Genome-wide linkage analysis of congenital heart defects using MOD score analysis identifies two novel loci Antònia Flaquer1,2*, Clemens Baumbach1,2, Estefania Piñero3, Fernando García Algas4, María Angeles de la Fuente Sanchez4, Jordi Rosell5, Jorge Toquero6, Luis Alonso-Pulpon6, Pablo Garcia-Pavia6, Konstantin Strauch1,2 and Damian Heine-Suñer5 Abstract Background: Congenital heart defects (CHD) is the most common cause of death from a congenital structure abnormality in newborns and is often associated with fetal loss. There are many types of CHD. Human genetic studies have identified genes that are responsible for the inheritance of a particular type of CHD and for some types of CHD previously thought to be sporadic. However, occasionally different members of the same family might have anatomically distinct defects — for instance, one member with atrial septal defect, one with tetralogy of Fallot, and one with ventricular septal defect. Our objective is to identify susceptibility loci for CHD in families affected by distinct defects. The occurrence of these apparently discordant clinical phenotypes within one family might hint at a genetic framework common to most types of CHD. Results: We performed a genome-wide linkage analysis using MOD score analysis in families with diverse CHD. Significant linkage was obtained in two regions, at chromosome 15 (15q26.3, Pempirical = 0.0004) and at chromosome 18 (18q21.2, Pempirical = 0.0005). Conclusions: In these two novel regions four candidate genes are located: SELS, SNRPA1, and PCSK6 on 15q26.3, and TCF4 on 18q21.2. The new loci reported here have not previously been described in connection with CHD. Although further studies in other cohorts are needed to confirm these findings, the results presented here together with recent insight into how the heart normally develops will improve the understanding of CHD. Keywords: Congenital heart defects, Genetics, Linkage analysis, Genome-wide study Background fects (ASD), ventricular septal defects (VSD), bicuspid Congenital heart defects (CHD) refer to abnormalities in aortic valve (BAV), and Ebstein’s anomaly, among many the heart structure or function that arise at the fetal others. stages and affect approximately 1% of newborns [1]. Normal heart development involves many regula- Multiple surgeries are almost always required to correct tory pathways including receptor-ligand interactions many of the anatomical defects, and quality of life is (JAGGED/NOTCH, TGFB-BMP/TGFBR, VEGF/FLT1- often greatly compromised. There are many types of FLK1, NODAL/ACVRA-ACVRB and RTK/RAS), signal CHD. Examples include transposition of the great ar- transduction (kinases or phosphatases such as MAPK, teries/vessels (TGA/TGV), tetralogy of Fallot (TOF), ERK1/2, calcineurin, or GSK), and transcription factors double outlet right ventricle (DORV), atrial septal de- that determine the expression of cardio-specific genes (protein families with a T-box domain TBX1, TBX5, and * Correspondence: [email protected] TBX20, the GATA family GATA4 and FOG2 or homeobox 1 Institute of Medical Informatics, Biometry, and Epidemiology, Chair of domain NKX2.5 and NKX2.6). Mutations that show a Genetic Epidemiology, Ludwig-Maximilians-Universität, Munich, Germany 2Institute of Genetic Epidemiology, Helmholtz Zentrum München, German greater penetrance and that therefore approximate a Research Center for Environmental Health, Neuherberg, Germany Full list of author information is available at the end of the article © 2013 Flaquer et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Flaquer et al. BMC Genetics 2013, 14:44 Page 2 of 8 http://www.biomedcentral.com/1471-2156/14/44 monogenic inheritance are those affecting transcription ful method for detecting gene effects [18,19]. Especially factors or genes transcribed by them [2]. when looking at rare variants, linkage has the advantage Although the major underlying defects that cause over association of not being prone to allelic heteroge- CHD are thought to be mutations in regulators of heart neity. That is, the combination of many weak association development during embryogenesis, epidemiological data signals obtained in a certain region for single variants by also indicate an environmental influence [2-4]. However, means of collapsing methods is automatically performed these epidemiological studies mostly suggest risk factors in the context of linkage analysis. rather than underlying disease mechanisms. Genetic fac- In this study we performed a detailed genome-wide lin- tors for some of the CHD include Mendelian mutations, kage analysis using MOD scores and 6 families affected by copy number variants, translocations, and single nucleo- different CHD to test for a common genetic background tide polymorphisms (SNPs) [5-7]. among different types of heart defects. A genetic component of CHD diseases