Genetic Heterogeneity of Heart-Hand Syndromes
Background Heart-hand syndromes compose a class of combined congenital cardiac and limb deformities. The prototypical heart-hand disorder is Holt-Oram syndrome, which is characterized by cardiac septation defects and radial ray limb deformity. We have recently mapped the Holt-Oram syndrome gene defect to the long arm of human chromosome 12 in two families. The role of this disease locus in the pathogenesis of related conditions such as heart-hand syndrome type III (cardiac conduction disease accompanied by skeletal malformations) or familial atrial septal defects is unknown.
Methods and Results Clinical evaluations and genetic linkage analyses were performed in five additional kindreds with Holt-Oram syndrome and also in one kindred with heart-hand syndrome type III and one kindred with familial atrial septal defect and conduction disease. Holt-Oram syndrome in all five kindreds mapped to chromosome 12q2. These studies and previous data provide odds of greater than 1025:1 that the Holt-Oram syndrome disease gene is at chromosome 12q2. In contrast, neither the phenotypically similar disorder heart-hand syndrome type III nor the locus responsible for a familial atrial septal defect with atrioventricular block maps to chromosome 12q2.
Conclusions We demonstrate that heart-hand syndromes are genetically heterogeneous. Conditions that clinically appear to be partial phenocopies of Holt-Oram syndrome arise from distinct disease genes.
Congenital cardiac and upper-limb malformations frequently occur in association and are classified as heart-hand syndromes. Despite multiple similarities in the clinical presentations of these syndromes, it remains unknown whether hereditary heart-hand syndromes arise from common or distinct genetic defects. A variety of morphological and anatomic criteria have led others to postulate that congenital heart diseases arise from a limited number of shared genetic defects.1 2 Elucidation of the genetic etiologies of these autosomal dominant heart-hand syndromes will improve diagnosis and management of a complex group of disorders with highly variable phenotypic expression.
The most common heart-hand syndrome is the Holt-Oram syndrome3 (MIM No. 142900). Diagnosis is based on skeletal preaxial radial ray abnormalities that may be unilateral and asymmetrical and can vary from subtle, subclinical findings to frank phocomelia. More than 85% of affected individuals also have cardiac malformations that typically include atrial and/or ventricular septal defects and atrioventricular nodal disease. The rare Heart-Hand syndrome Type II (Tabatznik’s syndrome)4 5 has no known etiology and is characterized by upper-limb abnormalities (hypoplastic deltoids; skeletal anomalies in the humeri, radii, ulnae, and thenar bones; and brachydactyly type D) and congenital cardiac arrhythmias (junctional rhythms and atrial fibrillation). The poorly understood Heart-Hand syndrome Type III3 6 (MIM No. 140450) is phenotypically similar and is characterized by cardiac conduction disease (intraventricular delays and sick sinus syndrome). Skeletal malformations are limited to the hands and feet (brachydactyly type C). Septal defects have not yet been identified in patients with Heart-Hand syndrome Type III. Clinical similarities also exist between the heart-hand syndromes and a variety of other less complex autosomal dominant “partial phenocopy” conditions, including familial atrial septal defects (ASDs) with conduction disease3 7 (MIM No. 108900) that occur without limb deformities, and familial limb malformations that occur without cardiac defects.
Identification of the chromosomal location for a disease gene permits studies to ascertain whether phenotypically similar disorders are genetically related. We have previously demonstrated that a disease locus responsible for Holt-Oram syndrome in two families maps to human chromosome 12q2.8 Analyses of five additional families reported here demonstrate that the same genetic locus causes Holt-Oram syndrome in more than 60 affected individuals. To determine if the cardiac and/or limb abnormalities found in other heart-hand syndromes or in partial phenocopy syndromes are also due to a gene defect on chromosome 12q2, we have applied genetic linkage analysis to kindreds affected by Heart-Hand syndrome Type III or familial ASD with conduction disease. (The small size of families with Heart-Hand syndrome Type II/Tabatznik’s syndrome precludes informative linkage analyses for this disorder.) We demonstrate that heart-hand syndromes are genetically heterogeneous, and partial phenocopy disorders arise from mutations at genomic loci distinct from that which causes Holt-Oram syndrome.
