Mapping of a novel gene for familial hypertrophic cardiomyopathy to chromosome 11.

Familial hypertrophic cardiomyopathy (FHC) is a cardiac disorder transmitted as an autosomal dominant trait. FHC has been shown to be genetically heterogeneous with less than 50% of published pedigrees being associated with mutations in the beta myosin heavy chain (beta-MHC) gene on chromosome 14q11-q12. A second locus has recently been reported on chromosome 1. We examined the segregation of microsatellite markers in a French pedigree for which the disease is not linked to beta-MHC gene. We found significant linkage of the disease locus to several (CA)n repeats located on chromosome 11 (lod scores between +3.3 and +4.98). The data suggest the localization of the novel FHC gene in a region spanning 17 centiMorgans.

Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene.

Familial hypertrophic cardiomyopathy (FHC) is a clinically and genetically heterogeneous disease. The first identified disease gene, located on chromosome 14q11-q12, encodes the beta-myosin heavy chain. We have performed linkage analysis of two French FHC pedigrees, 720 and 730, with two microsatellite markers located in the beta-myosin heavy chain gene (MYO I and MYO II) and with four highly informative markers, recently mapped to chromosome 14q11-q12. Significant linkage was found with MYO I and MYO II in pedigree 720, but results were not conclusive for pedigree 730. Haplotype analysis of the six markers allowed identification of affected individuals and of some unaffected subjects carrying the disease gene. Two novel missense mutations were identified in exon 13 by direct sequencing, 403Arg-->Leu and 403Arg-->Trp in families 720 and 730, respectively. The 403Arg-->Leu mutation was associated with incomplete penetrance, a high incidence of sudden deaths and severe cardiac events, whereas the consequences of the 403Arg-->Trp mutation appeared less severe. Haplotyping of polymorphic markers in close linkage to the beta-myosin heavy chain gene can, thus, provide rapid analysis of non informative pedigrees and rapid detection of carrier status. Our results also indicate that codon 403 of the beta-myosin heavy chain gene is a hot spot for mutations causing FHC.

A colorimetric nanosensor based on a selective target-responsive aptamer kissing complex.

Herein, we report a novel approach for the design of a colorimetric aptasensor based on functionalized gold nanoparticle probes. This approach relies on the conjugation of nanoparticles by two functional DNA and RNA hairpins that engage specific kissing (loop-loop) interactions in response to the addition of a small analyte ligand, leading to particle aggregation and then red-to-purple colour change of the colloidal solution.

Design of a dual aptamer-based recognition strategy for human matrix metalloproteinase 9 protein by piezoelectric biosensors.

MMP-9, human matrix metalloproteinase 9, belongs to the family of zinc-dependent peptide-bond hydrolases and is involved in the degradation of the extracellular matrix (ECM). In clinics, it is well known that elevated MMP-9 serum levels are associated with cardiovascular dysfunctions, several aspects of the physiology and pathology of the central nervous system, neuropsychiatric disorders and degenerative diseases related to brain tumors, and excitotoxic/neuroinflammatory processes. Due to the large interest of diagnostics in this protein, efforts to set up sensitive methods to detect MMP-9 for early diagnosis of a number of metabolic alterations are rapidly increasing. In this panorama, biosensors could play a key role; therefore we explored for the first time the development of an aptamer-based piezoelectric biosensor for a sensitive, label free, and real time detection of MMP-9. The detecting strategy involved two different aptamers in a sandwich-like approach able to detect down to 100 pg mL(-1) (1.2 pM) of MMP-9 as detection limit in standard solution. As proof of principle, commercial serum was investigated in terms of possible interferents, their identification and role in MMP-9 detection. The estimated detection limit for MMP-9 is about 560 pg mL(-1) (6.8 pM) in untreated serum.

