José M. Fernández

Molecular heterogeneity of myophosphorylase deficiency (McArdle's disease): a genotype-phenotype correlation study.

We report on 54 Spanish patients with McArdles disease from 40 unrelated families. Molecular analysis revealed that the most common R49X mutation was present in 70% of patients and 55% of alleles. The G204S mutation was less frequent and found in 14.8% of patients and 9% of mutant alleles. The W797R mutation was observed in 16.5% of patients, accounting for 13.7% of mutant alleles. Moreover, 78% of mutant alleles among Spanish patients can be identified by using polymerase chain reaction‐restriction fragment length polymorphism analysis for the R49X, G204S, and W797R mutations, which makes noninvasive diagnosis possible through molecular genetic analysis of blood DNA. Six novel mutations were found. Three were missense mutations, E348K, R601W, and A703V; two nonsense mutations, E124X and Q754X; and one single base pair deletion, 533 delA. No clear genotype‐phenotype correlation emerges from our study. Most of the mutations of uncharged and solvent inaccessible residues and the truncations must disrupt the basic structure of the protein. The mutations of charged residues would be expected to interfere with internal hydrogen bonding networks, introducing severe incompatible partnering that is caused by poor packing or electrostatic repulsions.

LG2 agrin mutation causing severe congenital myasthenic syndrome mimics functional characteristics of non-neural (z−) agrin

We describe a severe form of congenital myasthenic syndrome (CMS) caused by two heteroallelic mutations: a nonsense and a missense mutation in the gene encoding agrin (AGRN). The identified mutations, Q353X and V1727F, are located at the N-terminal and at the second laminin G-like (LG2) domain of agrin, respectively. A motor-point muscle biopsy demonstrated severe disruption of the architecture of the neuromuscular junction (NMJ), including: dispersion and fragmentation of endplate areas with normal expression of acetylcholinesterase; simplification of postsynaptic membranes; pronounced reduction of the axon terminal size; widening of the primary synaptic cleft; and, collection of membranous debris material in the primary synaptic cleft and in the subsynaptic cytoplasm. Expression studies in heterologous cells revealed that the Q353X mutation abolished expression of full-length agrin. Moreover, the V1727F mutation decreased agrin-induced clustering of the acetylcholine receptor (AChR) in cultured C2 muscle cells by >100-fold, and phosphorylation of the MuSK receptor and AChR beta subunit by ~tenfold. Surprisingly, the V1727F mutant also displayed increased binding to α-dystroglycan but decreased binding to a neural (z+) agrin-specific antibody. Our findings demonstrate that agrin mutations can associate with a severe form of CMS and cause profound distortion of the architecture and function of the NMJ. The impaired ability of V1727F agrin to activate MuSK and cluster AChRs, together with its increased affinity to α-dystroglycan, mimics non-neural (z−) agrin and are important determinants of the pathogenesis of the disease.

A novel MYH7 mutation links congenital fiber type disproportion and myosin storage myopathy

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patients biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy.

Evaluation of heart involvement in gamma-sarcoglycanopathy (LGMD2C). A study of ten patients

Thorough non-invasive cardiovascular studies were conducted in a series of ten gamma-sarcoglycanopathy Gypsy patients with the founder C283Y mutation in 13q12. Results were compared with those obtained in an age-matched group of normal boys and girls. The studies included electrocardiographic and echocardiographic evaluations using pulsed wave Doppler tissue imaging to assess regional diastolic function and myocardial velocities at various levels. This study confirms the significant electrocardiographic abnormalities described in previous studies. Furthermore, measurement of myocardial velocity at different levels demonstrated an abnormal relaxation pattern in the tricuspid annulus in four of the oldest patients, which strongly suggests intrinsic myocardial involvement of the right ventricle. To our knowledge, these specific studies have not been previously performed in a clinically and genetically homogeneous group of gamma-sarcoglycanopathy patients and suggest primary myocardial involvement probably due to gamma-sarcoglycan deficiency in cardiac muscle fibres. Our results could be of interest in the follow-up of these patients and the prevention and treatment of late cardiological complications.

Molecular and clinical study of McArdle's disease in a cohort of 123 European patients. Identification of 20 novel mutations.

McArdles disease is the most common muscle glycogenosis. It is caused by the deficiency of myophosphorylase, encoded by the PYGM gene. We studied 123 patients previously diagnosed with McArdles disease and we identified 20 novel mutations (10 missense and 3 nonsense mutations, 3 small deletions, 2 gross deletions and 2 small insertions). Most patients of this cohort belong to Spanish and French populations. This allowed us to determine the differences between the allelic frequencies of the common mutations R50X and G205S of these populations. The R50X has an allelic frequency in this cohort of about 61.7%, being 68.5% in French and 53.7% in Spanish patients. The G205S had a higher allelic frequency in the Spanish (10.2%) than in the French population (3.2%). Moreover, a clinical study of 91 patients was performed to establish both genotype-phenotype correlation and gender influence in the severity of the disease. We conclude that no genotype-phenotype correlation is evident and that no gender effect is related to the phenotype.