Informed consent was obtained from all participants in accordance with the Brigham and Women’s Hospital Committee for the Protection of Human Subjects from Research Risks. Family members were screened without knowledge of genotype status by a thorough history and physical examination, ECG, and transthoracic echocardiography with color-flow Doppler. Families with Holt-Oram syndrome were evaluated as described previously.8 9 All individuals at risk for Heart-Hand syndrome Type III (family C, Fig 1⇓) and selected individuals with familial ASDs and atrioventricular block (family P, Fig 1⇓) had radiographic studies of the upper limbs. Heart-Hand syndrome Type III was diagnosed in family C if an individual had gross or radiographic evidence of hand deformity with or without associated cardiac disease. Individuals in family P were diagnosed as affected on the basis of echocardiographic and ECG evidence of ASD and atrioventricular block.
Genomic DNA was isolated from peripheral lymphocytes as previously described8 or from whole blood with QIAamp columns (Qiagen). Polymorphic short tandem repeats (STRs, also termed microsatellites) were amplified with the polymerase chain reaction (PCR) with published nucleotide primer sequences and analyzed on denaturing polyacrylamide-urea gels as previously described.8
Two-point linkage analyses were performed with mlink (V5.1), and multipoint analyses were performed with linkmap.10 Penetrance was set at 0.95 for all analyses. Multipoint linkage analyses were performed with polymorphic loci D12S84, DAO, and D12S79. The Holt-Oram syndrome locus is flanked by D12S84 and DAO on the centromeric side and D12S79 on the telomeric side. Loci DAO and D12S84 map 5.5 centimorgans (cM) and 6 cM, respectively, centromeric to D12S79.8 Allele frequencies were taken from published data where available and otherwise estimated from at least 30 chromosomes. The homog program was used to test for heterogeneity.10
Holt-Oram Syndrome (Heart-Hand Syndrome Type I)
Evaluations of multigenerational families F, L, M, O, and Q revealed 25 individuals typically affected3 5 8 9 by the autosomal dominant Holt-Oram syndrome. All exhibited upper-arm skeletal anomalies that ranged in severity from subclinical radiographic findings of the radii or thenar bones to frank phocomelia. Cardiac manifestations, when present, included atrial (secundum type) and ventricular septal defects, an atrioventricular canal defect, and various degrees of atrioventricular block.
Heart-Hand Syndrome Type III
Family C (Fig 1⇑) is a Spanish kindred previously described6 by one of us (F.P.) with skeletal and cardiac disease that is inherited as an autosomal dominant trait. Three living affected individuals had brachydactyly type C manifested as hypoplasia of hand metacarpals and phalanges as well as an extra ossicle of the index finger. Individuals II-1 and III-1 also exhibited hypoplasia and aplasia of foot phalanges. Each affected individual also had cardiac conduction system disease: individual II-1 had sick sinus syndrome; individuals III-1 and III-4 had right bundle-branch block. None had any evidence of cardiac septation defects.
ASD With Atrioventricular Block
Family P (Fig 1⇑) is a North American kindred of Anglo-Saxon descent previously described7 by one of us (W.E.P.). ASDs and atrioventricular block are inherited in this family in an autosomal dominant pattern. Eight of 14 affected individuals in this kindred were alive and available for study. All had preoperative first-degree atrioventricular block. Each affected individual, with the exception of individual IV-7, had an ostium secundum ASD that required surgical intervention. In addition, individual V-1 had a ventricular septal defect. There were neither gross nor radiographic skeletal abnormalities in any individuals in family P with cardiac abnormalities.
Analyses of polymorphic STRs (“Methods”) in five families (F, L, M, O, and Q) demonstrated linkage of these Holt-Oram syndromes to the locus previously identified on chromosome 12q2. The homog program provided further evidence of homogeneity (>99% confidence; data not shown). Compilation of these data with previous genetic studies8 yielded a combined multipoint LOD (Logarithm of the Odds) score of 25 (Fig 2⇓) and provided odds of greater than 1025:1 that the Holt-Oram syndrome disease gene is located on chromosome 12q2.
