Vincenzo Nigro

Retinoic acid induces stage-specific antero-posterior transformation of rostral central nervous system

We report a time-course analysis of the effect of retinoic acid (RA) on the development of the mouse central nervous system (CNS) from the beginning of gastrulation throughout induction and patterning of the neural tube. RA administration induces three different, stage-specific alterations of brain development, indicating perturbation of different morphogenetic steps during the establishment of a neural pattern. In particular, treatment at mid-late streak stage (7.2-7.4 days post coitum (d.p.c.)) results in early repression of Otx2 expression in the posterior neuroectoderm of the head fold and in the ventral mid line, including the prechordal plate and the rostralmost endoderm, followed by loss of forebrain morphological and molecular identities, as revealed by analysis of the expression of regionally-restricted brain genes (Otx2, Otx1, Emx2, Emx1 and Dlx1). In these embryos, reduction of the Otx2 expression domain correlates with hindbrain expansion marked by rostral extension of the Hoxb-1 expression domain. Our analysis indicates that RA interferes with the correct definition of both planar and vertical morphogenetic signals at specific developmental stages by affecting gene expression in the regions which are likely either to produce or to respond to these signals. We suggest that retinoids may contribute to early definition of head from trunk structures by selecting different sets of regulatory genes.

Orthopedia, a novel homeobox-containing gene expressed in the developing CNS of both mouse and drosophila

A novel homeobox-containing gene has been identified. Its name, Orthopedia (Otp), exemplifies the homology shared by both the orthodenticle and Antennapedia homeodomains. Otp is highly conserved in evolution. In mouse, Otp is expressed only in restricted domains of the developing forebrain, hindbrain, and spinal cord. In Drosophila, otp first appears at gastrulation in the ectodermal proctodeum and later in the hindgut, anal plate, and along the CNS. Here, we compare the Otp-, Distal-less homeobox 1-(DIx1-), Orthodenticle homolog 1-(Otx1-), Otx2-, and Empty spiracles homolog 2-expressing domains. Our results indicate that Otp is expressed along the CNS both in mouse and Drosophila; Otp could specify regional identities in the development of the forebrain and spinal cord; transcription of Otp and DIx1 takes place in alternating hypothalamic regions reminiscent of a segment-like pattern; and the structural and functional conservation could correspond to a conserved function maintained in evolution.

Muscle MRI findings in patients with limb girdle muscular dystrophy with calpain 3 deficiency (LGMD2A) and early contractures.

Limb girdle muscular dystrophy 2A is a common variant secondary to mutations in the calpain 3 gene. A proportion of patients has early and severe contractures, which can cause diagnostic difficulties with other conditions. We report clinical and muscle magnetic resonance imaging findings in seven limb girdle muscular dystrophy 2A patients (four sporadic and three familial) who had prominent and early contractures. All patients showed a striking involvement of the posterior thigh muscles. The involvement of the other thigh muscles was variable and was related to clinical severity. Young patients with minimal functional motor impairment showed a predominant involvement of the adductors and semimembranosus muscles while patients with restricted ambulation had a more diffuse involvement of the posterolateral muscles of the thigh and of the vastus intermedius with relative sparing of the vastus lateralis, sartorius and gracilis. At calf level all patients showed involvement of the soleus muscle and of the medial head of the gastrocnemius with relative sparing of the lateral head. MRI findings were correlated to those found in two patients with the phenotype of limb girdle muscular dystrophy 2A without early contractures and the pattern observed was quite similar. However, the pattern observed in limb girdle muscular dystrophy 2A is different from that reported in other muscle diseases such as Emery-Dreifuss muscular dystrophy and Bethlem myopathy which have a significant clinical overlap with limb girdle muscular dystrophy 2A once early contractures are present. Our results suggest that muscle MRI may help in recognising patients with limb girdle muscular dystrophy 2A even when the clinical presentation overlaps with other conditions, and may therefore, be used as an additional investigation to target the appropriate biochemical and genetic tests.

