Isabel Fineza

l-2-Hydroxyglutaric aciduria: clinical, biochemical and magnetic resonance imaging in six Portuguese pediatric patients

We present clinical, biochemical and cranial magnetic resonance imaging data of six pediatric patients with L-2-hydroxyglutaric aciduria. All the children have the same ethic origin and lived in the northern area of Portugal. Our findings reinforce the described phenotype of this rare metabolic disease with mental deficiency, severe cerebellar dysfunction, mild extrapyramidal and pyramidal symptoms, progressive macrocephaly and seizures. Magnetic resonance imaging revealed subcortical leukoencephalopathy, cerebellar atrophy and signal changes in the putamina and dentate nuclei. These were similar to those of the previous reports in all patients. The urinary excretion of L-2-hydroxyglutaric acid was variably increased in all patients. The other persistent biochemical abnormality was hyperlysinemia. We have found a strong correlation between the severity of the clinical manifestations and the extension of the lesions in the neuroimaging studies. There was no correlation between the clinical findings and the amount of urinary excretion of L-2-hydroxyglutaric acid. We report the second case in the literature of a cerebral thalamic tumor in L-2-hydroxyglutaric aciduria; neuropathological examination of the surgical biopsy demonstrated a diffuse fibrillary astrocytoma.

LAMA2 gene analysis in a cohort of 26 congenital muscular dystrophy patients

Congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin‐α2. We describe the molecular study of 26 patients with clinical presentation, magnetic resonance imaging and/or laminin‐α2 expression in muscle, compatible with MDC1A. The combination of full genomic sequencing and complementary DNA analysis led to the particularly high mutation detection rate of 96% (50/52 disease alleles). Besides 22 undocumented polymorphisms, 18 different mutations were identified in the course of this work, 14 of which were novel. In particular, we describe the first fully characterized gross deletion in the LAMA2 gene, encompassing exon 56 (c.7750‐1713_7899‐2153del), detected in 31% of the patients. The only two missense mutations detected were found in heterozygosity with nonsense or truncating mutations in the two patients with the milder clinical presentation and a partial reduction in muscle laminin‐α2. Our results corroborate the previous few genotype/phenotype correlations in MDC1A and illustrate the importance of screening for gross rearrangements in the LAMA2 gene, which may be underestimated in the literature.

New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing

Muscular dystrophies (MDs) are a group of hereditary muscle disorders that include two particularly heterogeneous subgroups: limb-girdle MD and congenital MD, linked to 52 different genes (seven common to both subgroups). Massive parallel sequencing technology may avoid the usual stepwise gene-by-gene analysis. We report the whole-exome sequencing (WES) analysis of a patient with childhood-onset progressive MD, also presenting mental retardation and dilated cardiomyopathy. Conventional sequencing had excluded eight candidate genes. WES of the trio (patient and parents) was performed using the ion proton sequencing system. Data analysis resorted to filtering steps using the GEMINI software revealed a novel silent variant in the choline kinase beta (CHKB) gene. Inspection of sequence alignments ultimately identified the causal variant (CHKB:c.1031+3G>C). This splice site mutation was confirmed using Sanger sequencing and its effect was further evaluated with gene expression analysis. On reassessment of the muscle biopsy, typical abnormal mitochondrial oxidative changes were observed. Mutations in CHKB have been shown to cause phosphatidylcholine deficiency in myofibers, causing a rare form of CMD (only 21 patients reported). Notwithstanding interpretative difficulties that need to be overcome before the integration of WES in the diagnostic workflow, this work corroborates its utility in solving cases from highly heterogeneous groups of diseases, in which conventional diagnostic approaches fail to provide a definitive diagnosis.

Novel synonymous substitution in POMGNT1 promotes exon skipping in a patient with congenital muscular dystrophy

AbstractWalker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, congenital muscular dystrophy type 1C, and congenital muscular dystrophy type 1D are overlapping clinical entities belonging to a subgroup of the congenital muscular dystrophies (CMD), collectively designated dystroglycanopathies, in which the common underlying defect is hypoglycosylation of alfa-dystroglycan. Currently, six different genes are known to be implicated in these diseases: POMT1, POMT2, POMGNT1, FCMD, FKRP, and LARGE. We report the molecular characterization of a patient presenting clinical features of CMD and reduced immunostaining for alfa-dystroglycan in muscle. Three candidate genes (FCMD, POMT1 and POMGNT1) were analyzed, and a total of 18 sequence variants were detected: 15 polymorphisms in POMT1 [including three unreported single nucleotide polymorphisms (SNPs)], two polymorphisms in FCMD, and the exonic silent mutation c.636C > T in POMGNT1. Expression analysis revealed that this apparently silent mutation compromises correct premessenger RNA (mRNA) splicing, promoting skipping of the entire exon 7, with a consequent frameshift. In silico analysis of this mutation did not predict alterations in the canonical splice sequences, but rather the creation of a new exonic splice silencer. The recognition of such disease-causing elements may contribute to the further understanding of RNA processing and assist mutation screening in routine diagnosis, where such changes may be underestimated. To aid clinical diagnosis, we generated publicly available LOVD-powered Locus Specific Databases for these three genes and recorded all known sequence variants (http://www.dmd.nl).

Autosomal Recessive Axonal Neuropathy With Neuromyotonia: A Rare Entity

BACKGROUNDnAutosomal recessive axonal neuropathy with neuromyotonia is a recently described entity associated to the HINT1 gene, encoding histidine triad nucleotide-binding protein 1.nnnPATIENTnThe authors report a Portuguese 16-year-old girl of Roma ethnicity, descendant of consanguineous parents, with progressive distal muscular atrophy and weakness, beginning at age 6. After several years of extensive investigation with inconclusive results, clinical myotonia was identified. Electrophysiologic studies revealed neuromyotonia associated with a severe chronic predominantly motor axonal neuropathy and homozygous mutation (c.334 C > A, p.H112 N) in HINT1 was detected.nnnCONCLUSIONnThis report emphasizes the late onset of clinical myotonia essential to the diagnosis.

