SCA Tethering-PCR: A Rapid Genetic Test for the Diagnosis of SCA1-3, 6, and 7 by PCR and Capillary Electrophoresis

Spinocerebellar ataxias (SCA) type 1, 2, 3, 6, and 7, associated with a (CAG)n repeat expansion in coding sequences, are the most prevalent autosomal dominant ataxias worldwide (approximately 60% of the cases). In addition, the phenotype of SCA2 expansions has been now extended to Parkinson's disease and amyotrophic lateral sclerosis. Their diagnosis is presently based on a PCR to identify small expanded alleles, followed by a second-level test whenever the suspect of false normal homozygous, or a CAT interruption in SCA1 needs to be verified. Next-generation sequencing still does not allow efficient detection of these repeats. Here, we show the efficacy of a novel, rapid, and cost-effective method to identify and size pathogenic expansions in SCA1-3, 6, and 7 and recognize large alleles or interruptions without a second-level test. Twenty-five healthy controls and 33 expansion carriers were analyzed: alleles migrated consistently in different PCRs/capillary runs, and homozygous subjects were always distinguishable from heterozygous carriers of both common and large (>100 repeats) pathogenic CAG expansions. Repeat number could be calculated counting the number of peaks, except for the largest SCA2 and SCA7 alleles. Interruptions in SCA1 were always visible. Overall, our method allows a simpler, cost-effective, and sensibly faster SCA diagnostic protocol compared to the standard technique and to the still unadapted next-generation sequencing

Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes

Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders

Nusinersen in type 1 spinal muscular atrophy: Twelve-month real-world data

Abstract Objective: The aim of the study was to report 12-month changes after treatment with nusinersen in a cohort of 85 type I spinal muscular atrophy patients of ages ranging from 2 months to 15 years and 11 months. Methods: All patients were assessed using the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) and the Hammersmith Infant Neurological Examination-Section 2 (HINE-2). Results: Two of the 85 patients had 1 SMN2 copy, 61 had 2 copies, and 18 had 3 copies. In 4 patients the SMN2 copy number was not available. At baseline, the mean CHOP INTEND scores ranged between 0 and 52 (mean = 15.66, standard deviation [SD] = \ub113.48), and the mean HINE-2 score was between 0 and 5 (mean = 0.69, SD = \ub11.23). There was a difference between baseline and the 12-month scores on both the CHOP INTEND and the HINE-2 for the whole group (p < 0.001), the subgroups with 2 SMN2 copies (p < 0.001), and those with 3 SMN2 copies (p < 0.001). The difference was found not only in patients younger than 210 days at baseline (p < 0.001) but also in those younger than 5 years on the CHOP INTEND and younger than 2 years on the HINE-2. Interpretation: Our results, expanding the age range and the severity of type I patients treated with nusinersen over 1 year, provide additional data on the range of efficacy of the drug that will be helpful in making an informed decision on whether to start treatment in patients of different ages and severity

Fcγ receptor IIA genotype and susceptibility to P. aeruginosa infection in patients with cystic fibrosis

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An observational study of functional abilities in infants, children, and adults with type 1 SMA

ObjectiveTo report cross-sectional clinical findings in a large cohort of patients affected by type 1 spinal muscular atrophy.MethodsWe included 122 patients, of age ranging between 3 months and 22 years, 1 month. More than 70% (85/122) were older than 2 years and 25% (31/122) older than 10 years. Patients were classified according to the severity of phenotype and to the number of SMN2 copies.ResultsPatients with the more common and the most severe phenotype older than 2 years were, with few exceptions, on noninvasive ventilation and, with increasing age, more often had tracheostomy or >16-hour ventilation and a gastrostomy inserted. In contrast, 25 of the 28 patients with the mildest phenotype older than 2 years had no need for tracheostomy or other ventilatory or nutritional support. In patients older than 2 years, the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders scores were generally lower compared to those found in younger patients and showed distinct levels of functional abilities according to the severity of the phenotype. Similar findings were also observed on the Hammersmith Infant Neurological Examination.ConclusionsOur findings confirm that, after the age of 2 years, patients with type 1 spinal muscular atrophy generally survive only if they have gastrostomy and tracheostomy or noninvasive ventilation >16 hours and have low scores on the functional scales. More variability, however, can be expected in those with the mildest phenotype, who achieve head control. These data provide important baseline information at the time treatments are becoming available

Technical Design Report for PANDA Electromagnetic Calorimeter (EMC)

This document presents the technical layout and the envisaged performance of the Electromagnetic Calorimeter (EMC) for the PANDA target spectrometer. The EMC has been designed to meet the physics goals of the PANDA experiment, which is being developed for the Facility for Antiproton and Ion Research (FAIR) at Darmstadt, Germany. The performance figures are based on extensive prototype tests and radiation hardness studies. The document shows that the EMC is ready for construction up to the front-end electronics interface