Mutations in human TBX5 [corrected] cause limb and cardiac malformation in Holt-Oram syndrome.

Holt-Oram syndrome is characterized by upper limb malformations and cardiac septation defects. Here, we demonstrate that mutations in the human TBX5 gene underlie this disorder. TBX5 was cloned from the disease locus on human chromosome 12q24.1 and identified as a member of the T-box transcription factor family. A nonsense mutation in TBX5 causes Holt-Oram syndrome in affected members of one family; a TBX5 missense mutation was identified in affected members of another. We conclude that TBX5 is critical for limb and heart development and suggest that haploinsufficiency of TBX5 causes Holt-Oram syndrome.

A gene defect that causes conduction system disease and dilated cardiomyopathy maps to chromosome 1p1-1q1.

Longitudinal evaluation of a seven generation kindred with an inherited conduction system defect and dilated cardiomyopathy demonstrated autosomal dominant transmission of a progressive disorder that both perturbs atrioventricular conduction and depresses cardiac contractility. To elucidate the molecular genetic basis for this disorder, a genome-wide linkage analysis was performed. Polymorphic loci near the centromere of chromosome 1 demonstrated linkage to the disease locus (maximum multipoint lod score = 13.2 in the interval between D1S305 and D1S176). Based on the disease phenotype and map location we speculate that gap junction protein connexin 40 is a candidate for mutations that result in conduction system disease and dilated cardiomyopathy.

Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events.

Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.

Congenital heart disease caused by mutations in the transcription factor NKX2-5.

Mutations in the gene encoding the homeobox transcription factor NKX2-5 were found to cause nonsyndromic, human congenital heart disease. A dominant disease locus associated with cardiac malformations and atrioventricular conduction abnormalities was mapped to chromosome 5q35, where NKX2-5, a Drosophila tinman homolog, is located. Three different NKX2-5 mutations were identified. Two are predicted to impair binding of NKX2-5 to target DNA, resulting in haploinsufficiency, and a third potentially augments target-DNA binding. These data indicate that NKX2-5 is important for regulation of septation during cardiac morphogenesis and for maturation and maintenance of atrioventricular node function throughout life.

Mutations in the protein kinase A R1alpha regulatory subunit cause familial cardiac myxomas and Carney complex.

Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1alpha gene encoding the R1alpha regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1alpha frameshift mutations in three unrelated families result in haploinsufficiency of R1alpha and cause Carney complex. We did not detect any truncated R1alpha protein encoded by mutant PRKAR1alpha. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1alpha deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1alpha alleles and that wild-type R1alpha protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1alpha to R2beta regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1alpha haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation.

Familial Hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome maps to a locus on chromosome 7q3.

We have mapped a disease locus for Wolff-Parkinson-White syndrome (WPW) and familial hypertrophic cardiomyopathy (FHC) segregating in a large kindred to chromosome 7 band q3. Although WPW syndrome and FHC have been observed in members of the same family in prior studies, the relationship between these two diseases has remained enigmatic. A large family with 25 surviving individuals who are affected by one or both of these conditions was studied. The disease locus is closely linked to loci D7S688, D7S505, and D7S483 (maximum two point LOD score at D7S505 was 7.80 at theta = 0). While four different FHC loci have been described this is the first locus that can be mutated to cause both WPW and/or FHC.

Identification of a chromosome 11q23.2-q24 locus for familial aortic aneurysm disease, a genetically heterogeneous disorder.

