Nicola Carboni

Epidemiology of multiple sclerosis in south-western Sardinia

Background: Sardinia is a known high-risk area for multiple sclerosis (MS), but no data for south-western Sardinia (SWS) are available. SWS has a genetically homogeneous population, apart from St Peter Island, and represents a peculiar environment related to the industrial, mineralogical and military economy. Objective: To estimate prevalence and incidence and to evaluate temporal trends and geographical distribution of MS in SWS. Methods: MS prevalence was evaluated on 31 December 2007 and crude mean annual incidence rate was defined between 2003 and 2007. Temporal trend in MS incidence was assessed using the Armitage test. To identify MS clusters, Standard Morbidity Ratio (SMR) was calculated for each village and geographical distribution prevalence by means of a Bayesian hierarchical model. Results: Total crude prevalence rate was 210.4 (95% CI 186.3–234.5): 280.3 (95% CI 241.4–319.3) for females, 138 (95% CI 110.1–165.8) for males. The crude mean annual incidence rate was 9.7/100,000 (95% CI 3.4–13.2): 4.7/100,000 (95% CI 2.4–17.0) and 14.6/100,000 (95% CI 11.8–34.8) for males and females respectively. MS incidence has increased over the last 50 years. Cluster analysis showed an SMR of 0.2 (95% CI 0.05–0.68, p = 0.002) on the island of San Pietro, and 2.0 (95% CI 1.35–2.95, p = 0.001) in Domusnovas. Spatial distribution of MS was confirmed by Bayesian geographical analysis. Conclusions: Our data confirm Sardinia as a high-risk area for MS and support the relevance of genetic factors in MS, as evidenced in St Peter Island. However, we found an unexpectedly high MS prevalence in one village, in particular in males, suggesting an environmental influence on MS occurrence.

MUSCLE IMAGING ANALOGIES IN A COHORT OF PATIENTS WITH DIFFERENT CLINICAL PHENOTYPES CAUSED BY LMNA GENE MUTATIONS

Laminopathies are a heterogeneous group of LMNA‐gene‐mutation–related clinical disorders associated with alterations of cardiac and skeletal muscle and peripheral nerves, metabolic defects, and premature aging. Leg muscle imaging investigations were performed in a cohort of patients with LMNA gene alterations who were suffering from Emery–Dreifuss muscular dystrophy, limb‐girdle muscular dystrophy type 1B, isolated cardiac disorders or a phenotype of cardiac disorders, and lipodystrophy, including one individual with peripheral neuropathy. Leg muscle imaging revealed varying degrees of alteration in the soleus and medial head of gastrocnemius in each subject. This study demonstrates that LMNA‐gene‐mutated patients devoid of any clinically detectable skeletal muscle involvement have the same pattern of leg muscle involvement as patients with overt skeletal muscle compromise. This finding suggests the presence of a continuum of skeletal muscle involvement among phenotypes of LMNA‐gene‐mutation–related skeletalmyopathy and cardiomyopathy. Muscle Nerve, 2010

LMNA-associated myopathies The Italian experience in a large cohort of patients

Objectives: Our aim was to conduct a comparative study in a large cohort of myopathic patients carrying LMNA gene mutations to evaluate clinical and molecular features associated with different phenotypes. Methods: We performed a retrospective cohort study of 78 myopathic patients with LMNA mutation and 30 familial cases with LMNA mutation without muscle involvement. We analyzed features characterizing the various forms of LMNA-related myopathy through correlation statistics. Results: Of the 78 patients, 37 (47%) had limb-girdle muscular dystrophy 1B (LGMD1B), 18 (23%) congenital muscular dystrophy (MDCL), 17 (22%) autosomal dominant Emery-Dreifuss muscular dystrophy 2 (EDMD2), and 6 (8%) an atypical myopathy. The myopathic phenotypes shared a similar cardiac impairment. Cardioverter defibrillator or pacemaker was implanted in 41 (53%) myopathic patients compared to 7 (23%) familial cases without muscle involvement (p = 0.005). Heart transplantation was performed in 8 (10.3%) myopathic patients and in none of the familial cases. Ten (12.8%) myopathic patients died; there were no deaths among the familial cases (p = 0.032). Missense mutations were found in 14 patients (82%) with EDMD2 and 14 patients (78%) with MDCL compared to 17 patients (45%) with LGMD1B and 4 (67%) atypical patients. Frameshift mutations were detected in 17 (45%) LGMD1B compared to 3 (18%) EDMD2, 1 (6%) MDCL, and 2 (33%) with atypical myopathy (p = 0.021). Furthermore, frameshift mutations were found in 30 of 73 patients (41%) with heart involvement compared to 4 of 35 (11%) without heart involvement (p = 0.004). Conclusions: Our data provided new insights in LMNA-related myopathies, whose natural history appears to be dominated by cardiac involvement and related complications.

