GATA4 sequence variants in patients with congenital heart disease.

BACKGROUND: Recent reports have identified mutations in the transcription factor GATA4 in familial cases of cardiac septal defects. The prevalence of GATA4 mutations in the population of patients with septal defects is unknown. Given that patients with septal and conotruncal defect can share a common genetic basis, it is unclear whether patients with additional types of CHD might also have GATA4 mutations. AIMS: To explore these questions by investigating a large population of 628 patients with either septal or conotruncal defects for GATA4 sequence variants. METHODS: The GATA4 coding region and exon-intron boundaries were investigated for sequence variants using denaturing high-performance liquid chromatography or conformation-sensitive gel electrophoresis. Samples showing peak or band shifts were reamplified from genomic DNA and sequenced. RESULTS: Four missense sequence variants (Gly93Ala, Gln316Glu, Ala411Val, Asp425Asn) were identified in five patients (two with atrial septal defect, two with ventricular septal defect and one with tetralogy of Fallot), which were not seen in a control population. All four affected amino acid residues are conserved across species, and two of the sequence variants lead to changes in polarity. Ten synonymous sequence variants were also identified in 18 patients, which were not seen in the control population. CONCLUSIONS: These data suggest that non-synonymous GATA4 sequence variants are found in a small percentage of patients with septal defects and are very uncommonly found in patients with conotruncal defects.

Marfan phenotype variability in a family segregating a missense mutation in the epidermal growth factor-like motif of the fibrillin gene.

To examine the associations among fibrillin gene mutations, protein function, and Marfan syndrome phenotype, we screened for alterations in the fibrillin coding sequence in patients with a range of manifestations and clinical severity. A cysteine to serine substitution at codon 1409 (C1409S) was identified in an epidermal growth factor (EGF)-like motif from one fibrillin allele which segregates with the disease phenotype through three generations of a family affected with the Marfan syndrome. This alteration was not observed in 60 probands from other families or in 88 unrelated normal individuals. The altered cysteine is completely conserved in all EGF-like motifs identified in fibrillin, and in all proteins that contain this motif. These observations strongly indicate that C1409S is the disease-producing mutation in this family. The phenotype of individuals carrying C1409S varied widely with respect to onset of disease, organ-system involvement, and clinical severity; certain affected adults were unaware of their status before being diagnosed through this investigation. We conclude that fibrillin gene defects cause familial Marfan syndrome, that mutations in the EGF-like motif of the fibrillin gene are not uniformly associated with severe disease, and that fibrillin genotype is not the sole determinant of Marfan phenotype.

The molecular basis of Marfan syndrome.

The Marfan syndrome is an inherited, autosomal dominant disorder that affects the skeletal, ocular, and cardiovascular systems. Recent biochemical and genetic studies have demonstrated that this deadly genetic disorder arises from defects in the connective tissue protein fibrillin. Fibrillin is a component of microfibrils, structures found in the extracellular matrices of most tissues, including those affected in Marfan patients. The appearance of microfibrils in the matrix produced by Marfan patient fibroblasts is different from that of normal cells. Genetic linkage between the fibrillin gene and the Marfan phenotype has been established and the gene mapped to the same chromosomal position as the disease locus. In several instances, the disease has been associated with mutations in the fibrillin gene, confirming that defects in fibrillin cause the Marfan syndrome.

A simple PCR-based assay allows detection of a common mutation, IVS8-1G-->C, in DHCR7 in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive multiple malformation disorder. A deficiency of the enzyme 7-dehydrocholesterol delta 7-reductase (DHCR7) is the primary abnormality in SLOS. The gene encoding DHCR7 has been cloned, and we have identified a mutation affecting the splice acceptor site 5' of exon 9 that occurs frequently in affected individuals. We developed a novel PCR-based assay to detect this common mutation in DHCR7. Using this assay, heterozygosity was detected for this mutation in 18 of 26 and homozygosity in 1 of 26 unrelated affected individuals. The high frequency of this mutation is suggestive of either a founder effect in our group of patients or a mutational hotspot. The simplicity and reliability of this assay will allow it to be used as a clinical test to aid in diagnosis of atypical cases, in carrier testing, in prediction of prognosis based on genotype, and in prenatal molecular genetic diagnostic testing.

PTPN11 mutations play a minor role in isolated congenital heart disease.

