Mutations in the laminin alpha 2-chain gene (LAMA2) cause merosin-deficient congenital muscular dystrophy.

Congenital muscular dystrophies (CMDs), are heterogeneous autosomal recessive disorders. Their severe manifestations consist of early hypotonia and weakness, markedly delayed motor milestones and contractures, often associated with joint deformities. Histological changes seen in muscle biopsies consist of large variations in muscle fibre size, a few necrotic and regenerating fibres and a marked increase in endomysial collagen tissue. Diagnosis is based on clinical features and on morphological changes. In several CMD cases, we have demonstrated an absence of one of the components of the extracellular matrix around muscle fibres, the merosin M chain, now referred to as the alpha 2 chain of laminin-2 (ref.3). We localized this CMD locus to chromosome 6q2 by homozygosity mapping and linkage analysis. The laminin alpha 2 chain gene (LAMA2) maps to the same region on chromosome 6q22-23 (ref. 5). We therefore investigated LAMA2 for the presence of disease-causing mutations in laminin alpha 2 chain-deficient CMD families and now report splice site and nonsense mutations in two families leading presumably to a truncated laminin alpha 2 protein.

A novel mutation in the potassium channel gene KVLQT1 causes the Jervell and Lange-Nielsen cardioauditory syndrome.

The Jervell and Lange-Nielsen (JLN) syndrome (MIM 220400) is an inherited autosomal recessive disease characterized by a congenital bilateral deafness associated with a QT prolongation on the electrocardiogram, syncopal attacks due to ventricular arrhythmias and a high risk of sudden death. JLN syndrome is a rare disease, which seems to affect less than one percent of all deaf children. Linkage to chromosome 11p15.5 markers was found by analysing four consanguinous families. Recombinants allowed us to map the JLN gene between D11S922 and D11S4146, to a 6-cM interval where KVLQT1, a potassium channel gene causing Romano-Ward (RW) syndrome, the dominant form of long QT syndrome, has been previously localized. An homozygous deletion-insertion event (1244, -7 +8) in the C-terminal domain of this gene was detected in three affected children of two families. We found that KVLQT1 is expressed in the stria vascularis of mouse inner ear by in situ hybridization. Taken together, our data indicate that KVLQT1 is responsible for both JLN and RW syndromes and has a key role not only in the ventricular repolarization but also in normal hearing, probably via the control of endolymph homeostasis.

In vivo and in vitro examination of the functional significances of novel lamin gene mutations in heart failure patients.

CONTEXT: Lamin A/C (LMNA) gene variations have been reported in more than one third of genotyped families with dilated cardiomyopathy (DCM). However, the relationship between LMNA mutation and the development of DCM is poorly understood. METHODS AND RESULTS: We found that end stage DCM patients carrying LMNA mutations displayed either dramatic ultrastructural changes of the cardiomyocyte nucleus (D192G) or nonspecific changes (R541S). Overexpression of the D192G lamin C dramatically increased the size of intranuclear speckles and reduced their number. This phenotype was only partially reversed by coexpression of the D192G and wild type lamin C. Moreover, the D192G mutation precludes insertion of lamin C into the nuclear envelope when co-transfected with the D192G lamin A. By contrast, the R541S phenotype was entirely reversed by coexpression of the R541S and wild type lamin C. As lamin speckle size is known to be correlated with regulation of transcription, we assessed the SUMO1 distribution pattern in the presence of mutated lamin C and showed that D192G lamin C expression totally disrupts the SUMO1 pattern. CONCLUSION: Our in vivo and in vitro results question the relationship of causality between LMNA mutations and the development of heart failure in some DCM patients and therefore, the reliability of genetic counselling. However, LMNA mutations producing speckles result not only in nuclear envelope structural damage, but may also lead to the dysregulation of cellular functions controlled by sumoylation, such as transcription, chromosome organisation, and nuclear trafficking.

Genetic aspects of heart failure.