Muscle MRI findings in patients with an apparently exclusive cardiac phenotype due to a novel LMNA gene mutation

The case of a family in which several members displayed conduction defects inherited as a dominant trait is reported. The proband was a young woman with a 1st degree atrio-ventricular block and high serum creatine kinase. Several members of the family featured cardiologic symptoms. All adult family members were clinically evaluated and blood tests including serum creatine-kinase levels, standard and Holter ECG, echocardiogram and muscle MRI were performed. LMNA gene analysis was carried out and a novel missense mutation consisting in substitution of exon 4 c.799 T/C, p.Tyr267His was revealed. The mutation was present in seven family members, five of whom displayed cardiac defects alone with no involvement of the skeletal muscle. In all mutated individuals muscle MRI featured a pattern of skeletal muscle involvement similar to that observed in autosomal dominant Emery Dreifuss muscular dystrophy, suggesting that even patients bearing a LMNA gene mutation associated to an apparently selective cardiac phenotype may present subclinical skeletal muscle involvement.

Influence of treatments in multiple sclerosis disability: A cohort study

Background and objective: A critical aspect of multiple sclerosis (MS) treatments is understanding the effect of disease-modifying drugs (DMDs) on the long-term risk of disability and whether the effect is related to disability at start of treatment. Methods: We performed an observational study on 3060 MS patients. The effect of therapy on progression to Expanded Disability Status Scale (EDSS) 3.0 and 6.0 from onset was analysed in treated vs untreated (UTP) patients using Cox regression analysis adjusted for propensity score and immortal time bias. Results: Compared to UTP, the risks of EDSS 3.0 were 94% and 73% lower in immunomodulant (IMTP-) and immunosuppressant (ISTP-) treated patients, respectively, while the risk of EDSS 6.0 was 86% lower in IMTP. The risk of EDSS 6.0 was, respectively, 91% and 75% lower in 1275 IMTP before and 114 after EDSS 3.0 than in 539 UTP; the risk was higher in IMTP starting therapy after EDSS 3.0 than before (HR = 4.42). Conclusions: DMDs delayed long-term disability in MS patients treated either in the early or, to a lesser extent, in the later phase of the disease. Thus, the window of therapeutic opportunity is relatively extended, assuming that early is better than late treatment, but late is better than never.

Clinical and molecular characterization of limb-girdle muscular dystrophy due to LAMA2 mutations

In this study we describe the clinical and molecular characteristics of limb‐girdle muscular dystrophy (LGMD) due to LAMA2 mutations.

Skeletal Muscle Laminopathies: A Review of Clinical and Molecular Features

LMNA-related disorders are caused by mutations in the LMNA gene, which encodes for the nuclear envelope proteins, lamin A and C, via alternative splicing. Laminopathies are associated with a wide range of disease phenotypes, including neuromuscular, cardiac, metabolic disorders and premature aging syndromes. The most frequent diseases associated with mutations in the LMNA gene are characterized by skeletal and cardiac muscle involvement. This review will focus on genetics and clinical features of laminopathies affecting primarily skeletal muscle. Although only symptomatic treatment is available for these patients, many achievements have been made in clarifying the pathogenesis and improving the management of these diseases.

