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Neuromuscular diseases (NMDs) are a group of200 highly genetically as well as clinically heterogeneous inherited genetic disorders that affect the peripheral nervous and muscular systems, resulting in gross motor disability. The clinical and genetic heterogeneities of NMDs make disease diagnosis complicated and expensive, often involving multiple tests.To expedite the molecular diagnosis of NMDs, we designed and validated several next generation sequencing (NGS)-based comprehensive gene panel tests that include complementary deletion and duplication testing through comparative genomic hybridization arrays. Our validation established the targeted gene panel test to have 100% sensitivity and specificity for single nucleotide variant detection. To compare the clinical diagnostic yields of single gene (NMD-associated) tests with the various NMD NGS panel tests, we analyzed data from all clinical tests performed at the Emory Genetics Laboratory from October 2009 through May 2014. We further compared the clinical utility of the targeted NGS panel test with that of exome sequencing (ES).We found that NMD comprehensive panel testing has a 3-fold greater diagnostic yield (46%) than single gene testing (15-19%). Sanger fill-in of low-coverage exons, copy number variation analysis, and thorough in-house validation of the assay all complement panel testing and allow the detection of all types of causative pathogenic variants, some of which (about 18%) may be missed by ES.Our results strongly indicate that for molecular diagnosis of heterogeneous disorders such as NMDs, targeted panel testing has the highest clinical yield and should therefore be the preferred first-tier approach.

OBJECTIVE: The objective of this study is to evaluate genetic risks already present before pregnancy in a cohort of pregnant women referred for prenatal genetic counseling exclusively for advanced maternal age (AMA). METHOD: We retrospectively reviewed the records of 1353 women referred over 1 year (2010) for pre-test genetic counseling with the only indication of AMA at three Italian Clinical Genetic Services. RESULTS: Of the 1353 women fulfilling the inclusion criteria of the study, 87 (6.4%) had cumulatively 94 genetic risk factors not previously identified (one risk factor in 80 patients and two risk factors in seven). Twenty-six risk factors (27.7%) concerned heterogeneous or multifactorial conditions and 68 (72.3%) Mendelian or chromosomal disorders and consanguinity.In nine out of these 87 women, the estimated risk for the offspring of a genetic disease or a significant structural anomaly was >5%. Additional testing according to the identified risks was performed in 36 of these 87 women/families. CONCLUSIONS: The proportion of cases with additional risk factors is smaller than reported in previous studies, but it remains substantial and confirms the need for strategies to increase awareness of the public and health professionals responsible for the care of women in childbearing age.

Sickle cell disease is the commonest haemoglobinopathy in Africa, the Middle East and India. In recent years, its incidence has increased dramatically also in Europe and North America because of the high rate of migration of people from endemic areas. From January 2009 to January 2010 the number of foreign residents in the province of Ferrara (Italy) increased by 12.2%: most of the immigrants were from countries at high risk of sickle cell disease. Since neonatal screening and prophylactic penicillin in early childhood could reduce mortality by 10 years of age to less than 2%, the aim of this study was to establish a neonatal screening programme for haemoglobinopathies in Ferrara.First we assessed how many pregnant women underwent haemoglobin analysis by high performance liquid chromatography before or during pregnancy and how many of them were carriers of haemoglobinopathies. Subsequently, we verified the feasibility of neonatal screening for sickle cell disease and other haemoglobinopathies, analysing cord blood by high performance liquid chromatography. Neonates found to be positive were managed by a multidisciplinary team to implement all the appropriate prophylactic and therapeutic measures.We found that 59% of women who delivered at the University Hospital of Ferrara, from 2007 to 2009, had undergone high performance liquid chromatography. Of the 41% who were not tested, many were from areas in which sickle cell disease is common. Between September 26th 2010 and January 31st 2012, 1992 neonatal tests were performed and 24 carriers of haemoglobinopathies were identified (16 with HbS, 4 with HbC, 2 with HbE, 1 with HbD Punjab and 1 with HbD-Ouled Rabah); 42.6% of the mothers of these 1,992 neonates had not undergone high performance liquid chromatography during pregnancy.Currently prevention of haemoglobinopathies in Italy is provided during the pre-conception period but only to patients with abnormal blood counts. Neonatal screening is useful and cost-effective to ensure early diagnosis and appropriate treatment for infants with sickle cell disease or other haemoglobinopathies.