Heart failure is a major health problem and is associated with a high mortality and morbidity. Recently, the role of the genetic background in the onset and the development of the disease has been evidenced in both heart failure with and without systolic dysfunction and in familial and non-familial forms of this condition. Preliminary studies suggest that the I/D polymorphism of the Angiotensin Converting Enzyme gene influence the development of left ventricular hypertrophy, a major determinant of heart failure. Familial hypertrophic cardiomyopathy (FHC) is a highly heterogenous autosomal dominant disease. Seven genes have been identified which all encode proteins of the sarcomere or proteins involved in the regulation of contraction. More than one hundred mutations have been evidenced. Modifier genes such as the I/D polymorphism seem to play a role in the expression of the disease. Susceptibility genes have been searched for in sporadic forms of dilated cardiomyopathy and conflicting results have been published with regard to the I/D polymorphism. Finally, familial forms of dilated cardiomyopathy (FDC) are frequent. Various modes of inheritance and phenotypes have been reported and this condition appears genetically highly heterogenous. It has been postulated that the molecular defect involved in FDC is an abnormality in the transmission of contractile force. The analysis of genetic factors that predispose to heart failure looks promising: it should allow better understanding of the underlying mechanisms that promote the progression of the disease, to identify subjects at risk of the disease who would benefit from early medical management and promote the development of pharmacogenetics.

Familial dilated cardiomyopathy: clinical features in French families.

UNLABELLED: The aims of the study were to analyze the clinical features, the penetrance and the mode of inheritance of 13 French families with dilated cardiomyopathy using diagnostic criteria recently established by a European collaboration. METHODS: Screening consisted of physical examination, ECG and Echo of all the probands first degree relatives (n = 118). Using major Echo criteria [ejection fraction (EF) < 45% or FS < 25% and left ventricular diameter (LVD) > 117% of the predictive value], or combined minor Echo/ECG criteria, relatives were classified as affected, unknown or healthy. RESULTS: (1) Adult affected relatives (n = 31) were identified with major Echo criteria in 74% of cases, and with combined minor Echo/ECG criteria in 26% of cases. (2) In the unknown relatives (n = 21), the most common abnormality was an isolated left ventricular dilation (67%). (3) Mode of inheritance was autosomal dominant (AD) in 11 families and possibly autosomal recessive in two. (4) In AD families, the penetrance was incomplete in adults (72%), age-related (O.R.: 1.3 per 10 years; 95% CI 1.03-1.56) and sex-related [greater in men (87%) than in women (61%), actuarial survival curve: P<0.002]. (5) Mortality related to end stage heart failure was 2.2 times as high as mortality related to sudden death (11% vs. 5%). CONCLUSIONS: (1) In the absence of a specific phenotype of FDC, the characterization of relatives appears more accurate when minor criteria were added. (2) Since high mortality (16%) and incomplete penetrance frequently give rise to small nuclei of clinically affected and alive relatives per family, the accurate model of penetrance that we proposed might be helpful in the future to enhance the statistical power of linkage analysis in this disease.

Characterization of imidazoline-guanidinium receptive sites in renal medulla from human kidney.

Previous studies showed that alpha 2-adrenergic receptors and imidazoline-guanidinium receptive sites (IGRS) are colocalized in rabbit and human renal proximal tubule. In the present study we investigated the localization of these two binding sites in the renal medulla from human kidney. Binding studies performed with [3H]idazoxan (IGRS ligand) and [3H]rauwolscine (alpha 2-adrenergic ligand) showed that, in membrane preparations from renal medulla, the density of IGRS was 3.6-fold higher than that of alpha 2-adrenergic receptors (134 +/- 7 v 37 +/- 5 fmol/mg protein, respectively). These data indicate that imidazoline, guanidinium, and oxazoline derivatives could induce their therapeutic effects through the interaction with IGRS and/or alpha 2-adrenergic receptors located not only in the renal proximal tubule but also in other segments of the nephron.

Characterization of mitochondrial imidazoline-guanidinium receptive sites (IGRS) in liver.

Some imidazoline and guanidinium antihypertensive drugs display high affinity for a nonadrenergic membrane protein, the imidazoline-guanidinium receptive site (IGRS), which is insensitive to catecholamine and physically distinct from alpha 2-adrenoceptor. In the present report, we characterized IGRS in human and rabbit liver using [3H]idazoxan as radioligand. By performing subcellular fractionation, we showed a significant increase in [3H]idazoxan binding sites on membrane fractions enriched in cytochrome oxidase activity, a mitochondrial marker. A further enrichment in [3H]idazoxan binding (53-fold with respect to the homogenate) was found in a purified preparation of mitochondrial outer membranes. This localization of IGRS will facilitate the characterization of its functional activity in liver.