Genetic heterogeneity of familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is characterized by an autosomal dominant transmission, a left ventricular hypertrophy and a myocardial disorganization. A first morbid gene coding for the beta-myosin heavy chain (beta-MHC) located on chromosome 14q11-q12 has been identified and genetic heterogeneity has been demonstrated. We performed linkage and haplotype analysis of 17 French families with microsatellite markers located to the beta-MHC locus and we searched for a new locus in a family for which the beta-MHC locus was excluded. Two novel missense mutations were identified in exon 13 of the beta-MHC resulting in the replacement of 403 arginine by a leucine or a tryptophan in two different families. In one other family, systematic screening of the genome with highly informative markers allowed us to find a new locus for FHC on chromosome 11.

Dissociation of hypertension and genetically enhanced cell growth capacity in skin fibroblasts of F2 hybrid spontaneously hypertensive rats/Wistar-Kyoto rats.

Skin fibroblasts from newborn spontaneously hypertensive rats (SHR) grow faster in culture than Wistar-Kyoto rat (WKY) cells. Similar results have been described for vascular smooth muscle cells from prehypertensive and adult SHR. This suggests the existence of an intrinsic abnormality in vascular and nonvascular cells of mesodermal origin affecting cell growth control in those rats. In an attempt to determine the relation between high blood pressure and this trait, we cultured skin fibroblasts from adult SHR, WKY, F1, and F2 hybrid SHR/WKY populations by explant technique. Their growth capacity was determined by culture well DNA doubling time and by [3H]thymidine incorporation. Adult SHR fibroblasts grew more quickly (doubling time [DT] = 37.2 +/- 2.3 h, n = 8) than WKY ones (DT = 53.9 +/- 3.6 h, n = 6). Female SHR were crossed with male WKY to produce an F1 and an F2 hybrid generation presenting a Mendelian distribution of blood pressure. Skin fibroblasts were cultured from 21 rats belonging to the highest and the lowest blood pressure groups. No difference was observed between the two groups in either growth (DT = 47.5 +/- 4.1 h, n = 11 v DT = 44.6 +/- 3.2 h, n = 10) or epidermal growth factor-induced [3H]thymidine incorporation. These observations suggest that the increased growth capacity observed in SHR is not a determinant of high blood pressure initiation but may be involved in early cardiovascular enlargement.

Proliferation and Na+/H+ antiport activity in human fibroblasts from type I diabetic patients with nephropathy.

To investigate whether constitutive alterations of the Na+/H+ antiport or of cell proliferation control mechanisms are implicated in development of nephropathy in insulin-dependent diabetics (IDD), skin fibroblasts from controls, recent-onset IDD with normal or high glomerular filtration rates, and IDD with proteinuria were cultured by explant technique. The Na+/H+ antiport activity was studied using the pH sensitive fluorescent dye BCECF. The cell growth capacity was investigated by determination of cell DNA doubling time and by nuclear [3H]thymidine incorporation in response to serum. The Na+/H+ antiport activity and fibroblast growth capacity did not differ between fibroblasts from controls and IDD patients, suggesting the absence of a genetic alteration of the Na+/H+ antiport in the development of diabetic nephropathy.

Driving in vitro selection of anti-HIV-1 TAR aptamers by magnesium concentration and temperature.

In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.

DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes.

In vitro selection was performed in a DNA library, made of oligonucleotides with a 30-nucleotide random sequence, to identify ligands of the human immunodeficiency virus type-1 trans-activation-responsive (TAR) RNA element. Aptamers, extracted after 15 rounds of selection-amplification, either from a classical library of sequences or from virtual combinatorial libraries, displayed an imperfect stem-loop structure and presented a consensus motif 5'ACTCCCAT in the apical loop. The six central bases of the consensus were complementary to the TAR apical region, giving rise to the formation of RNA-DNA kissing complexes, without disrupting the secondary structure of TAR. The RNA-DNA kissing complex was a poor substrate for Escherichia coli RNase H, likely due to steric and conformational constraints of the DNA/RNA heteroduplex. 2'-O-Methyl derivatives of a selected aptamer were binders of lower efficiency than the parent aptamer in contrast to regular sense/antisense hybrids, indicating that the RNA/DNA loop-loop region adopted a non-canonical heteroduplex structure. These results, which allowed the identification of a new type of complex, DNA-RNA kissing complex, demonstrate the interest of in vitro selection for identifying non-antisense oligonucleotide ligands of RNA structures that are of potential value for artificially modulating gene expression.