γ1- and γ2-Syntrophins, Two Novel Dystrophin-binding Proteins Localized in Neuronal Cells

Dystrophin is the scaffold of a protein complex, disrupted in inherited muscular dystrophies. At the last 3′ terminus of the gene, a protein domain is encoded, where syntrophins are tightly bound. These are a family of cytoplasmic peripheral membrane proteins. Three genes have been described encoding one acidic (α1) and two basic (β1 and β2) proteins of ∼57–60 kDa. Here, we describe the characterization of two novel putative members of the syntrophin family, named γ1- and γ2-syntrophins. The human γ1-syntrophin gene is composed of 19 exons and encodes a brain-specific protein of 517 amino acids. The human γ2-syntrophin gene is composed of at least 17 exons, and its transcript is expressed in brain and, to a lesser degree, in other tissues. We mapped the γ1-syntrophin gene to human chromosome 8q11 and the γ2-syntrophin gene to chromosome 2p25. Yeast two-hybrid experiments and pull-down studies showed that both proteins can bind the C-terminal region of dystrophin and related proteins. We raised antibodies against these proteins and recognized expression in both rat and human central neurons, coincident with RNA in situ hybridization of adjacent sections. Our present findings suggest a differentiated role of a modified dystrophin-associated complex in the central nervous system.

Diagnostic value of muscle MRI in differentiating LGMD2I from other LGMDs

Mutations in the fukutin–related protein (FKRP) have recently been demonstrated to cause limb girdle muscular dystrophy type 2I (LGMD2I), one of the most common forms of the autosomal recessive LGMDs in Europe. We performed a systematic clinical and muscle MRI assessment in 6 LGMD2I patients and compared these findings with those of 14 patients with genetically confirmed diagnosis of other forms of autosomal recessive LGMDs or dystrophinopathies. All LGMD2I patients had a characteristic clinical phenotype with predominant weakness of hip flexion and adduction, knee flexion and ankle dorsiflexion. These findings were also mirrored on MRI of the lower extremities which demonstrated marked signal changes in the adductor muscles, the posterior thigh and posterior calf muscles. This characteristic clinical and MRI phenotype was also seen in LGMD2A. However, in LGMD2A there was a selective involvement of the medial gastrocnemius and soleus muscle in the lower legs which was not seen in LGMD2I. The pattern in LGMD2A and LGMD2I were clearly different from the one seen in alpha–sarcoglycanopathy and dystrophinopathy type Becker which showed marked signal abnormalities in the anterior thigh muscles. Our results indicate that muscular MRI is a powerful tool for differentiating LGMD2I from other forms of autosomal recessive LGMDs and dystrophinopathies.

Low frequency of melanocortin-4 receptor (MC4R) mutations in a Mediterranean population with early-onset obesity.

Background: Melanocortin-4 receptor (MC4R) mutations have been reported as the most common single genetic cause of obesity in some populations and it has been suggested that they may be responsible for more than 4% of early-onset obesity.Objectives: To verify the presence of mutations of the MC4R coding region in children from southern Italy with early-onset obesity.Subjects and Methods: Two-hundred and eight unrelated obese children and adolescents were included in the study. The average age at obesity onset was 4.5±2.6 y. MC4R coding region was screened using both single-strand conformation polymorphism (SSCP) analysis and denaturing high-performance liquid chromatography (DHPLC). Automatic sequencing of PCR products of all individuals that showed an aberrant SSCP and/or DHPLC pattern was performed.Results: One novel missense mutation and one previously described polymorphism (Vall03Ile) were identified. The missense mutation C142T, resulting in the substitution of proline with serine at codon 48, within the first MC4R transmembrane domain, was detected at the heterozygous state in a 15-y-old obese girl (body mass index (BMI)=35 kg/m2) who has been obese since she was 8 y old. The mutation co-segregated with the obesity phenotype for over three generations and was not found in the control population.Conclusions: Our data show MC4R obesity causing mutations in less than 0.5% of the patients (ie 1 out of 208 patients) and therefore indicate a low prevalence of MC4R variants in the obese population from southern Italy. The specific genetic background of the Mediterranean population could make it difficult for MC4R mutations to produce an essentially polygenic trait such as common obesity, at least during childhood.