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1(-/-) mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis.

Collagen (Col) VI is a major component of the extracellular matrix which, in skeletal muscle, is localized just outside the basement membrane. Deficiency of ColVI in humans due to mutations of COL6 genes gives rise to Bethlem Myopathy (BM), Ullrich Congenital Muscular Dystrophy (UCMD), and Myosclerosis Myopathy. About 70 different COL6 gene mutations have been associated to ColVI myopathies which, although present a wide range of clinical features, share a common pathogenesis. This mechanism, initially identified in the Col6a1-/- model (Irwin et al., 2003), and then in cultures from BM and UCMD patients (Angelin et al., 2007), involves a mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP). The pat hogenic role of PTP opening, causing the release of proapoptotic factors, has been confirmed by the normalizing effect of cyclosporine A (CsA) on the mitochondrial defect and on the increased apoptotic rate in both the mouse model and in a selected group of patients (Merlini et al., 2008). We have recently demonstrated that the persistence of abnormal mitochondria and apoptosis are amplified by defective autophagy (Grumati et al., 2010). In fact, forced activation of autophagy by genetic, dietary and pharmacological approaches restore myofiber survival and ameliorate the dystrophic phenotype in mice. Since also muscle cells of BM and UCMD patients present a defective activation of the autophagic machinery, it will be possible to restore this activity by using a low protein diet or drugs capable to reactivate autophagy. To monitor the effects of therapies on ColVI-related myopathies highly invasive muscle/skin biopsies have been so far utilized. We have recently obtained evidence that ColVI expression in blood macrophages from BM and UCMD patients can be detected at levels comparable to those observed in muscle biopsies and cultured skin fibroblasts (Gualandi et al., 2011). These data support the suitability of peripheral blood macrophages as a reliable, minimally invasive tool for supplementing or replacing highly invasive biopsies in the diagnosis and monitoring of ColVI myopathies. 1. Irwin WA et al., Nat Genet 2003;35:267-71 2. Angelin A et al., PNAS USA 2007;104:991-6 3. Merlini L et al., PNAS USA 2008;105:5225-9 4. Gualandi F et al., Muscle Nerve 2011;44:80-4, Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

International audience; Taxanes represent a group of anticancer drugs with a wide range of activity against breast cancer. Therapy side effects include haematologic toxicity (neutropenia, leucopenia), peripheral neuropathy and hypersensitivity, and demonstrate inter-individual variations. Since it is known that three genes are implicated in Taxane turnover, namely in the transport, in the metabolism and in the activity, we explored the association among polymorphisms (single nucleotide polymorphisms, SNPs) in these three genes and the occurrence of Taxane-induced toxicity. We studied 95 patients affected by breast cancer and under treatment with Taxanes as adjuvant, metastatic or neo-adjuvant therapy. We genotyped them for SNPs in the (alleles *1, *2, *3 and *4), (alleles *1 and *3) and (1236 C>T; 2677 G>T/A; 3435 C>T) genes by real-time PCR assay. We observed a significant association between the *3 allele and a lower occurrence of hypersensitivity reactions to Taxane treatment. We speculate that the highest production of 4-hydroxyestradiol (4-OHE2) metabolite by *3 allele could increase the formation of the 4-OHE2-Taxane adduct and possibly inhibit Taxane toxicity. We suggest that might affect Taxane hypersensitivity therefore representing, if confirmed in a large cohort of patients, an exploratory hypersensitivity predictive biomarker.