BACKGROUND: Aortic aneurysms cause significant mortality, and >20% relate to hereditary disorders. Familial aortic aneurysm (FAA) has been described in such conditions as the Marfan and Ehlers-Danlos type IV syndromes, due to defects in the fibrillin-1 and type III procollagen genes, respectively. Other gene defects that cause isolated aneurysms, however, have not thus far been described. METHODS AND RESULTS: We studied 3 families affected by FAA. No family met the diagnostic criteria for either Marfan or Ehlers-Danlos syndrome. Echocardiography defined involvement of both the thoracic and abdominal aorta. In family ANA, candidate gene analysis excluded linkage to loci associated with aneurysm formation, including fibrillin-1, fibrillin-2, and type III procollagen, and chromosome 3p24.2-p25. Genome-wide linkage analysis identified a 2.3-cM FAA locus (FAA1) on chromosome 11q23.3-q24 with a maximum multipoint logarithm of the odds score of 4.4. In family ANB, FAA was linked to fibrillin-1. In family ANF, however, FAA was not linked to any locus previously associated with aneurysm formation, including fibrillin-1 and FAA1. CONCLUSIONS: FAA disease is genetically heterogeneous. We have identified a novel FAA locus at chromosome 11q23.3-q24, a critical step toward elucidating 1 gene defect responsible for aortic dilatation. Future characterization of the FAA1 gene will enhance our ability to achieve presymptomatic diagnosis of aortic aneurysms and will define molecular mechanisms to target therapeutics.

Familial thoracic aortic aneurysms and dissections: genetic heterogeneity with a major locus mapping to 5q13-14.

BACKGROUND: Aneurysms and dissections affecting the ascending aorta are associated primarily with degeneration of the aortic media, called medial necrosis. Families identified with dominant inheritance of thoracic aortic aneurysms and dissections (TAA/dissections) indicate that single gene mutations can cause medial necrosis in the absence of an associated syndrome. METHODS AND RESULTS: Fifteen families were identified with multiple members with TAAs/dissections. DNA from affected members from 2 of the families was used for a genome-wide search for the location of the defective gene by use of random polymorphic markers. The data were analyzed by the affected-pedigree-member method of linkage analysis. This analysis revealed 3 chromosomal loci with multiple markers demonstrating evidence of linkage to the phenotype. Linkage analysis using further markers in these regions and DNA from 15 families confirmed linkage of some of the families to 5q13-14. Genetic heterogeneity for the condition was confirmed by a heterogeneity test. Data from 9 families with the highest conditional probability of being linked to 5q were used to calculate the pairwise and multipoint logarithm of the odds (LOD) scores, with a maximum LOD of 4.74, with no recombination being obtained for the marker D5S2029. In 6 families, the phenotype was not linked to the 5q locus. CONCLUSIONS: A major locus for familial TAAs and dissections maps to 5q13-14, with the majority (9 of 15) of the families identified demonstrating evidence of linkage to this locus. The condition is genetically heterogeneous, with 6 families not demonstrating evidence of linkage to any loci previously associated with aneurysm formation.

The evolving role of statins in the management of atherosclerosis.

Significant advances in the management of cardiovascular disease have been made possible by the development of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors--"statins." Initial studies explored the impact of statin therapy on coronary artery disease (CAD) progression and regression. Although the angiographic changes were small, associated clinical responses appeared significant. Subsequent large prospective placebo-controlled clinical trials with statins demonstrated benefit in the secondary and primary prevention of CAD in subjects with elevated cholesterol levels. More recently, the efficacy of statins has been extended to the primary prevention of CAD in subjects with average cholesterol levels. Recent studies also suggest that statins have benefits beyond the coronary vascular bed and are capable of reducing ischemic stroke risk by approximately one-third in patients with evidence of vascular disease. In addition to lowering low-density lipoprotein (LDL) cholesterol, statin therapy appears to exhibit pleiotropic effects on many components of atherosclerosis including plaque thrombogenicity, cellular migration, endothelial function and thrombotic tendency. Growing clinical and experimental evidence indicates that the beneficial actions of statins occur rapidly and yield potentially clinically important anti-ischemic effects as early as one month after commencement of therapy. Future investigations are warranted to determine threshold LDL values in primary prevention studies, and to elucidate effects of statins other than LDL lowering. Finally, given the rapid and protean effects of statins on determinants of platelet reactivity, coagulation, and endothelial function, further research may establish a role for statin therapy in acute coronary syndromes.