PTPN11 missense mutations cause approximately 50% of Noonan syndrome, an autosomal dominant disorder presenting with various congenital heart defects, most commonly valvar pulmonary stenosis, and hypertrophic cardiomyopathy. Atrioventricular septal defects and coarctation of the aorta occur in 15% and 9%, respectively. The aim of this study was to determine if PTPN11 mutations exist in non-syndromic patients with these two relevant forms of congenital heart disease. The 15 coding PTPN11 exons and their intron boundaries from subjects with atrioventricular septal defects (n = 24) and coarctation of the aorta (n = 157) were analyzed using denaturing high performance liquid chromatography and sequenced if abnormal. One subject with an atrioventricular septal defect but no other known medical problems had a c.127C > T transition in exon 2, predicting a p.L43F substitution. This mutation affected the phosphotyrosine-binding region in the N-terminal src homology 2 domain and was close to a Noonan syndrome mutation (p.T42A). An otherwise healthy patient with aortic coarctation had a silent c.540C > T change in exon 5 corresponding to p.D180D. Our study showed that PTPN11 mutations are rarely found in two isolated forms of congenital heart disease that commonly occur in Noonan syndrome. The p.L43F mutation belongs to a rare class of PTPN11 mutations altering the phosphotyrosine-binding region. These mutations are not predicted to alter the autoinhibition of the PTPN11 protein product, SHP-2, which is the mechanism for the vast majority of mutations causing Noonan syndrome. Future studies will be directed towards understanding these rare phosphotyrosine binding region mutants.

The oxidation of serum sulph-hydryl groups by hydrogen peroxide secreted by stimulated phagocytic cells in rheumatoid arthritis.

Levels of free sulph-hydryl (SH) groups are depressed in the sera of patients with rheumatoid arthritis, especially during active disease. However, the mechanism underlying this effect is not known. We have investigated several oxidative species generated during the inflammatory process for their ability to react with serum SH in vitro. Our results show that serum oxidase enzymes (e.g. caeruloplasmin) do not have this activity but that "active oxygen species" generated either by an enzymatic reaction (xanthine plus xanthine oxidase) or by neutrophils stimulated with heat-aggregated IgG cause rapid oxidation of serum SH groups. The use of selective inhibitors of active oxygen species has demonstrated that this reaction is mediated by hydrogen peroxide. This compound is secreted in considerable amounts by activated phagocytic cells, especially neutrophils. Thus, serum SH levels may reflect phagocytic activity in patients with rheumatoid arthritis. We suggest that serum SH groups act as important extracellular scavengers of peroxides and so help to protect cells from damage by these molecules.

Occurrence of a mutation associated with Wolman disease in a family with cholesteryl ester storage disease.

Cholesteryl ester storage disease (CESD) and Wolman disease (McKusick 278000) are two distinct autosomal recessive disorders, both attributable to a severe reduction in acid cholesteryl ester hydrolase/lysosomal acid lipase activity (EC 3.1.1.13). We have identified compound heterozygous mutations in a family with two siblings affected with CESD. Molecular genetic analysis revealed two mutations one of which has previously been seen only in Wolman disease. Analysis of these mutations acting in concert provides new insight into the correlation of genotype with phenotype in these allelic disorders.

Calcium binding, hydroxylation, and glycosylation of the precursor epidermal growth factor-like domains of fibrillin-1, the Marfan gene protein.

The extracellular matrix protein fibrillin-1 is a major component of elastic microfibrils, which are complex assemblies of several proteins and are found in most connective tissues, frequently associated with elastin. Fibrillin-1 contains 43 precursor epidermal growth factor-like (pEGF) domains that have a consensus sequence for calcium binding. The calcium binding potential of a fibrillin-1 pepsin fragment (PF2) was quantitatively analyzed using microvolume equilibrium dialysis. Peptide sequence data and pepsin fragment size determination indicate that PF2 contains seven pEGF domains, each with the calcium binding consensus sequence. Scatchard plot analysis of the calcium binding data shows that PF2 has six to seven high affinity binding sites with a Kd = 250 microM at pH 7.5. There is a second overlapping consensus sequence in the pEGF domains for beta-hydroxylation of a specific Asp/Asn residue. Five partially hydroxylated Asn residues have been identified by protein sequence analysis of fibrillin-1 fragments. This is the first demonstration of this modification in a connective tissue protein. The calcium binding consensus sequence also contains a conserved Ser residue with an apparently novel modification, which causes the Ser residue to behave like an Asp residue during protein sequencing. Marfan syndrome, a heritable disorder of connective tissue, is known to be associated with mutations in the FBN1 gene. Most of these mutations have been found in pEGF domains, frequently substituting Cys for another amino acid, destroying the pEGF motif secondary structure along with its calcium binding potential. Other mutations cause the substitution of single amino acids in the calcium binding consensus sequence, which could affect calcium binding but also the hydroxylation of Asp/Asn residues or the modification of Ser residues.