Identification of aptamers against the DNA template for in vitro transcription of the HIV-1 TAR element.

We have extracted from a random population of about 10(9) oligodeoxynucleotides a series of 21-mers that are able to bind to a folded DNA 76-mer used as a template for in vitro transcription of the TAR element of the retrovirus HIV-1, by the T7 RNA polymerase. Five aptastrucs, that is, aptamers able to bind to the structure, out of 15 analyzed sequences, share the consensus motif 5'-PyGGG(TG)PyC, complementary in part to a weak double-stranded region of the target. (The parentheses indicate that either T or G is missing in one of these aptastrucs.) A dissociation constant of about 3 microM was evaluated by electrophoretic mobility shift assay for the winner sequence. Interactions between the aptastruc and the target sequences involve more than Watson-Crick base pairing of the consensus octamer. The binding is chemistry dependent. Phosphorothioate oligodeoxyribonucleotides and 2'-O-methyl oligoribonucleotides derived from the selected aptastrucs exhibit a weak if any affinity for the target.

Rational and combinatorial strategies for designing oligonucleotides targeted to RNA structures.

Many RNA structures play a key role in the regulation of gene expression. We designed synthetic oligonucleotides able to interact with folded RNA regions (see Toulmé et al., Biochimie (1996) 78, 663-673, for a review). We have demonstrated that a decanucleotide can form a triple helix with the stem of the hairpin responsible for ribosomal frame-shifting of the gag-pro message of HTLV-I, leading to the inhibition of translation. We have isolated, through an in vitro selection procedure, from a library composed of oligonucleotides with a random part of 30 nucleotides, sequences able to bind to the TAR RNA element of HIV-1 with a dissociation constant of 20-50 nM. The association between the two partners involve non-canonical interactions. This extends the range of potential targets for antisense sequences to functional RNA structures.

Readjusting the localization of long QT syndrome gene on chromosome 11p15.

Long QT syndrome (LQT) is an autosomal dominant cardiac disease characterized by ventricular arrhythmia. A first locus for LQT has been identified on chromosome 11p15.5 (LQT1), closely linked to HRAS. To refine the location of LQT1, microsatellites were genotyped in 8 French families and the following order was determined: tel-HRAS-DRD4-D11S922-D11S4046- IGF2-INS-TH-D11S1318-D11S1323-D11S1338-D11S90 9-D11S1346-cen. By haplotype analysis, 12 crossing-over events were identified in affected and unaffected subjects, delineating the LQT1 candidate region to 7 cM. This new delineated localization between D11S1318 and D11S1323 is in a more centromeric region than previously thought and is 5 cM proximal to HRAS.

Exclusion of genes coding for proteins of the cytoskeleton and the extracellular matrix in familial hypertrophic cardiomyopathy using a candidate gene approach.

Familial hypertrophic cardiomyopathy (FHC), a primary cardiac pathology, is a genetically heterogeneous disease, with autosomal dominant inheritance. The first gene identified as responsible for FHC codes for beta-myosin heavy chain (beta-MHC). To find a second locus, a candidate gene approach was applied on two families for which the beta-MHC locus was excluded. Selection of candidate genes is based on the observation of tissular and cellular disorganisation in FHC, and included genes coding for proteins involved in human myocardium architecture: the extracellular matrix components and cytoskeleton proteins. Chromosomal areas containing the candidate genes were examined by linkage analysis with microsatellite markers. The genes coding for different types of collagens, laminins, fibronectin, fibrillins, desmin, titin, alpha-actinin, vinculin, cardiac and skeletal alpha-actins, ankyrin and spectrin were excluded as responsible for FHC.