New variants, challenges and pitfalls in DMD genotyping: implications in diagnosis, prognosis and therapy

Molecular characterization of patients with Duchenne or Becker muscular dystrophies is essential for establishing a differential diagnosis, allowing appropriate clinical follow-up, patient management and genetic counseling. In light of the recent mutation-based therapeutic approaches, DMD gene analysis has gained further relevance. Owing to the size and complexity of the DMD gene and the diversity of mutation types, molecular analysis is not always a straightforward task requiring the combination of several methodologies. Our national genetic diagnostic service genetically characterized 308 dystrophinopathy patients (284 unrelated families), leading to the identification of 175 distinct mutations, including 39 unpublished variants. These studies revealed several potential diagnostic pitfalls (because of technical limitations or related with DMD’s genetic heterogeneity) that may be overlooked even considering the international disease-specific diagnostic guidelines. Comprehensive analysis involved expression studies at the mRNA level, the identification of splicing changes and ultimately providing evidence for apparent exceptions to the reading-frame rule. Besides increasing the mutation detection rate, this detailed molecular characterization is indispensable for the identification of suitable candidates for the new mutation-centered therapies. As patient registries are internationally recognized as essential for clinical trial recruitment, this led us to develop the Portuguese Duchenne and Becker Muscular Dystrophy registry in collaboration with the Translational Research in Europe—Assessment and Treatment of Neuromuscular Diseases network.

Value of Brain Magnetic Resonance Imaging in Mitochondrial Respiratory Chain Disorders

Mitochondrial respiratory chain (MRC) disorders have variable clinical manifestations which are mainly neurologic. Diagnosis in children is more complex than in adults because the classic phenotype, ragged red fibers, and mtDNA mutations are rarely seen in children. Moreover, clinical manifestations of disease in developing brains are less explicit. Although not specific, neuroimaging may be contributory to the diagnosis of these disorders in pediatric patients. Brain magnetic resonance images were reviewed for 133 pediatric patients investigated for a MRC disorder at a single center over a period of 10 years (1997-2006), in an attempt to identify distinctive neuroimaging features of MRC defects. Patients fit into four groups, according to the Bernier criteria: definite (63 cases), probable (53 cases), possible (7 cases) and unlikely diagnosis (10 cases). Brain atrophy (41 cases), supratentorial white matter lesions (14 cases), basal ganglia involvement (9 cases), and delayed myelination (9 cases) were the most frequent anomalies in the definite group, and 8 patients presented Leigh syndrome. Neuroimaging findings of the 63 children in the definite group were compared with the remainder and with those in the possible and unlikely groups. There were no significant differences in brain images between the groups analyzed, and therefore no distinctive brain imaging features were identified specific for MRC disorders.

Reviewing Large LAMA2 Deletions and Duplications in Congenital Muscular Dystrophy Patients

BACKGROUNDnCongenital muscular dystrophy (CMD) type 1A (MDC1A) is caused by recessive mutations in laminin-α2 (LAMA2) gene. Laminin-211, a heterotrimeric glycoprotein that contains the α2 chain, is crucial for muscle stability establishing a bond between the sarcolemma and the extracellular matrix. More than 215 mutations are listed in the locus specific database (LSDB) for LAMA2 gene (May 2014).nnnOBJECTIVEnA limited number of large deletions/duplications have been reported in LAMA2. Our main objective was the identification of additional large rearrangements in LAMA2 found in CMD patients and a systematic review of cases in the literature and LSDB.nnnMETHODSnIn four of the fifty-two patients studied over the last 10 years, only one heterozygous mutation was identified, after sequencing and screening for a frequent LAMA2 deletion. Initial screening of large mutations was performed by multiplex ligation-dependent probe application (MLPA). Further characterization implied several techniques: long-range PCR, cDNA and Southern-blot analysis.nnnRESULTSnThree novel large deletions in LAMA2 and the first pathogenic large duplication were successfully identified, allowing a definitive molecular diagnosis, carrier screening and prenatal diagnosis. A total of fifteen deletions and two duplications previously reported were also reviewed. Two possible mutational hotspots for deletions may exist, the first encompassing exons 3 and 4 and second in the 3 region (exons 56 to 65) of LAMA2.nnnCONCLUSIONSnOur findings show that this type of mutation is fairly frequent (18.4% of mutated alleles) and is underestimated in the literature. It is important to include the screening of large deletions/duplications as part of the genetic diagnosis strategy.

A Portuguese case of Fukuyama congenital muscular dystrophy caused by a multi-exonic duplication in the fukutin gene.

Fukuyama congenital muscular dystrophy (FCMD) is one of the most common autosomal recessive diseases among the Japanese population, due to a founder mutation of the fukutin gene (FKTN). Mutations in FKTN are now being described in an increasing number of non-Japanese patients. We report a Portuguese child with FCMD. The diagnosis was supported by clinical, histological, magnetic resonance imaging (MRI) and genetic studies. Genetic analysis of FKTN by Multiplex Ligation Probe Amplification (MLPA) revealed a homozygous duplication from exon 4 to exon 7. This in-frame duplication was confirmed by cDNA analysis. To our knowledge this is the first report of a FCMD case caused by an intragenic gross exonic duplication in the FKTN gene. This report widens the clinical and mutational spectrum in FCMD and corroborates the importance of screening for large deletions and duplications in CMD patients.