Angiotensin-converting enzyme and cardiovascular disease risk.

The published studies of the association of the angiotensin-converting enzyme (ACE) genotype with cardiovascular disease have used many different diagnostic criteria for cardiovascular disease and have drawn their samples from different patient groups and different populations. This review examines the association of the ACE DD genotype with cardiovascular disease risk in studies grouped by their case criterion, the geographical region of the population samples, and by the cardiovascular risk level of the patient sample. In studies where the underlying odds ratios are determined to be homogeneous, the overall odds ratios for myocardial infarction and coronary artery disease with regard to the ACE DD genotype are estimated using the Mantel-Haenszel method.

Type VI collagen anchors endothelial basement membranes by interacting with type IV collagen.

Type VI collagen filaments are found associated with interstitial collagen fibers, around cells, and in contact with endothelial basement membranes. To identify type VI collagen binding proteins, the amino-terminal domains of the alpha1(VI) and alpha2(VI) chains and a part of the carboxyl-terminal domain of the alpha3(VI) chain were used as bait in a yeast two-hybrid system to screen a human placenta library. Eight persistently positive clones were identified, two coding the known matrix proteins fibronectin and basement membrane type IV collagen and the rest coding new proteins. The amino-terminal domain of alpha1(VI) was shown to interact with the carboxyl-terminal globular domain of type IV collagen. The specificity of this interaction was further studied using the yeast two-hybrid system in a one-on-one format and confirmed by using isolated protein domains in immunoprecipitation, affinity blots, and enzyme-linked immunosorbent assay-based binding studies. Co-distribution of type VI and type IV collagens in human muscle was demonstrated using double labeling immunofluorescent microscopy and immunoelectron microscopy. The strong interaction of type VI collagen filaments with basement membrane collagen provided a possible molecular pathogenesis for the heritable disorder Bethlem myopathy.

Localization of the fibrillin (FBN) gene to chromosome 15, band q21.1.

Fibrillin (FBN), a large extracellular matrix glycoprotein, is an important component of structures called microfibrils. Because fibrillin microfibrils appear to be abnormal in patients with the Marfan syndrome, fibrillin is a candidate for the gene defect in the Marfan syndrome. Derived clones from fibrillin cDNA were used as probes in isotopic and nonisotopic in situ hybridization studies to map the chromosomal location of the fibrillin gene. Fluorescent signals were found on chromosome 15 band q21.1; an excess of silver grains was noted over a similar region of chromosome 15 following in situ hybridization with a tritium-labeled probe. These results are consistent with linkage studies that localize the Marfan gene to chromosome 15.

Angiotensin-converting enzyme DD genotype and cardiovascular disease in heterozygous familial hypercholesterolemia.

BACKGROUND: Controversy exists as to whether the deletion/deletion genotype (DD) of the ACE gene polymorphism increases the risk of myocardial infarction (MI). Studies have suggested that the ACE DD genotype is associated with increased plaque instability. We hypothesized that the ACE DD genotype may increase the risk of myocardial infarction and coronary heart disease (CHD) in patients with heterozygous familial hypercholesterolemia (FH) or familial defective apolipoprotein B-100 (FDB) who, as a group, are at high risk of having lipid-rich plaques in their coronary arteries. METHODS AND RESULTS: We determined the ACE genotypes and incidence of MI or surgical intervention for CHD in 213 adult patients with heterozygous FH or FDB. The incidence of MI in 35 male patients who carried the ACE DD genotype was 2.5 times that observed in male patients with the II or DI genotypes, and the incidence of CHD in male patients with the DD genotype was 2.2 times higher than in those who had ACE DI+II. The potential effects of ACE genotype on CHD could not be directly compared in female patients because of a disparity in the smoking history of the genotypic groups. From logistic regression analysis, the estimated odds ratio associated with the ACE DD genotype was 2.57 for MI and 2.21 for CHD adjusted for age, sex, and smoking history. CONCLUSIONS: The ACE DD genotype is associated with an increased risk of MI and CHD in patients with heterozygous FH or FDB. Determination of the ACE genotype in asymptomatic FH and FDB patients provides an additional means to identify those patients at greatest risk for the premature development of CHD.

Enhanced oxidative metabolism of neutrophils from patients with systemic sclerosis.