Atrial form and function: lessons from human molecular genetics.

Molecular genetic analyses of human hereditary disorders that affect cardiac atrial structure and function have recently identified several genes that regulate atrial morphogenesis. Mutations of the TBX5, NKX2.5, EVC, and PRKAR1 alpha genes all result in abnormalities of human atrial growth and development, and mutations in at least one gene results in familial atrial fibrillation and is as yet unidentified. Ongoing studies to find interactions between these transcription factors and intracellular signaling molecules and other as yet unknown genes are establishing critical pathways in human cardiogenesis. Human investigation and experimental animal models of heart development synergize to elucidate etiologies of common congenital heart disease.

Carney complex, Peutz-Jeghers syndrome, Cowden disease, and Bannayan-Zonana syndrome share cutaneous and endocrine manifestations, but not genetic loci.

Carney complex (CC), Peutz-Jeghers syndrome (PJS), Cowden disease (CD), and Bannayan-Zonana syndrome (BZS) share clinical features, such as mucocutaneous lentigines and multiple tumors (thyroid, breast, ovarian, and testicular neoplasms), and autosomal dominant inheritance. A genetic locus has been identified for CC on chromosome 2 (2p16), and the genes for PJS, CD, and BZS were recently identified; genetic heterogeneity appears likely in both CC and PJS. The genes for PJS and CD/BZS, STK11/LKB1 and PTEN, respectively, may act as tumor suppressors, because loss of heterozygosity (LOH) of the PJS and CD/BZS loci has been demonstrated in tumors excised from patients with these disorders. We studied 2 families with CC in whom the disease could not be shown to segregate with polymorphic markers from the 2p16 locus. Their members presented with lesions frequently seen in PJS and the other lentiginosis syndromes. We also tested 16 tumors and cell lines established from patients with CC for LOH involving the PJS and CD/BZS loci. DNA was extracted from peripheral blood, tumor cell lines, and tissues and subjected to PCR amplification with primers from microsatellite sequences flanking the STK11/LKB1 and PTEN genes on 19p13 and 10q23, respectively, and a putative PJS locus on 19q13. All loci were excluded as candidates in both families with LOD scores less than 2 and/or by haplotype analysis. LOH for these loci was not present in any of the tumors that were histologically identical to those seen in PJS. The overall rate of LOH for the PJS and CD/BZS loci in tumors from patients with CC was less than 10%. We conclude that despite substantial clinical overlap among CC, PJS, CD, and BZS, LOH for the STK11 and PTEN loci is an infrequent event in CC-related tumors. Linkage analysis excluded the PJS and CD/BZS loci on chromosomes 19 (19p13 and 19q13) and 10 (10q23) from harboring the gene defect(s) responsible for the phenotype in these 2 families.

Genetic heterogeneity of familial atrial myxoma syndromes (Carney complex).

Familial atrial myxomas may present as a component of the Carney complex, an autosomal dominant multiple neoplasia and lentiginosis syndrome. Although a gene defect for the Carney complex in some families maps to chromosome 2p, this syndrome is genetically heterogeneous, and other genetic loci also produce familial atrial myxomas.

Molecular determinants of atrial and ventricular septal defects and patent ductus arteriosus.

Septation defects and patent ductus arteriosus are the most common human cardiovascular malformations (CVMs). Genetic factors play a major part in the origin of these malformations. Recent molecular analyses have shed light on several mendelian forms. In the autosomal dominant Holt-Oram syndrome, both atrial and ventricular septal defects are inherited in association with limb deformity as a result of mutations in the gene encoding the TBX5 transcription factor. Mutations in the NKX2.5 transcription factor gene cause autosomal dominant familial atrial septal defects in association with progressive atrioventricular block as well as complex congenital heart disease. Common atrial syndromes in autosomal dominant Ellis-van Creveld syndrome arise in the context of axial skeletal and limb malformation as a result of mutations in the EVC gene, whose function is unknown. Patent ductus arteriosus occurs in several syndromic forms of congenital heart disease, including Holt-Oram syndrome. Recent analyses of autosomal dominant Char syndrome, which includes, with variable penetrance, patent ductus arteriosus as well as craniofacial and hand malformations, have shown that the syndrome is caused by mutations in the TFAP2B transcription factor gene. Ongoing analyses are poised to determine the contribution of these genes as well as others yet to be identified to common, sporadic forms of congenital heart disease.

