Jesús Molano

Genetic basis of end‐stage hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by a heterogeneous presentation and clinical course. A minority of HCM patients develop end‐stage HCM and require cardiac transplantation. The genetic basis of end‐stage HCM is unknown but small series, isolated case reports and animal models have related the most aggressive heart failure course with the presence of multiple mutations.

Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

Screening for mutations in Spanish families with myotonia. Functional analysis of novel mutations in CLCN1 gene

Myotonia congenita is an inherited muscle disorder caused by mutations in the CLCN1 gene, a voltage-gated chloride channel of skeletal muscle. We have studied 48 families with myotonia, 32 out of them carrying mutations in CLCN1 gene and eight carry mutations in SCN4A gene. We have found 26 different mutations in CLCN1 gene, including 13 not reported previously. Among those 26 mutations, c.180+3A>T in intron 1 is present in nearly one half of the Spanish families in this series, the largest one analyzed in Spain so far. Although scarce data have been published on the frequency of mutation c.180+3A>T in other populations, our data suggest that this mutation is more frequent in Spain than in other European populations. In addition, expression in HEK293 cells of the new missense mutants Tyr137Asp, Gly230Val, Gly233Val, Tyr302His, Gly416Glu, Arg421Cys, Asn567Lys and Gln788Pro, demonstrated that these DNA variants are disease-causing mutations that abrogate chloride currents.

Electroretinogram in Duchenne/Becker Muscular Dystrophy

The authors studied all cases with dystrophinopathy consecutively reviewed between May 1995 and December 1996 by means of electroretinography (ERG), which was recorded using skin eyelid electrodes and with standard flash stimulation. This methodology can detect the functional abnormalities associated with dystrophinopathies. The most valuable parameter is the ratio of B-wave amplitude to A-wave amplitude (B/A amplitude ratio), which was greater than 2 in all normal control patients (n = 10) and nondystrophinopathic muscular dystrophy (MD) patients (n = 2). It was less than 2 in 100% (n = 16) of Duchenne muscular dystrophy (DMD) patients (mean ratio 0.73; range 0.4-1.26). It was less than 2 in 71% (n = 7) of Becker muscular dystrophy (BMD) patients (mean ratio 1.12; range 0.88-1.37), and less than 2 in 50% (n = 4) of definitive DMD carriers. Twenty-nine percent of BMD, 50% of DMD carriers, and the only case with asymptomatic dystrophinopathy had normal ratios (greater than 2). The differences between the mean ratios of control, DMD, and BMD groups were statistically significant, all of them with P < 0.001. ERG abnormalities of dystrophinopathies were associated with the more severe muscular phenotype but not with the presence or location of gene deletion. ERG is an easy and simple technique that is useful in cases of suspected dystrophinopathy with a nonconclusive molecular study. It is less useful in patients who are DMD carriers.

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

The G397A (E133K) change in the AGGF1 (VG5Q) gene is a single nucleotide polymorphism in the Spanish population

Silvia Gutierrez, Luis Magano, Alicia Delicado, Marı́a A. Mori, Marı́a L. de Torres, Luis Fernández, Marı́a Palomares, Eva Fernández, Gemma R. Tarduchy, Jesús Molano, Ricardo Gracia, Isidora López Pajares, and Pablo Lapunzina* Department of Molecular Genetics, Hospital Universitario La Paz, Madrid, Spain Department of Medical Genetics, Hospital Universitario La Paz, Madrid, Spain Sequencing Unit, IBB, Madrid, Spain The Spanish Overgrowth Syndrome Registry

High-Risk Hypertrophic Cardiomyopathy Associated With a Novel Mutation in Cardiac Myosin-Binding Protein C

Hypertrophic cardiomyopathy is an autosomal dominant inherited disease characterized by ventricular hypertrophy and myofibril disarray. Mutations responsible for hypertrophic cardiomyopathy have been identified in 11 genes that encode for cardiac sarcomere proteins. Traditionally, hypertrophic cardiomyopathy due to mutation of the myosin-binding protein C gene (MYBPC3) has been thought to follow a benign course. We report a family with several members affected by hypertrophic cardiomyopathy in which there was a high incidence of sudden death. Disease was presumably caused by the substitution of cytosine by guanine at nucleotide 269 of MYBPC3 mRNA. This mutation, which has not previously been described, modifies codon 79, which encodes for the incorporation of a tyrosine, and gives rise to a stop codon. The mutation described here appears to confer a higher risk than that previously associated with hypertrophic cardiomyopathy due to MYBPC3 gene mutation.

Miocardiopatía hipertrófica: baja frecuencia de mutaciones en el gen de la cadena pesada de la betamiosina cardiaca

El objetivo del presente estudio es la busqueda de mutaciones en el gen de la cadena pesada de la betamiosina cardiaca ( MYH7b ) en pacientes espanoles con miocardiopatia hipertrofica. Se incluyo en el estudio a 36 familias con al menos un individuo enfermo de miocardiopatia hipertrofica. Se secuencio el ADN de los exones 3 al 24 del gen de la cadena pesada de la betamiosina cardiaca (gen MYH7b ). Se ha identificado 2 mutaciones (Arg858Cys y Met515Val) en 2 familias distintas, una de ellas de origen marroqui, lo que representa una frecuencia de mutaciones en el gen MYH7b inferior al 5%. Al contrario de lo que ocurre en otras poblaciones caucasicas, las mutaciones en el gen MYH7b en este grupo de familias espanolas con miocardiopatia hipertrofica son poco frecuentes.

Miocardiopatía hipertrófica de alto riesgo asociada con una nueva mutación en la proteína C fijadora de miosina

La miocardiopatia hipertrofica (MCH) es una enfermedad genetica de herencia autosomica dominante caracterizada por incremento en la masa ventricular y desorganizacion miofibrilar. Se han identificado mutaciones causantes de MCH en 11 genes de proteinas del sarcomero cardiaco. La MCH causada por mutaciones en el gen de la proteina C fijadora de miosina (MYBPC3) tradicionalmente se ha considerado de curso benigno. Presentamos a una familia con varios miembros afectados de MCH y alta incidencia de muerte subita, presumiblemente como consecuencia de la sustitucion de citosina por guanina en el nucleotido 269 del ARNm del gen MYBPC3. Esta mutacion, no descrita previamente, modifica el codon 79, que codifica la incorporacion del aminoacido tirosina y da lugar a un codon de terminacion. La mutacion descrita parece conferir un riesgo mayor que el atribuido hasta el momento a la MCH secundaria a mutaciones en el gen MYBPC3.

Hypertrophyc Cardiomyopathy: Infrequent Mutation of the Cardiac Beta-Myosin Heavy-Chain Gene

The aim of this study was to identify mutations in the cardiac heavy-chain beta-myosin gene (MYH7b) in a group of Spanish patients with hypertrophic cardiomyopathy. The study included 36 families with at least one member who had hypertrophic cardiomyopathy. DNA from exons 3 to 24 of the MYH7b gene was sequenced. Two mutations were identified: Arg858Cys and Met515Val. They occurred in two families, one of which was of Moroccan origin. This corresponds to a MYH7b gene mutation frequency of less than 5%. In contrast to findings in other Caucasian populations, MYH7b gene mutation occurred infrequently in this group of Spanish families with hypertrophic cardiomyopathy.