Application of a Clinical Workflow May Lead to Increased Diagnostic Precision in Hereditary Spastic Paraplegias and Cerebellar Ataxias: A Single Center Experience

The molecular characterization of Hereditary Spastic Paraplegias (HSP) and inherited cerebellar ataxias (CA) is challenged by their clinical and molecular heterogeneity. The recent application of Next Generation Sequencing (NGS) technologies is increasing the diagnostic rate, which can be influenced by patients\u2019 selection. To assess if a clinical diagnosis of CA/HSP received in a third-level reference center might impact the molecular diagnostic yield, we retrospectively evaluated the molecular diagnostic rate reached in our center on 192 unrelated families (90 HSP and 102 CA) (i) before NGS and (ii) with the use of NGS gene panels. Overall, 46.3% of families received a genetic diagnosis by first-tier individual gene screening: 43.3% HSP and 50% spinocerebellar ataxias (SCA). The diagnostic rate was 56.7% in AD-HSP, 55.5% in AR-HSP, and 21.2% in sporadic HSP. On the other hand, 75% AD-, 52% AR- and 33% sporadic CA were diagnosed. So far, 32 patients (24 CA and 8 HSP) were further assessed by NGS gene panels, and 34.4% were diagnosed, including 29.2% CA and 50% HSP patients. Eleven novel gene variants classified as (likely) pathogenic were identified. Our results support the role of experienced clinicians in the diagnostic assessment and the clinical research of CA and HSP even in the next generation era

Correlation between GJB2 mutations and audiological deficits: personal experience

Mutations in GJB2 gene are the most common cause of genetic deafness. More than 100 mutations have been described. The aim of this work is to describe the personal experience in genetic hearing loss, investigating the audiological and genetical characteristics of Cx26 deafness and correlating genotype and phenotype. We performed audiological and genetical evaluation in 154 patients affected by non-syndromic deafness of different degree. All patients showed a bilateral symmetrical sensorineural hearing loss. From the genetical analysis 127 probands resulted as negatives while 27 as positives (51.8% homozygous for 35 delG, 14.8% compound heterozygosis and 33.3% single mutation); 7.5% of patients had a mild deafness, 37% moderate, 33.3% severe and 22.2% profound. The c.35 delG mutation was detected in 66.6% of patients. Three mutations were found in compound heterozygosis with 35 delG, six different single mutations already described, and a new mutation S138G were also found. Correlation between genotype and phenotype confirmed the high variability of hearing loss

The chromosome analysis of the miscarriage tissue. Miscarried embryo/fetal crown rump length (CRL) measurement: A practical use

Objective To investigate whether miscarried embryo/fetal crown rump length (CRL) measurement may yield a practical application for predicting a conclusive result at the cytogenetic analysis of miscarriage tissue. Our study might help in improving the cytogenetic method, the results of which may be affected by maternal cell contamination (MCC). In particular, we aimed at establishing whether the miscarried embryo/fetal CRL measurement shows accuracy in predicting the possibility of MCC and the scan cut-off value useful to this purpose and, as a result, suggest a multi-step procedure for the genetic ascertainment. Methods Women experiencing at least two miscarriages of less than 20 weeks size at the Pregnancy Loss Unit at Fondazione Policlinico A. Gemelli underwent a scan before surgery. The CRL value was recorded. After the dilatation and courettage (D&C) procedure, miscarriage tissue was processed through the proposed multi-step procedure before performing oligo-nucleotide- based and SNP (single nucleotide polymorphisms)-based comparative genomic hybridization (CGH+SNP) microarray analysis. Results 63 women and 63 miscarriages met the criteria. By using the Receiving Operator Characteristic (ROC) curves, CRL showed an AUC of 0.816 (95%CI:0.703\ub10.928,p<0.001). A CRL24.5 mm cut-off value showed a higher positive likelihood ratio (5.27) but, conversely, a higher negative likelihood ratio (0.64) in predicting the possibility of MCC. Microarray analysis was successful in the totality of cases in which the embryo/fetal origin of miscarriage tissues was proven

Molecular, clinical, and muscle studies in myotonic dystrophy type 1 (DM1) associated with novel variant CCG expansions

We assessed clinical, molecular and muscle histopathological features in five unrelated Italian DM1 patients carrying novel variant pathological expansions containing CCG interruptions within the 3'-end of the CTG array at the DMPK locus, detected by bidirectional triplet primed PCR (TP-PCR) and sequencing. Three patients had a negative DM1 testing by routine long-range PCR; the other two patients were identified among 100 unrelated DM1 cases and re-evaluated to estimate the prevalence of variant expansions. The overall prevalence was 4.8 % in our study cohort. There were no major clinical differences between variant and non-variant DM1 patients, except for cognitive involvement. Muscle RNA-FISH, immunofluorescence for MBNL1 and RT-PCR analysis documented the presence of ribonuclear inclusions, their co-localization with MBNL1, and an aberrant splicing pattern involved in DM1 pathogenesis, without any obvious differences between variant and non-variant DM1 patients. Therefore, this study shows that the CCG interruptions at the 3'-end of expanded DMPK alleles do not produce qualitative effects on the RNA-mediated toxic gain-of-function in DM1 muscle tissues. Finally, our results support the conclusion that different patterns of CCG interruptions within the CTG array could modulate the DM1 clinical phenotype, variably affecting the mutational dynamics of the variant repeat

Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine

In fragile X syndrome, hypermethylation of the expanded CGG repeat and of the upstream promoter leads to transcriptional silencing of the FMR1 gene. Absence of the FMR1 protein results in mental retardation. We previously proved that treatment with 5-azadeoxycytidine (5-azadC) of fragile X cell lines results in reactivation of the FMR1 gene. We now show that this treatment causes passive demethylation of the FMR1 gene promoter. We employed the bisulfite-sequencing technique to detect the methylation status of individual CpG sites in the entire promoter region, upstream of the CGG repeat. Lymphoblastoid cell lines of fragile X males with full mutations of different sizes were tested before and after treatment with 5-azadC at various time points. We observed that individual cells are either completely unmethylated or not, with few relevant exceptions. We also investigated the extent of methylation in the full mutation (CGG repeat) itself by Southern blot analysis after digestion with methylation-sensitive enzymes Fnu4HI and McrBC and found that the CGG repeat remains at least partially methylated in many cells with a demethylated promoter. This may explain the quantitative discrepancy between the large extent of promoter demethylation and the limited levels of FMR1 transcriptional reactivation estimated by quantitative real-time fluorescent RT–PCR analysis

Simpson–Golabi–Behmel syndrome in a female: A case report and an unsolved issue

Simpson–Golabi–Behmel syndrome is an X-linked recessive overgrowth condition caused by alterations in GPC3 gene, encoding for the cell surface receptor glypican 3, whose clinical manifestations in affected males are well known. Conversely, there is little information regarding affected females, with very few reported cases, and a clinical definition of this phenotype is still lacking. In the present report we describe an additional case, the first to receive a primary molecular diagnosis based on strong clinical suspicion. