Mutational spectrum of Duchenne muscular dystrophy in Spain: Study of 284 cases. / Espectro mutacional de la distrofia muscular de Duchenne en España: estudio de 284 casos.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive neuromuscular disease that affects one in 3500 live-born males. The total absence of dystrophin observed in DMD patients is generally caused by mutations that disrupt the reading frame of the DMD gene, and about 80% of cases harbour deletions or duplications of one or more exons. METHODS: We reviewed 284 cases of males with a genetic diagnosis of DMD between 2007 and 2014. These patients were selected from 8 Spanish reference hospitals representing most areas of Spain. Multiplex PCR, MLPA, and sequencing were performed to identify mutations. RESULTS: Most of these DMD patients present large deletions (46.1%) or large duplications (19.7%) in the dystrophin gene. The remaining 34.2% correspond to point mutations, and half of these correspond to nonsense mutations. In this study we identified 23 new mutations in DMD: 7 large deletions and 16 point mutations. CONCLUSIONS: The algorithm for genetic diagnosis applied by the participating centres is the most appropriate for genotyping patients with DMD. The genetic specificity of different therapies currently being developed emphasises the importance of identifying the mutation appearing in each patient; 38.7% of the cases in this series are eligible to participate in current clinical trials.

Isolated cardiomyopathy caused by a DMD nonsense mutation in somatic mosaicism: genetic normalization in skeletal muscle.

X-linked dilated cardiomyopathy is a pure cardiac dystrophinopathy phenotype mainly caused by DMD mutations that present a specific transcription effect in cardiac tissue. We report a 26-year-old male who presented with severe dilated cardiomyopathy and high creatine kinase. The patient did not complain of skeletal muscle weakness. A muscle biopsy showed mild dystrophic changes and a low proportion of dystrophin-negative fibres. A molecular study identified a nonsense DMD mutation (p.Arg2098X) in somatic mosaicism. The ratio of mutant versus normal allele in blood and skeletal muscle suggests selective pressure against mutant muscle cells, a process known as genetic normalization. We hypothesize that this process may have mitigated skeletal muscle symptoms in this patient. This is the second report of a DMD somatic mosaic with evidence of genetic normalization in muscle. Somatic DMD mutations should be considered in patients presenting with idiopathic dilated cardiomyopathy.

Abnormal expression of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele.

BACKGROUND: In some cases, a definitive confirmation of dysferlinopathy cannot be achieved by DNA test, because the mutation is detected in one allele only. PATIENTS AND METHODS: DYSFERLIN expression in skeletal muscle and peripheral blood monocytes (PBM) was studied by Western blot in two unrelated adult patients. The comparative C(T) method (ΔΔC(T) ) was used to calculate relative changes in dysferlin mRNA determined from real-time quantitative PCR experiments. The dysferlin gene was studied by direct sequencing of cDNA and genomic DNA and by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: A comparable severe reduction in dysferlin was demonstrated in both skeletal muscle and PBM. The expression of dysferlin mRNA was significantly reduced. A novel mutation in exon 47 (c.5289G>C) of the dysferlin gene in the heterozygous state, causing an amino acid change (p.Glu1763Asp), was detected in both patients. The MLPA analysis did not reveal any deletion or duplication. CONCLUSIONS: Dysferlin and/or dysferlin mRNA abnormalities are diagnostic for dysferlinopathy when mutational analysis detects a mutation in one allele only. Analysis of dysferlin mRNA can be helpful for distinguishing symptomatic heterozygotes from such patients.

A new phenotype of dysferlinopathy with congenital onset.

We report two patients with a new phenotype of dysferlinopathy presenting as congenital muscular disease. Both patients showed weakness in proximal lower limbs and neck flexor muscles at birth. The presence of normal CK levels during the first years should be noted. Initial MRI showed no abnormalities but short-time-inversion-recovery (STIR) sequences revealed a striking myoedema in gastrocnemius and hamstring muscles at the age of 5. Muscle biopsy showed mild dystrophic features and the absence of dysferlin. Dysferlin gene (DYSF) analysis revealed a p.Ala927LeufsX21 mutation in a homozygous state in both siblings. This new phenotype widens the clinical spectrum of dysferlin myopathies.