Fabry disease: an ultrastructural comparative study of skin in hemizygous and heterozygous patients

Fabry disease is a rare X-linked lysosomal storage disorder due to alpha galactosidase A deficiency, better known after the advent of a promising treatment, a periodical enzyme replacement. As other hereditary X-linked disorders, females have historically been considered non-affected carriers, although they are, actually, clinically and pathologically affected to a variable degree. Some women are asymptomatic, but the majority present milder forms of the disease and later onset. This wide range of disease expression is supposed to be related to the levels of enzymatic activity, probably in accordance with a skewing of X inactivation. Lysosomal deposits of ceramide trihexoside have been repeatedly documented in a wide range of tissues, including those found in angiokeratoma, the characteristic cutaneous lesion which allowed the definition of Fabry disease. The aim of this study was to investigate whether there was any difference in the amount of dermal lysosomal storage in males and females, thus accounting for the difference in clinical severity of both groups. For that purpose, with electron microscopy and quantitative methods, we studied the extent of lysosomal deposits in dermal fibroblasts of normal-appearing skin in six females and nine men, enzymatically and genetically proven as to have Fabry disease, and results were compared. Our results indicate a statistically significant difference between the two groups regarding both the percentage of dermal fibroblasts bearing stored material, and the storage surface occupied in 100 fibroblasts per case. We suggest that periodical ultrastructural examination of normal-appearing skin could be an indicator of the efficacy of enzyme replacement therapy and could help to evaluate results.

Adult glycogenosis II with paracrystalline mitochondrial inclusions and Hirano bodies in skeletal muscle

Hirano bodies constitute eosinophilic intracytoplasmic inclusions, typically seen in the central nervous system, where they are related to senility and certain dementias such as Alzheimers disease or the Parkinson-dementia complex. They have been found in different tissues of experimental animals and, on rare occasions, in extraocular muscles of elderly individuals. However, to our knowledge they have not been described in skeletal muscle in locations other than extraocular muscles or associated with muscle pathology. Glycogenosis II or Pompes disease, is a metabolic disorder caused by acid maltase deficiency and is characterized by glycogen accumulation in lysosomes in various tissues, including skeletal muscle. There are three clinical forms depending on age at onset, the most frequent being the childhood form. We present the histopathological and ultrastructural findings of a muscle biopsy performed in a case of the adult form of glycogenosis II which showed, in addition to characteristic lysosomal glycogen storage, paracrystalline mitochondrial inclusions and, as an exceptional finding, intracytoplasmic Hirano bodies in some muscle fibres.

Myoadenylate deaminase deficiency: clinico-pathological and molecular study of a series of 27 Spanish cases.

Myoadenylate deaminase deficiency (MADD) is the most common metabolic muscle disorder. Here we report the largest study to date of MADD in Spanish patients, including clinical, histological, and molecular data. Most of the patients presented with moderate clinical symptoms of exercise intolerance, including myalgia, fatigability and cramps. In 70% of the patients, serum creatine kinase (CK) was elevated. Muscle biopsy showed mild, nonspecific alterations with absent histochemical reaction for MAD. Eight cases ofMADD were coincidental with other associated diseases, and had more severe tissue alterations upon muscle biopsy. The mutation C34T in the MAD gene was present in a homozygous state in 26 of the 27 patients. One patient was a compound heterozygote for the C34T/G468T mutations. We conclude that MADD should be suspected in patients with exercise intolerance and with idiopathic hyperCKemia. Since symptoms may be subtle, we recommend routine histochemical analysis of MAD in all muscle biopsies, followed by molecular analysis in MAD-negative cases.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) Report of a Spanish case with extended clinicopathological follow-up.

Background: Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a clinically and genetically distinct and uncommon variant of SMA that results from irreversible degeneration of α-motor neurons in the anterior horns of the spinal cord and in ganglion cells on the spinal root ganglia. Aims: To describe the clinical, electrophysiological, neuropathological, and genetic findings, at different stages from birth to death, of a Spanish child diagnosed with SMARD1. Patient and methods: We report the case of a 3-month-old girl with severe respiratory insufficiency and, later, intense hypotonia. Paraclinical tests included biochemistry, chest X-ray, and electrophysiological studies, among others. Muscle and nerve biopsies were performed at 5 and 10 months and studied under light and electron microscopy. Post-mortem examination and genetic investigations were performed. Results: Pre- and post-mortem histopathological findings demonstrated the disease progression over time. Muscle biopsy at 5 months of age was normal, however a marked neurogenic atrophy was present in post-mortem samples. Peripheral motor and sensory nerves were severely involved likely due to a primary axonal disorder. Automatic sequencing of IGHMBP2 revealed a compound heterozygous mutation. Conclusions: The diagnosis of SMARD1 should be considered in children with early respiratory insufficiency or in cases of atypical SMA. Direct sequencing of the IGHMBP2 gene should be performed.

Histopathology of Skin in Fabry Disease

Fabry disease (FD) is a lysosomal storage disorder (LSD) with multisystem manifestations among which skin lesions are common and well-recognized. They may appear early in childhood in both hemizygous males and heterozygous females, their frequency increasing with age. In addition to dermatological lesions, normal-looking skin, as occurs with other tissues, accumulates unmetabolized products in multiple cells of different embryonic origin with variable distribution, biochemical and staining properties and morphological features, according to the type of stored material and the physiopathology of the disease. Globotryaosil-ceramide (Gb3), the most abundant glycosphyngolipid accumulated in FD, can be specifically documented with immunocytochemical methods using a particular monoclonal antibody. On the other hand, ultrastructural characteristics of Gb3 and its distribution in dermal cells are well-defined. Furthermore, the study of unmyelinated and small myelinated fibers in epidermal nerve endings is a reflection of the impaired sensory innervation and the characteristic painful neuropathy present in FD. Therefore, a combination of different technologies in tissues obtained with noninvasive techniques, such as skin biopsy, is advisable and should be considered in order to obtain better insights into FD. This is essential in the case of heterozygous women in whom biochemical diagnosis is often controversial.