Identification of an imidazoline-guanidinium receptive site in mitochondria from rabbit cerebral cortex.

In the present report, we used [3H]idazoxan to characterize imidazoline-guanidinium receptive sites (IGRS) in mitochondria from rabbit cerebral cortex. When compared to the starting homogenate, [3H]idazoxan binding was higher (1.161 +/- 0.159 vs. 0.102 +/- 0.024 pmol/mg of protein) in a membrane fraction 6-fold enriched in cytochrome oxidase activity, a specific marker for mitochondria. In addition, the enrichment of [3H]idazoxan binding sites positively correlates with cytochrome oxidase activity in different membrane preparations (r = 0.977, P less than 0.001). In competition studies, [3H]idazoxan binding was completely inhibited by imidazoline and guanidinium derivatives but not affected by 10 microM epinephrine. Taken together, these data show the localization of IGRS in the mitochondria from rabbit cerebral cortex.

Tissue-specific localization of mitochondrial imidazoline-guanidinium receptive sites.

In the present report, we studied the distribution of the imidazoline-guanidinium receptive site in mitochondrial fractions from different rabbit and human tissues. Binding studies of the imidazoline-guanidinium receptive site ligand [3H]idazoxan, allowed to distinguish two groups of tissues: the first one, including kidney, brain and liver, displays a high density of imidazoline-guanidinium receptive site; the second one, consisting of striated and smooth muscle, enterocytes, lung, spleen and heart, is characterized by 4- to 16-fold lower binding site density. The demonstration that mitochondrial imidazoline-guanidinium receptive sites are not equally expressed in all tissues can be considered as a further progress towards the characterization of their functional activity.

Cell-associated and soluble phospholipases A2 increase during carrageenan and zymosan-induced pleurisy in rat.

Extra-cellular and cell-associated Ca(2+)-dependent phospholipases A2 and released thromboxane B2 were correlated to exudation and cell migration during rat pleurisy induced by carrageenan or zymosan. Extra-cellular phospholipase A2 was delayed with respect to acute inflammation, while cell-associated phospholipase A2 closely correlated with cell migration and thromboxane B2 levels. This confirms that the subcellular localization of phospholipases A2 is linked to their physiological action and, in particular, suggests that the cell-associated, rather than the extracellular enzyme, accounts for the production of eicosanoids.

Penetrance of familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant cardiac disease for which the penetrance remains a much-debated issue. Since the recent identification of the genes involved in the disease, the penetrance of FHC has not been reassessed in a large genotyped population. The aim of our study was therefore to evaluate it, according to age and sex, in ten families with previously identified mutations. Among 178 individuals we studied, 90 were genetically affected (9 different mutations in 3 genes). We found that penetrance, assessed by classical echocardiographic and electrocardiographic criteria, was (1) incomplete: 69%; (2) age-related: 55% between 10 and 29 years old, 75% between 30 and 49 y. and 95% over 50 y.; (3) greater in males than in females: 77% vs 58%, age-adjusted odds ratio: 3.98, CI 95%: 1.34 to 11,48; (4) similar for the genes analyzed. The consequences of these results for genetic counseling and linkage analyses are discussed.

[Imidazoline-guanidine site: a subtype of imidazoline receptors]. / Le site imidazolinique-guanidinique: un sous-type de récepteurs imidazoliniques.

Since the demonstration that imidazoline and guanidinium alpha-2 adrenergic agonists induce some of their functional effects by a "nonadrenergic" mechanism, many efforts have been done to identify an imidazoline receptor. Binding studies have allowed to characterize two classes of potential imidazoline receptors: the "(p-amino)clonidine" and the "idazoxan" binding sites. These last, that we named "imidazoline-guanidinium receptive sites" (IGRS) on the basis of their ligand-recognition properties, have been identified, for the first time, in the proximal tubule from rabbit and human kidney. In the present report we will summarize the studies that led us to the characterization of IGRS.

Influence of Pro12Ala peroxisome proliferator-activated receptor gamma2 polymorphism on glucose response to exercise training in type 2 diabetes.