Analyses of polymorphic STRs in family C (Heart-Hand syndrome Type III) and family P (familial ASD with atrioventricular block) excluded linkage to the Holt-Oram syndrome locus. Multipoint LOD scores in each family were less than −2.0 across the entire interval between D12S79 and DAO (Fig 2⇑). Analyses with the homog program indicated that neither Heart-Hand syndrome Type III nor familial ASD with atrioventricular block is likely to be due to mutations in the Holt-Oram syndrome gene on chromosome 12q2 (>99% confidence; data not shown). Because a previous report suggested possible linkage of a gene defect responsible for familial ASDs to the HLA locus on human chromosome 6,11 12 additional studies were performed in family P. LOD scores less than −2.0 were obtained for 10 cM flanking STR D6S105, excluding this as the genomic location of the disease gene in family P.
We demonstrate that two disorders, Heart-Hand syndrome Type III and familial ASD accompanied by atrioventricular conduction abnormalities, are genetically distinct from the Holt-Oram syndrome. While clinically these disorders appear to be partial phenocopies of Holt-Oram syndrome, neither is genetically linked to chromosome 12q2. Combined with previous establishment of genetic loci for isolated preaxial limb deformity,13 14 these data provide clear evidence for genetic heterogeneity among syndromes that share findings of skeletal radial ray deformity, cardiac septation defects, and/or conduction system disease.
Genetic studies in family P clearly excluded the HLA locus on chromosome 611 12 as the cause of both conduction system disease and ASDs. While genetic heterogeneity of ASDs may exist, the previously proposed HLA linkage may reflect a spurious finding based on composite data derived from kindreds in which independent analyses did not provide proof of linkage.
The cardiac and limb malformations that characterize the relatively rare heart-hand syndromes frequently occur in isolation or in the setting of other multiorgan system congenital disorders that are likely to be genetically heterogeneous. For instance, patients with Fanconi’s anemia may be affected by hypoplastic radii in the presence or absence of conotruncal or septal defects.15 However, since the Fanconi anemia gene defect causes increased DNA fragility, cardiac and skeletal deformity may represent direct effects of the Fanconi mutations or secondary effects on other genes such as the Holt-Oram syndrome gene. The cooccurrence of cardiac and limb deformity with genitourinary, hematological, and vascular anomalies in Kaufman-McKusick syndrome, Thrombocytopenia-Absent Radius syndrome, SC Phocomelia, or Roberts syndrome3 suggests at least some of the genes involved in these syndromes have ubiquitous actions or affect early developmental pathways that comprise a regulatory cascade in cardiac and skeletal morphogenesis.
Despite the marked genetic heterogeneity of heart-hand syndromes, genetic heterogeneity of Holt-Oram syndrome itself is at most minimal. Of 22 kindreds that we8 and others16 17 have genetically evaluated, the Holt-Oram syndrome has been shown not to be linked to the chromosome 12q2 locus in only one family. Because Holt-Oram syndrome may manifest as subtly as unilateral asymmetric radiographic carpal bone malformation,3 18 19 Terrett et al’s17 diagnostic criterion of bilateral radial ray defects may account for nonlinkage in this one family. Moreover, it is possible that the unlinked disorder segregating in this family is actually a non–Holt-Oram phenocopy heart-hand syndrome. We conclude that the predominant locus for Holt-Oram syndrome is located on chromosome 12q2.
Several genes, such as homeobox genes, peptide growth factors, and retinoic acid receptors, have been proposed to contribute to cardiac and limb development. Although genomic localization excludes such known genes as the Holt-Oram syndrome gene, each remains an important candidate for the Heart-Hand syndrome Type III and the familial ASD with atrioventricular block syndrome studied here. Ongoing efforts to identify cardiac and limb morphogens will contribute to genetic analysis of all heart and hand syndromes and improve our understanding of normal development as well as congenital malformations.
The authors are indebted to the members of the families, without whose generous assistance these studies would not have been possible, and to Mohammed Miri for technical assistance. This work was supported by grants from the American Heart Association (Dr Basson, Bugher Fellowship), the Wellcome Trust (Dr MacRae), the Peabody Foundation, Inc (Dr Holmes), the Howard Hughes Medical Institute (Drs J.G. and Christine Seidman), and an unrestricted grant from Bristol-Myers Squibb (Drs J.G. and Christine Seidman).
- Received October 31, 1994.
- Revision received January 3, 1995.
- Accepted January 10, 1995.
- Copyright © 1995 by American Heart Association
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