Dysferlin expression in monocytes: a source of mRNA for mutation analysis.

Dysferlin protein is expressed in peripheral blood monocytes. The genomic analysis of the DYSF gene has proved to be time consuming because it has 55 exons. We designed a mutational screening strategy based on cDNA from monocytes to find out whether the mutational analysis could be performed in mRNA from a source less invasive than the muscle biopsy. We studied 34 patients from 23 families diagnosed with dysferlinopathy. The diagnosis was based on clinical findings and on the absence of protein expression using either immunohistochemistry or Western blot of skeletal muscle and/or monocytes. We identified 28 different mutations, 13 of which were novel. The DYSF mutations in both alleles were found in 30 patients and only in one allele in four. The results were confirmed using genomic DNA in 26/34 patients. This is the first report to furnish evidence of reliable mutational analysis using monocytes cDNA and constitutes a good alternative to genomic DNA analysis.

Molecular characterization of congenital myasthenic syndromes in Spain.

Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders, all of which impair neuromuscular transmission. Epidemiological data and frequencies of gene mutations are scarce in the literature. Here we describe the molecular genetic and clinical findings of sixty-four genetically confirmed CMS patients from Spain. Thirty-six mutations in the CHRNE, RAPSN, COLQ, GFPT1, DOK7, CHRNG, GMPPB, CHAT, CHRNA1, and CHRNB1 genes were identified in our patients, with five of them not reported so far. These data provide an overview on the relative frequencies of the different CMS subtypes in a large Spanish population. CHRNE mutations are the most common cause of CMS in Spain, accounting for 27% of the total. The second most common are RAPSN mutations. We found a higher rate of GFPT1 mutations in comparison with other populations. Remarkably, several founder mutations made a large contribution to CMS in Spain: RAPSN c.264C > A (p.Asn88Lys), CHRNE c.130insG (Glu44Glyfs*3), CHRNE c.1353insG (p.Asn542Gluf*4), DOK7 c.1124_1127dup (p.Ala378Serfs*30), and particularly frequent in Spain in comparison with other populations, COLQ c.1289A > C (p.Tyr430Ser). Furthermore, we describe phenotypes and distinguishing clinical signs associated with the various CMS genes which might help to identify specific CMS subtypes to guide diagnosis and management.

Severe limb girdle muscular dystrophy in Spanish gypsies: further evidence for a founder mutation in the gamma-sarcoglycan gene.

Limb-girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive muscular dystrophy with primary gamma-sarcoglycan deficiency, generally associated with a severe clinical course. gamma-sarcoglycan, a 35kDa dystrophin-associated protein, is encoded by a single gene on chromosome 13q12. Six different mutations have been described in that gene, and it has been proved they are the origin of the disease. One of these mutations (C283Y), a G-->A transition in codon 283, was recently and exclusively identified in Gypsy patients from different European countries. We report the study of 11 LGMD2C unrelated Gypsy families (nine Spanish and two Portugese). The muscle biopsies of these patients showed a drastically decreased immunostaining with alpha and gamma-sarcoglycan antibodies. All the patients were homozygous for C283Y missense mutation, and all affected chromosomes (patients and heterozygous relatives) carried the allele 5 (112 bp) of the intragenic microsatellite D13S232. Unexpectedly, this allele is most frequent in the Caucasian population but not in the normal Gypsy population. The clinical severity of all patients demonstrates that the C283Y missense mutation in a homozygous state causes a severe LGMD2C (DMD-like). The elevated number of families ascertained let us assume that LGMD2C is prevalent in the Gypsy population, and that all the families have inherited a founding mutation.

Homogeneous phenotype of the gypsy limb-girdle MD with the gamma-sarcoglycan C283Y mutation.