AIMS/HYPOTHESIS: Exercise training improves glycaemic control in some but not all individuals and little research has been done regarding genetic impact on the exercise training response in type 2 diabetes. The purpose of this study was to investigate the influence of the Pro(12)Ala variant of the peroxisome proliferator-activated receptor (PPAR) gamma2 gene on changes in fasting plasma glucose in response to exercise training. METHODS: The study population comprised 139 sedentary type 2 diabetic patients (age: 54.4+/-7.2; HbA(1)c: 7.7+/-0.9%) who completed 3 months of supervised exercise training. The primary outcome variable in our analysis was the post-intervention change in blood glucose. Other assessments included measures of body composition, insulin sensitivity indices and maximal oxygen uptake (VO(2max)). RESULTS: The frequency of the Ala allele was 8.3% and the genotypes were in Hardy-Weinberg equilibrium. At baseline, neither body composition variables (weight, BMI, waist circumference), glucose homeostasis variables (glucose, insulin, HbA(1)c, homeostasis model assessment method) nor VO(2max) were different between genotypes (wild-type: Pro(12)Pro n=117, Ala carriers: X(12)Ala n=22). The exercise-training intervention led to similar improvements in body composition and glucose homeostasis variables in both genotype groups (p<0.05). The change in fasting plasma glucose was significantly different between PPARgamma2 genotypes (-1.66 mmol/l vs -0.54 mmol/l, Ala carriers and wild-type, respectively) (p=0.034 unadjusted and p=0.089 including baseline glucose) and the significant association between genotype and glucose response remained after adjusting for statistically significant predictors (age, changes in insulin and BMI [p=0.015]) and including baseline glucose, insulin and BMI (p=0.031). CONCLUSIONS/INTERPRETATION: These data suggest that the Pro(12)Ala polymorphism may influence the glycaemic response to exercise in type 2 diabetes.

Subcellular distribution of imidazoline-guanidinium-receptive sites in human and rabbit liver. Major localization to the mitochondrial outer membrane.

Imidazoline-guanidinium-receptive site (IGRS) is a membrane protein that, even if recognized by a series of imidazoline and guanidinium alpha 2-adrenergic compounds, is insensitive to catecholamine and physically distinct from alpha 2 receptors (Parini, A., Coupry, I., Graham, R. M., Uzielli, I., Atlas, D., and Lanier, S. M. (1989) J. Biol. Chem. 264, 11874-11878). In the present report, we defined the subcellular localization of IGRS by performing binding studies with the imidazoline radioligand [3H]idazoxan. Binding studies on subcellular fractions of homogenates from human and rabbit liver showed a significant increase in [3H]idazoxan binding in a membrane fraction enriched in cytochrome oxidase activity, a specific marker for mitochondria. The enrichment in [3H]idazoxan binding sites correlates closely with cytochrome oxidase activity in the nuclear, mitochondrial, plasma membrane, microsomal, and soluble fractions (r = 0.966, p less than 0.002) but not with the specific markers for other cell compartments, suggesting a major localization of IGRS in mitochondria. Separation of inner and outer mitochondrial membranes by digitonin treatment showed that [3H]idazoxan binding correlates positively with monoamine oxidase (r = 0.960) and negatively with cytochrome oxidase (r = -0.950) activities. In addition, in highly purified preparations of outer mitochondrial membranes obtained by hypotonic shock, [3H]idazoxan binding activity was 12.5-fold enriched with respect to intact mitochondria. Taken together, these data show, for the first time, that IGRS in human and rabbit liver are mainly associated with the outer mitochondrial membranes. This demonstration of the major mitochondrial localization of IGRS will facilitate the characterization of its functional activity in liver.

I2-imidazoline binding sites: relationship with different monoamine oxidase domains and identification of histidine residues mediating ligand binding regulation by H+1.