Molecular genetic diagnosis of the familial myxoma syndrome (Carney complex).

We describe an individual in whom molecular genetic testing provided a diagnosis of the Carney complex, an autosomal dominant syndrome comprising cutaneous and cardiac myxomas, spotty pigmentation of the skin, and endocrinopathy. Recently, we localized the Carney complex disease gene to chromosome region 17q2. Our patient was a member of a family segregating the Carney complex, but was not, himself, initially thought to be affected. Haplotype analysis based on genotyping studies with 17q2 microsatellites predicted that this individual was, in fact, affected by Carney complex and was at risk for development of myxomas. Further clinical evaluation and re-review of prior pathologic studies, then, confirmed the DNA-based diagnosis. This report highlights the difficulty in establishing a diagnosis of Carney complex based on clinical and pathologic findings alone, and we suggest that molecular genetic analyses provide an important diagnostic method for this familial myxoma syndrome.

Do statins afford neuroprotection in patients with cerebral ischaemia and stroke?

An emerging body of evidence indicates that beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or 'statins', provide neuroprotection in addition to reducing ischaemic stroke. Statins reduce the incidence of ischaemic stroke by stabilising atherosclerotic plaques in the precerebral vasculature and through antithrombotic actions, and the neuroprotective effects of statins may confer significant clinical benefit. Some of these neuroprotective effects are likely to be cholesterol independent and mediated by the interruption of isoprenoid biosynthesis. Therapy with statins may modulate endothelial function and preserve blood flow to regions exposed to an ischaemic insult. In particular, statin-mediated preservation of endothelial nitric oxide synthase activity in cerebral vasculature, especially in the ischaemic penumbra, may limit neurological deficit. Moreover, putative anti-inflammatory and antioxidant properties of statins may confer additional neuroprotection. Further large clinical trials are necessary to address the role of statin therapy in the primary prevention of stroke, small vessel cerebrovascular disease and vascular dementia.

Advances in the molecular genetics of congenital structural heart disease.

Molecular genetic analyses have generated significant advances in our understanding of congenital heart disease. Techniques of genetic mapping with polymorphic microsatellites and fluorescence in situ hybridization (FISH) have provided informative tools for localization and identification of disease genes. Some cardiovascular diseases have proven to result from single gene defects. Others relate to more complex etiologies involving several genes and their interactions. Elucidation of the molecular genetic etiologies of congenital heart disease prompts consideration of DNA testing for cardiac disorders. Future integration of these diagnostic modalities with improved treatments may ultimately decrease morbidity and mortality from congenital heart diseases.

New understandings in the genetics of congenital heart disease.

In spite of the amazing success during the past half century in diagnosis and treatment of congenital heart disease, very little is known of its cause. However, a genetic cause has been clearly established for many forms of cardiovascular disease, and new understandings in the molecular genetics of congenital heart disease provide further insight. Progress has been quite impressive for some cardiovascular abnormalities, whereas in other areas the findings are more preliminary. For example, the molecular genetic cause of supravalvular aortic stenosis and the heart disease associated with Marfan syndrome has been clearly established. Impressive progress has been made in conotruncal defects, Holt-Oram syndrome, Alagille syndrome, and total anomalous pulmonary venous connection. In other areas, such as patent ductus arteriosus and atrioventricular septal defect, the findings are more preliminary. Taken as a whole, the prospect of understanding the genetic basis of congenital heart disease has never been better. Understanding the cause of congenital heart disease will provide new insights into both normal cardiac development and the pathophysiologic basis of congenital heart defects.