OBJECTIVE: To characterize the clinical phenotype of LGMD2C in gypsies. BACKGROUND: Limb-girdle muscular dystrophy (LGMD) in gypsies of Western Europe is caused by a homozygous C283Y mutation on the same haplotype, suggesting a founder effect. METHODS: We performed clinical, laboratory, and muscle imaging studies of 40 patients. RESULTS: Mean age at onset was 5.3 years. One half of the patients had loss of ambulation by the age of 12; 13% still could walk after age 16. Calf hypertrophy, scapular winging, macroglossia, and lumbar hyperlordosis were common. Girdle, trunk, and proximal limb flexor muscles had earlier and more severe involvement. Cardiomyopathy was not observed. Five patients in the third decade of life required mechanical ventilation. Scoliosis was common in the nonambulatory stage. CONCLUSIONS: LGMD2C in gypsy patients with C283Y mutation presents a rather homogeneous phenotype, characterized by an initial Duchenne-like progressive course followed by a more prolonged survival rate possibly due to the absence of early respiratory impairment and cardiac failure.

Novel mutations in the muscle chloride channel CLCN1 gene causing myotonia congenita in Spanish families.

Mutations in the muscular voltage-dependent chloride channel gene (CLCN1), located at 7q35, lead to recessive and dominant myotonia congenita. We report four novel mutations identified in this gene, after clinical, electromyographic, and genetic studies performed on 13 unrelated families. Two of the four mutations (2512insCTCA and A218T) were identified in families with Thomsen's disease, one (Q658X) in a family with Becker's disease, and the fourth (R669C) in a presumably sporadic patient with the Becker phenotype. Although identification of the mutations allows us to establish some genotype/phenotype correlations, this does not wholly account for the clinical heterogeneity and the inheritance patterns of the disease.

[Muscular dystrophy due to a mutation in the gene of alpha-sarcoglycan subunit of dystrophin associated protein complex]. / Distrofia muscular por déficit de la subunidad alfa-sarcoglucano del complejo de proteínas relacionadas con la distrofina.

Linkage studies have confirmed the existence of clinical an genetic heterogeneity among the muscular dystrophies due to adhalin deficiency. We present the clinical, histological and genetic characteristics in a case of primary adhalinopathy (deficiency of the 50 kD subunit or alpha-sarcoglycan). It was a 19 years-old woman, born of non consanguineous parents, who shows a long evolution myopathy with onset before age 7, a severe evolution and becoming wheelchair bound at 10 years. She showed evident calf pseudohypertrophy and serum creatinkinase (CK) levels were elevated (40-180 times the standard level). The histological pattern showed a destructed fascicular architecture in agreement with severe muscular dystrophy, normal staining with anti-dystrophin monoclonal antibodies and abnormal staining pattern with anti-adhalin antibodies. The molecular study evidenced an homozygous point mutation (Arg-->Cys) at position 77 of exon 3 of the gene coding for the 50 kD subunit of the alpha-sarcoglycan complex localised in chromosome 17. In the light of this case, we suggest a revision of the diagnostic orientation in the muscular dystrophies and we review the new taxonomy of the limb-girdle muscular dystrophies, remarking the clinical signs which could indicate a given genetic locus.

[Muscular dystrophy due to a deficit of gamma-sarcoglycan. A report of three patients with the Delta-521t mutation]. / Distrofia muscular por déficit de gamma-sarcoglicano. Aportación de tres pacientes con la mutación Delta-521T.