Vitamin D Responsive Elements within the HLA-DRB1 Promoter Region in Sardinian Multiple Sclerosis Associated Alleles

Vitamin D response elements (VDREs) have been found in the promoter region of the MS-associated allele HLA-DRB1*15∶01, suggesting that with low vitamin D availability VDREs are incapable of inducing *15∶01 expression allowing in early life autoreactive T-cells to escape central thymic deletion. The Italian island of Sardinia exhibits a very high frequency of MS and high solar radiation exposure. We test the contribution of VDREs analysing the promoter region of the MS-associated DRB1 *04∶05, *03∶01, *13∶01 and *15∶01 and non-MS-associated *16∶01, *01, *11, *07∶01 alleles in a cohort of Sardinians (44 MS patients and 112 healthy subjects). Sequencing of the DRB1 promoter region revealed a homozygous canonical VDRE in all *15∶01, *16∶01, *11 and in 45/73 *03∶01 and in heterozygous state in 28/73 *03∶01 and all *01 alleles. A new mutated homozygous VDRE was found in all *13∶03, *04∶05 and *07∶01 alleles. Functionality of mutated and canonical VDREs was assessed for its potential to modulate levels of DRB1 gene expression using an in vitro transactivation assay after stimulation with active vitamin D metabolite. Vitamin D failed to increase promoter activity of the *04∶05 and *03∶01 alleles carrying the new mutated VDRE, while the *16∶01 and *03∶01 alleles carrying the canonical VDRE sequence showed significantly increased transcriptional activity. The ability of VDR to bind the mutant VDRE in the DRB1 promoter was evaluated by EMSA. Efficient binding of VDR to the VDRE sequence found in the *16∶01 and in the *15∶01 allele reduced electrophoretic mobility when either an anti-VDR or an anti-RXR monoclonal antibody was added. Conversely, the Sardinian mutated VDRE sample showed very low affinity for the RXR/VDR heterodimer. These data seem to exclude a role of VDREs in the promoter region of the DRB1 gene in susceptibility to MS carried by DRB1* alleles in Sardinian patients.

Interaction between HLA-DRB1-DQB1 Haplotypes in Sardinian Multiple Sclerosis Population

We performed a case-control study in 2,555 multiple sclerosis (MS) Sardinian patients and 1,365 healthy ethnically matched controls, analyzing the interactions between HLA-DRB1-DQB1 haplotypes and defining a rank of genotypes conferring a variable degree of risk to the disease. Four haplotypes were found to confer susceptibility (*13∶03-*03∶01 OR = 3.3, Pc 5.1×10−5, *04∶05-*03∶01 OR = 2.1, Pc 9.7×10−8, *15∶01-*06∶02 OR = 2.0, Pc = 9.1×10−3, *03∶01-*02∶01 OR = 1.7 Pc = 7.9×10−22) and protection (*11, OR = 0.8, Pc = 2.7×10−2, *16∶01-*05∶02 OR = 0.6, Pc = 4.8×10−16, *14∶01-4-*05∶031 = OR = 0.5, Pc = 9.8×10−4 and *15∶02-*06∶01 OR = 0.4, Pc = 5.1×10−4). The relative predispositional effect method confirms all the positively associated haplotypes and showed that also *08 and *04 haplotypes confers susceptibility, while the *11 was excluded as protective haplotype. Genotypic ORs highlighted two typologies of interaction between haplotypes: i) a neutral interaction, in which the global risk is coherent with the sum of the single haplotype risks; ii) a negative interaction, in which the genotypic OR observed is lower than the sum of the OR of the two haplotypes. The phylogenic tree of the MS-associated DRB1 alleles found in Sardinian patients revealed a cluster represented by *14∶01, *04∶05, *13∶03, *08∶01 and *03∶01 alleles. Sequence alignment analysis showed that amino acids near pocket P4 and pocket P9 differentiated protective from predisposing alleles under investigation. Furthermore, molecular dynamics simulation performed on alleles revealed that position 70 is crucial in binding of MBP 85–99 peptide. All together, these data suggest that propensity to MS observed in Sardinian population carried by the various HLA-DRB1-DQB1 molecules can be due to functional peculiarity in the antigen presentation mechanisms.

Dilated cardiomyopathy with conduction defects in a patient with partial merosin deficiency due to mutations in the laminin-α2-chain gene: A chance association or a novel phenotype?

Patients with a partial reduction of merosin due to mutations in the laminin‐α2 chain gene usually present with a mild form of congenital muscular dystrophy or a limb‐girdle–like muscular dystrophy. To our knowledge, cardiac impairment has never been reported in such patients. A longitudinal study of a patient with partial laminin‐α2 deficiency secondary to mutations in the LAMA2 gene revealed dilated cardiomyopathy with ventricular arrhythmias. Is this a chance association or a novel phenotype? Muscle Nerve, 2011