We have shown that I2-imidazoline binding sites (I2BSs) are located on both monoamine oxidases A (MAO-A) and B (MAO-B) and are selectively regulated by H+ and K+ in vitro. In the present study we used chemical modifying agents to investigate the localization of I2BSs with respect to different MAO domains and the mechanisms of ligand binding regulation by K+ and H+. In mitochondrial or solubilized preparations from rabbit kidney and liver, modification of cysteine residues, which are critical for MAO activity, did not affect [3H]idazoxan binding, indicating that I2BS is not associated to the cysteine-containing flavin adenine dinucleotide (FAD) prosthetic group or to the catalytic site of MAOs. Among various chemical modifying agents, only diethylpyrocarbonate and 4-bromophenacyl bromide, two histidine modifying agents, inhibited [3H]idazoxan binding to I2BS. The pH profile of diethylpyrocarbonate effect was consistent with the specific modification of histidine residues. In protection experiments, the effect of diethylpyrocarbonate was not prevented in the presence of saturating concentrations of amiloride, guanabenz or KCl, suggesting that these residues are not located within the ligand or K+ binding sites. In contrast, histidine residues appear to be within a MAO domain involved in regulation of [3H]idazoxan binding by H+. Indeed, the pH-dependent increase in [3H]idazoxan binding was fully abolished after treatment of solubilized material with diethylpyrocarbonate. In conclusion, our results show that MAO I2BSs are not located within the flavin adenine dinucleotide prosthetic group or the catalytic site. Histidine(s) residue(s) involved in the regulation of ligand binding to I2BS by H+ also has been identified.

Localization of I2-imidazoline binding sites on monoamine oxidases.

Imidazoline binding sites (IBS) were proposed to be responsible for some of the pharmacological and therapeutic activities of imidazoline and related compounds and have been classified into two subtypes, I1BS and I2BS. Convergent studies attribute a role in central blood pressure regulation to the I1BS. In contrast, the function of I2BS remains unknown. In the present study, by combining biochemical and molecular biology approaches, we show that 1) microsequencing of I2BS purified from rabbit kidney mitochondria allowed the recovery of four peptide sequence stretches displaying up to 85.7% similarity with human, rat, and bovine monoamine oxidases (MAO)-A and -B; 2) I2BS and MAO displayed identical biophysical characteristics as their activities, measured by [3H]idazoxan binding and [14C]tyramine oxidation, respectively, could not be separated using various chromatographic procedures; and 3) heterologous expression of human placenta MAO-A and human liver MAO-B in yeast, inherently devoid of I2BS and MAO activities, led to the coexpression of [3H]idazoxan binding sites displaying ligand-recognition properties typical of I2BS. These results show definitely that I2BS is located on both MAO-A and -B. The fact that I2BS ligands inhibited MAO activity independently of the interaction with the catalytic region suggests that I2BS might be a previously unknown MAO regulatory site.

Exclusion of KCNE1 (IsK) as a candidate gene for Jervell and Lange-Nielsen syndrome.

The KCNE1 gene encodes a small protein, IsK, of 14.4 kDa, with a single transmembrane domain, and is part of a potassium channel expressed in the heart. This channel is thought to underly the very slow component of the cardiac delayed rectifying current which controls the duration and the degree of ventricular repolarization. This suggested that KCNE1 could be the morbid gene responsible for an autosomal recessive cardio-auditory disease, the Jervell and Lange-Nielsen syndrome, characterized by ventricular repolarization abnormalities and recurrent syncopes leading eventually to sudden death associated with a bilateral congenital deafness. By linkage analysis in four consmanguinous families, using microsatellite markers of chromosome 21 as well as KCNE1 intragenic polymorphisms, we excluded KCNE1 as a candidate gene for Jervell and Lange-Nielsen syndrome. In addition, we described a new polymorphism, a G-to-A substitution at position 253, in the KCNE1 coding sequence detectable by SSCP analysis or RFLP.

Notechis 11'2, a non-toxic phospholipase A2 from the venom of Notechis scutatus scutatus.

Previously, we deduced the amino acid sequence of a novel phospholipase-A2-like protein (PLA2) from the nucleotide sequence of a cDNA isolated from a library prepared from the venom gland of the Australian elapid Notechis scutatus scutatus. The corresponding protein has now been identified, purified from the venom and named Notechis 11'2. Its complete amino acid sequence has been determined by automated Edman degradation of both the whole protein and peptides generated by Staphylococcus aureus protease digestion and chemical cleavage at a tryptophan residue. As predicted from its sequence which contains all the residues putatively required for PLA2 activity, Notechis 11'2 exhibits an esterase activity, preferentially against neutral phospholipids. However, despite its sequence homology with other highly toxic PLA2 present in the venom of Notechis scutatus scutatus, notechis 11'2 has no lethal activity. This observation further supports the view that the lethal activity of PLA2 from Notechis scutatus scutatus is not due to the esterasic activity only.