INTRODUCTION: gamma-sarcoglicanopathies, also classified as limb girdle muscular dystrophy type 2C (LGMD2C) are a group of autosomal recessive muscular dystrophies due to mutations in 13q12 and subsequent g sarcoglican deficiency. The protein is one of the components of the dystrophin associated glycoprotein complex and is thought to impart structural integrity to the myofibre. The clinical course of the disease may be heterogeneous, ranging from severe forms with onset in the first decade and rapid progression resembling Progressive Duchenne muscular dystrophy (DMD) to milder forms with later onset and slower course. Cases hitherto reported in Spain corresponds to gypsie patients, homozygous for C283Y missense mutation. CASE REPORTS: Here, we report three new galician (Northwest Spain) patients (one male and one female sibling cases) with a severe DMD like muscular dystrophy homozygous for D 521T. In the first male familial case, initial diagnosis of DMD was made. On reevaluation fourteen years later, inmunohistochemical and molecular studies allowed for a definitive g sarcoglicanopathy diagnosis. CONCLUSIONS: Patients with a primary sarcoglycanopathy may be clinically indistinguishable from those with the primary dystrophinopathies. Probably, the diagnosis of LGMD are underestimated and a number of male patients diagnosed as DMD really corresponds to a recessive form o muscular dystrophy. Consequently, a definitive diagnosis rests on appropriate inmunohistochemical and molecular analysis, specially in those patients showing a normal pattern of dystrophin and/or suggestive for an autosomal recessive mode of inheritance.

[Early onset adhalinopathy (LGMD2D) mimicking congenital muscular dystrophy]. / Adhalinopatía primaria (LGMD2D) de inicio en los primeros meses de la vida que simula una distrofia muscular congénita.

INTRODUCTION: The recent discovery of the dystrophin-associated complex of glycoproteins led to the delineation of sarcoglycanopathies, a phenotypically similar to dystrophinopathies group of clinically heterogeneous and progressive muscular dystrophies. The objective of this paper is to report the clinical, biochemical, histological, immunohistochemical and molecular genetics characteristics observed in a case of adhalinopathy (alpha-sarcoglycanopathy or LGMD2D) presenting in early months of life and resembling congenital muscular dystrophy. CLINICAL CASE: An 12-year old school boy, the third son of a healthy, young, non consanguineous couple, presented at birth with bilateral cleft lip, cleft palate and mild hypotonia. At age 6 months it was believed he suffered from a benign form of congenital muscular dystrophy on the basis of clinical, biochemical, electrophysiological and histological findings. From 5 years onwards he had frequent falls and climbing stairs had become increasingly difficult. Also, a positive Gowers 'sign, mild calf hypertrophy, high serum creatine-phosphokinase level and myopathic electromyographic features were present; otherwise, cardiological evaluation and intelligence were normal. A repeated muscular biopsy at 10 years showed dystrophic features as well as selective deficiency of adhalin on immunostaining. DNA analysis demonstrated the patient being homozygote for a R77C mutation. Actually, a marked lumbar lordosis and waddling gait, an impossibility of climbing stairs and arising from the floor in addition to absent rotulian reflexes and mild Achilles retraction are present. CONCLUSIONS: LGMD2D may present in the first months of life mimicking congenital muscular dystrophy. It seems reasonable that biopsies of all new cases of muscular dystrophies be selectively immohistochemical analyzed, and when it is possible the diagnosis should be confirmed by DNA analysis.

[Alterations in functional proteins. Calpaine-3 deficiency]. / Alteraciones en las proteínas funcionales. Déficit de calpaína 3.

INTRODUCTION: Muscular dystrophies due to calpain deficiency are the first example of a muscular dystrophy due to the mutation of a gene codifying for a non-structural enzymatic protein of unknown function and substrate. DEVELOPMENT: More than 70 mutations have been described in the gene structure, localized to chromosome 15. Although the time course and topography is fairly homogeneous, correlation between the different mutations and the phenotype has still to be analyzed. The age of onset of symptoms is usually between 8 and 14, with no difference between the sexes. There is a slow but uniformly progressive course starting in the pelvis and extending to the shoulder and the distal musculature. Almost all patients are confined to a wheelchair twenty years after onset of the disease. There is no facial, oculomotor or bulbar involvement and gemellar pseudohypertrophy is rare. However, a winged scapula and marked lumbar hyperlordosis is universal. No cardiac or cognitive changes have been observed. Muscle CT shows a pattern of atrophy, mainly of the posterior and medial muscle compartments and of the posterosuperficial group of the legs, which varies depending on the time the disorder has been present. This condition is the commonest etiological group of the dystrophy syndromes, especially of those of late infancy or juvenile onset, in the open populations studied to date. Muscle biopsy, stained by all methods available, is essential to rule out other types of progressive dystrophies secondary to deficiencies of structural proteins.