was initially im- plicated by recurrence in families, and by studies showing Results and discussion a co-segregation of CHD with the deletion 22q11.2 [8]. It The genome-wide results for MOD scores are plotted in has also been shown that individuals with an affected pa- Figure 1. No significant results were obtained when mo- rent are at twofold greater risk, which is even greater if deling parent-specific heterozygote penetrances, resulting siblings are affected [9]. While many studies have investi- in no evidence of parental genomic imprinting in our set gated the role of genes in the etiology of inherited and of pedigrees. The best results were obtained on chromo- sporadic CHD [10-14] these studies have been focusing some 15 and 18. The results for these two chromosomes on specific cardiac lesions separately. Recently, rare vari- are plotted in Figures 1A and 1B, respectively. The highest ants have also been reported to be associated with CHD scores (MOD = 3.9) were detected on chromosome 18 [15]. These rare variants are usually present in less than (18q21.2-18q21.3; 51197–52023 kb) comprising 57 SNPs 1% of the normal population but are overrepresented in with Pempirical = (0.0005-0.0006), where Pempirical refers to selected patients. They are typically inherited from an the P value obtained after simulations were performed. asymptomatic parent and are therefore not believed to be The associated parametric model is a recessive mode of a sufficient cause of CHD in these patients; other muta- inheritance with incomplete penetrance with a probability tions need to be present to lead to CHD. of 0.27 to be affected for individuals carrying two copies However, occasionally, different members of one family of the disease allele. Incomplete penetrance refers to the are observed to suffer from anatomically distinct defects — failure of individuals who carry the high risk genotype to for example, one member with ASD, one with TOF, and exhibit the trait. This phenomenon is often observed in one with VSD. These apparently discordant clinical phe- complex diseases. Besides different ways of gene action or notypes arising within one family are difficult to ratio- degrees of expression, incomplete penetrance can arise nalize. Benson et al. [16] observed a coinheritance of from further genetic or environmental factors. The second different NKX2.5 mutations, i.e., compound heterozygotes, significant region was at chromosome 15 (15q26.3; in members of families with ASD, VSD, and cardiac con- 99749–99883 kb) comprising 27 SNPs, all of them with a duction abnormalities, suggesting that mutations in the MOD score of 3.5 and Pempirical = (0.0004-0.0005). The same gene can affect different parts of the heart, and best-fitting model points to a recessive mode of inheri- therefore cause different types of CHD. McGregor et al. tance, with a probability of 0.98 to be affected for indivi- [17] reported a statistically significant linkage evidence for duals carrying two copies of the disease allele. It should be a locus on chromosome 14 (within the HOMEZ gene) in noted that the estimate of the disease allele frequency South Indian cases born to consanguineous parents. How- obtained by a MOD-score analysis has the largest variance ever, the linkage finding was not robust in a genetic of all trait-model parameters. Furthermore, in some cases, association follow-up study in a general United States the estimated disease allele frequency will be markedly population. Despite the successes over the past few years, higher than the true value. This is due to the fact that spe- much remains unknown about the genetics of CHD. It is cifying a higher disease allele frequency can compensate believed that much of the missing heritability is likely to for a general model misspecification and hence lead to be due to rare variants with larger effect sizes as well as robustness in a multipoint analysis [20]. epigenetic effects. Their systematic characterization is be- To corroborate these findings, family-based association yond the means of studies that currently rely on linkage analysis was performed in these two significant regions disequilibrium (LD) patterns such as genome-wide associ- with SNPs displaying a MOD >2.5 (P value ≤ 0.01). Using ation analysis. In such situations taking family information this criterion a total number of 304 SNPs (P values bet- into consideration using a direct mapping approach ween 5 × 10-04 and 1 × 10-02) and 211 SNPs (P values bet- complemented by segregation techniques will be a useful ween 4 × 10-04 and 4 × 10-03)wereincludedinthe strategy. For complex diseases linkage analysis is a power- association analysis for 18q21.2-18q21.3 and 15q26.3, Flaquer et al. BMC Genetics 2013, 14:44 Page 3 of 8 http://www.biomedcentral.com/1471-2156/14/44 − ( ) − ( ) Figure 1 Genome-wide results for the MOD score analyses.