Pathological features of systemic sclerosis (SS) such as endothelial cell injury may be associated with damage caused by active oxygen species, especially peroxides. Neutrophils from SS patients express more Fc(IgG) receptors and generate significantly more peroxide than control cells when cultured alone or with heat-aggregated IgG, but not with F-Met-Leu-Phe. We suggest that circulating neutrophils are activated in SS and may, through secretion of toxic peroxides, contribute to the early vascular pathology of this disease.

Seven polymorphic loci mapping to human chromosomal region 11q22-qter.

Seven polymorphic loci that map to human chromosomal region 11q22-qter are revealed by DNA probes isolated from a chromosome-specific phage library constructed from a human X mouse somatic cell hybrid that has retained an 11q;16q translocation as the only human DNA. Three probes, each of which reveals a two-allele polymorphism, and four probes, each of which detects two linked RFLPs, have been characterized. Using a somatic cell hybrid mapping panel that divides 11q into four discrete sections, the seven clones have been localized to specific chromosomal regions. Localization of one of the clones has been confirmed and refined by in situ hybridization.

Partial sequence of a candidate gene for the Marfan syndrome.

Fibrillin is a large (relative molecular mass 350,000) glycoprotein which can be isolated from fibroblast cell cultures and is a component of the microfibrils that are ubiquitous in the connective tissue space. The microfibrils of the suspensory ligament of the lens as well as the elastic fibre microfibrils of the blood vessel wall are composed of fibrillin. The ocular and cardiovascular manifestations of the Marfan syndrome are consistent with a defect in the gene coding for a structural constituent of these connective tissues. Immunohistological experiments have recently implicated fibrillin microfibrils in the pathogenesis of the Marfan syndrome. Genetic linkage data localizing the Marfan gene to chromosome 15 and the in situ hybridization of fibrillin complementary DNA to 15q21.1 together support fibrillin as a candidate Marfan gene. As a first step towards investigating the function of fibrillin in the architecture and development of connective tissues and its relationship to the Marfan syndrome, we report the cloning and partial sequencing of fibrillin cDNA.

Carrier frequency of the common mutation IVS8-1G>C in DHCR7 and estimate of the expected incidence of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a multiple congenital anomaly/mental retardation syndrome of variable severity with an incidence previously estimated at 1 in 20,000-60,000 based on case frequency surveys. Identification of the gene defect in SLOS has made it possible to calculate the carrier frequency and estimate disease incidence using molecular methods to identify carriers. Using a previously described PCR-RFLP assay we screened 1503 anonymous blood samples from random newborn screening blood spot cards for the presence of the common SLOS mutation IVS8-1G>C in order to determine the carrier frequency. Sixteen carriers were identified in the 1503 samples. Since the frequency of the IVS8-1G>C mutation among all SLOS gene mutations is known, the overall carrier frequency for all mutations can be calculated. The calculated carrier frequency for all mutations based on this result is 1 in 30, predicting an SLOS incidence of 1 in 1590 to 1 in 13,500. The current incidence estimate may, therefore, significantly underestimate the true incidence of SLOS. This discrepancy between calculated and observed incidence could be due to undiagnosed mild cases, misdiagnosed severe cases, death prior to diagnosis, or fetal loss. More comprehensive incidence studies are needed to determine if SLOS is as common as predicted by the very high (1 in 30) carrier frequency determined in this study.

Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene.

Marfan syndrome is an inherited disorder of connective tissue manifested in the ocular, skeletal and cardiovascular systems. It is inherited as an autosomal dominant with high penetrance, but has great clinical variability. Linkage studies have mapped the Marfan locus to chromosome 15q15-21.3. There have been no reports of genetic heterogeneity in the syndrome. Following the identification of fibrillin (a glycoprotein component of the extracellular microfibril), immunohistopathological quantification of the protein in skin and fibroblast culture, and examination of fibrillin synthesis, extracellular transport, and incorporation into the extracellular matrix (D. M. Milewicz, R.E.P., E. S. Crawford and P. H. Byers, manuscript in preparation) have demonstrated abnormalities of fibrillin metabolism in most patients. A portion of the complementary DNA encoding fibrillin has been cloned and mapped by in situ hybridization to chromosome 15. Here we report that the fibrillin gene is linked to the Marfan phenotype (theta = 0.00; logarithm of the odds (lod) = 3.9) and describe a de novo missense mutation in the fibrillin gene in two patients with sporadic disease. We thus implicate fibrillin as the protein defective in patients with the Marfan syndrome.