Mutation of dystrophin gene and cardiomyopathy

The correlations between the type of gene mutation and the cardiac clinical picture were examined in 284 patients with dystrophinopathy (200 Duchenne and 84 Becker). The subjects with normal heart showed deletions including exons 48-49 in 21.4% DMD and in 25% BMD, and other deletions in 35.7% DMD and 25% BMD; vice versa the cases with severe cardiac involvement showed deletions including 48-49 in 38.8% DMD and 37.5% BMD and other deletions in 32.9% DMD and 20% BMD. The age of death was 18 years in DMD patients with deletions including 48-49 whereas the age was about 22 in the cases with other deletions. The differences were statistically significant.

Clinical variability in calpainopathy: What makes the difference?

Limb girdle muscular dystrophies (LGMD) are a heterogeneous group of genetic disorders characterised by progressive weakness of the pelvic and shoulder girdle muscles and a great variability in clinical course. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the calpain-3 gene (CAPN-3). More than 100 pathogenic mutations have been identified to date, however few genotype : phenotype correlation studies, including both DNA and protein analysis, have been reported. In this study we screened 26 unrelated LGMD2A Brazilian families (75 patients) through Single-Stranded Conformation Polymorphism (SSCP), Denaturing high-performance liquid chromatography (DHPLC) and sequencing of abnormal fragments which allowed the identification of 47 mutated alleles (approximately 90%). We identified two recurrent mutations (R110X and 2362-2363AG>TCATCT) and seven novel pathogenic mutations. Interestingly, 41 of the identified mutations (approximately 80%) were concentrated in only 6 exons (1, 2, 4, 5, 11 and 22), which has important implications for diagnostic purposes. Protein analysis, performed in 28 patients from 25 unrelated families showed that with exception of one patient (with normal/slight borderline reduction of calpain) all others had total or partial calpain deficiency. The effects of type of mutation, amount of calpain in the muscle, gender and ethnicity of affected patients on clinical course (age of onset and ascertainment) were analysed. Interestingly, it was observed that, on average, African–Brazilian calpainopathy patients are more severely affected than Caucasians.

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes

Background: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. Objective: To obtain unbiased information on the consequences of CAPN3 mutations. Patients: 530 subjects with different grades of symptoms and 300 controls. Methods: High throughput denaturing HPLC analysis of DNA pools. Results: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. Conclusions: A non-invasive and cost–effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

Limb girdle muscular dystrophies: update on genetic diagnosis and therapeutic approaches

PURPOSE OF REVIEW This review is an up-to-date analysis of the genetic diagnosis and therapeutic strategies for limb girdle muscular dystrophies (LGMDs). RECENT FINDINGS LGMDs are an example of both clinical and genetic heterogeneity. Clinically, by the description of non-LGMD phenotypes associated with LGMD genes and of LGMD phenotypes associated with originally non-LGMD disease genes; and genetically, by the description of new LGMD genes that further increase the diagnostic complexity. Moreover, new powerful approaches for DNA analysis, such as exome sequencing, promise to revolutionize the field of heterogeneous genetic diseases, also providing information about the true penetrance of LGMD mutations. The recent inputs on novel pathogenic mechanisms and pathways in LGMD will suggest novel therapeutic approaches and future clinical trials. In addition, therapeutic approaches of gene and cell delivery into animal models show promising results that will be translated into clinical trials. SUMMARY The genetic diagnosis of LGMD from the present home-made algorithms will move toward high-throughput diagnostic strategies based on next-generation sequencing (NGS) technologies. As therapy, new powerful drug approaches based on recent pathogenetic findings will be pushed to clinical trials. In addition, novel more efficient and safer viral vectors for gene delivery will be proposed.