LMNA mutation in progeroid syndrome in association with strokes.

Hutchinson-Gilford progeria syndrome is a very rare but well-characterized genetic disorder that causes premature ageing. Clinical features affect growth, skeleton, body fat, skin, hair and the cardiovascular system. It is caused by mutations in LMNA gene, the most frequent being p.Gly608Gly (c.1824C > T) in exon 11. Here we present a four-year-old HGPS patient who presented several severe strokes and carried a heterozygous LMNA missense mutation in exon 2: p.Glu138Lys. This mutation is located far from the C-terminal region implicated in the posttranslational processing of prelamin A, but it lies within the rod domain of lamin A/C that represents a highly conserved domain specific to nuclear lamins. We hypothesize that this region could be involved in early and severe strokes in HGPS, such as those presented by our patient.

Symptomatic dysferlin gene mutation carriers: characterization of two cases.

OBJECTIVE: To describe two symptomatic dysferlin gene mutation carriers. METHODS: One patient had limb girdle weakness. His brother was diagnosed with limb girdle muscular dystrophy 2B with two mutations in the dysferlin gene (D625Y and E1734G). The second patient had distal weakness. He had two sons with Miyoshi myopathy with a homozygous mutation (G519R). We performed immunofluorescence (dystrophin, DAG proteins, dysferlin, caveolin-3), Western blot (dysferlin, caveolin-3, calpain-3), and real-time PCR (dysferlin) using skeletal muscle samples. We also studied dysferlin in peripheral blood monocytes (PBMs) by Western blot. RESULTS: In addition to the muscle weakness, both patients showed elevated creatine kinase and abnormal muscle MRI. They presented a mutation in only one allele after screening of the whole gene (skeletal muscle and monocyte mRNA and genomic DNA). A muscle biopsy specimen showed moderate dystrophic changes and patchy dysferlin expression in the sarcolemma. Western blot of both PBMs and skeletal muscle demonstrated a significant reduction in dysferlin. All the other proteins including caveolin-3 and calpain-3 were normal. Real-time PCR showed normal levels of dysferlin mRNA vs the patients' affected relatives. CONCLUSIONS: The diagnosis of symptomatic carriers of dysferlin mutations should be considered when a pathologic pattern of dysferlin protein is observed.

A collaborative study of the segregation of inherited chromosome structural rearrangements in 1356 prenatal diagnoses.

A collaborative study involving 71 European prenatal diagnosis centres has collected 1356 observations on the karyotype of fetal cells in diagnoses performed in couples in which a parent had a balanced structural rearrangement. The segregations of the inherited chromosome structural rearrangement were analysed in relation to the types of rearrangement, to the sex of the carrier parent, the methods of ascertainment of the anomaly in the family, the chromosomes involved and the potential imbalance of the anomaly. Wide differences in the incidence of unbalanced fetuses were observed in relation to these criteria, and these results can be used in counselling parents with balanced rearrangements. Distortions in segregation of normal versus balanced karyotypes were observed in some types of anomalies.

Prospective risk in reciprocal translocation heterozygotes at amniocentesis as determined by potential chromosome imbalance sizes. Data of the European Collaborative Prenatal Diagnosis Centres.