Identification of a mutation near a functional site of the beta cardiac myosin heavy chain gene in a family with hypertrophic cardiomyopathy.

Several mutations within the gene coding for the cardiac beta myosin heavy chain (designed MYH7) have been shown to be responsible for Familial Hypertrophic Cardiomyopathy (FHC) in several families, and evidence of genetic heterogeneity has been reported. To investigate the MYH7 gene as the cause of the disease in a small family with FHC, inheritance of the disease and chromosome 14 q11-q12 markers haplotype were studied, exons coding for the head domain of the cardiac beta myosin heavy chain (beta MHC) were analysed for mutations by MDE gel electrophoresis, and sequenced. We report a mutation within exon eight of the MYH7 gene at a very conserved amino acid at position 232, which results in the conversion of an asparagine to serine. This residue Asn-232 is located in a MHC area that has been recently identified as a critical site for ATPase activity. According to recent results on the three-dimensional structure of the myosin head or subfragment-1 (S1), Asn-232 is located in an alpha-helix which forms part of the nucleotide binding pocket. Although this mutation affects an active site, it seems to be associated with a favourable prognosis and a weak penetrance in this family.

A mutation in HERG associated with notched T waves in long QT syndrome.

Long QT syndrome (LQT) is a genetically heterogeneous inherited disorder that causes sudden death from cardiac arrhythmia. Four loci have been mapped to chromosomes 3, 4, 7 and 11 and three specific mutated genes for LQT syndrome have been identified. LQT2 results from mutations in the human ether-a-gogo-related gene, HERG, a cardiac potassium channel, whose protein product likely underlies Ikd the rapidly activating delayed rectifier current. By SSCP analysis and direct sequencing, we determined a new missense mutation in the HERG coding sequence, a G to A transition at position 1681 resulting in the substitution of threonine for a highly conserved alanine at codon 561. This mutation, Ala561Thr, in the coding sequence of the fifth membrane-spanning domain (S5) of the HERG protein seems to convey a risk of cardiac events in affected family members. In addition to a prolonged T wave of low amplitude on the surface ECG, a distinctive biphasic T-wave pattern was found in the left precordial leads of all affected subjects with the Ala561Thr mutation regardless of age, gender and beta blocking therapy.

Diagnostic performance of QT interval variables from 24-h electrocardiography in the long QT syndrome.

AIMS: The long QT syndrome is mainly defined by QT interval prolongation (QTc > 0.44s). However, data obtained in genotyped patients showed that resting QTc measurement alone may be inaccurate for ascertaining the phenotype. The aim of this study was to evaluate the diagnostic performance of QT interval rate-dependence in untreated chromosome 11-linked patients. METHODS: The study population consisted of 25 untreated long QT patients linked to chromosome 11 and 25 age- and gender-matched controls. QTc intervals were measured on 12-lead resting ECG recordings. From 24-h Holter recordings, the slope of the relationship between ventricular repolarization and heart rate was studied separately day and night to assess neural modulation. Mean heart rates and rate-dependences of QT and Q-maximum of T (QTm) intervals were compared between long QT patients and controls for both time periods. RESULTS: In both groups, the rate-dependences were modulated by day-night influences. When compared to controls, long QT patients showed a significant increase at night in QT/RR slopes (0.158 +/- 0.05 vs 0.117 +/- 0.03, P = 0.002) and QTm/RR slopes (0.163 +/- 0.05 vs 0.116 +/- 0.04, P = 0.0006). Multivariate analysis, adjusting QTc interval on age and gender, discriminated between long QT patients and controls with a 76% sensitivity and a 84% specificity. A 96% sensitivity and a 96% specificity were reached by taking into account the QTm/RR slope at night, the QTc interval and the mean heart rate during the day. CONCLUSION: QT interval variables obtained from 24-h ECG recordings improve long QT syndrome diagnosis by showing an increased nocturnal ventricular repolarization rate-dependence in genotyped chromosome 11-linked patients.