The Date of the European Cooperative Prenatal Diagnosis Laboratories (Boué and Gallano, 1984) of 596 prenatal (amniocyte) diagnoses of familial rcp was examined as to relationships between balanced/unbalanced result and ascertainment, carrier parent and chromosome imbalance size (percentage haploid autosome length). Each rearrangement was graphed once with actual (unbalanced result) or potential (normal or balanced result) imbalances plotted with trisomy as the ordinate and monosomy as the abscissa. The graphed data was divided into 15 regions, each of 2.0 per cent trisomy and 0.75 per cent monosomy and the rate of unbalanced pregnancies determined for each region. The highest rates of chromosomally unbalanced progeny (excluding regions with inadequate data) were found closest to the origin (i.e. associated with the smallest imbalances) and these were for ascertainment category 1 (previous rcp unbalanced child) 22.3 per cent for maternal carriers and 39 per cent for paternal carriers. Overall in pooled data for this ascertainment category (without reference to the imbalance graphs) there were for paternal carriers 28.6 per cent unbalanced pregnancies and for maternal carriers 18.1 per cent. The graphed data, therefore, revealed the higher rates associated with some of the rcp with small potential (combined duplication/deficiency) imbalances. Lesser rates were observed for ascertainment category 2 (carrier parent with a history of recurrent miscarriage) with overall percentages of imbalanced progeny ranging from 2.7 (paternal carriers) to 4.7 (maternal carriers). Again, higher rates were revealed in graphed data for small potential imbalances. All unbalanced results for this group (ascertainment category 2) plotted in the region closest to the origin with rates of 16 per cent (maternal carriers) and 9.5 per cent (paternal carriers) in this region. Remarkably in both ascertainment groups 1 and 2 there was no significant difference in the size of the imbalanced segments for unbalanced progeny. In ascertainment group 1 this was (dup/def; mean +/- S.D.): 1.09 +/- 0.77/0.47 +/- 0.45 and in ascertainment group 2: 1.09 +/- 0.80/0.66 +/- 0.71. From the graphed data which arguably denote viability relationships, a trisomy was approximately 2.7 times as likely to survive until amniocentesis as a monosomy of equivalent size. It is proposed that given further data, risk estimates could be determined for rcp heterozygotes using the present approach where empiric data (from the family history or an analysed series of similar rcp) is not available.

[Dystrophinopathies]. / Distrofinopatías.

In 1987 a new concept in the X-linked muscular dystrophies was born with the identification of dystrophin, a cytoskeletal protein responsible for several muscular diseases previously grouped as Duchenne's or Becker's muscular dystrophies (DMD and BMD, respectively). Under the new concept these entities are referred to as dystrophinopathies. The discovery of dystrophin was made possible when polymerase chain reaction and reverse genetics opened the door to DNA examination. The DMD/BMD gene was located and sequenced and the protein product, dystrophin, was fully identified. The genetic study of dystrophinopathies was extended in the 1990's to include DNA analysis aimed at 1) discovering points of deletion or mutation that give rise to Xp21 myopathies, as dystrophinopathies are also known; 2) identifying the rules of protein translation (interruption or reading "signals" of the protein's genetic code), and 3) identifying atypical clinical phenotypes that can be diagnosed on a molecular level using anti-dystrophin antibodies or DNA probes.

[Use of dystrophin c-DNA for the direct diagnosis of Duchenne muscular dystrophy in female carriers]. / Empleo del c-ADN de la distrofina para el diagnóstico directo de portadoras de distrofia muscular de Duchenne.

DNA polymorphisms have been widely used as genetic markers for identification of the X chromosome that carries the mutation for Duchenne Muscular Dystrophy (DMD) in affected families, but serious limitations are associated with this approach. The complementary DNA (c-ADN) of the DMD gene has recently been isolated and shown to detect partial gene deletions in a large proportion of patients. We present the study of five DMD families in which we have shown the existence of a partial deletion in the DMD gene in the affected boys. We have evaluated the usefulness of this direct approach to diagnose DMD carriers in these families using the c-DNA derived probes. In all cases we have obtained satisfactory results. The method seems to be more reliable, more rapid and less expensive than linkage studies with DNA polymorphisms.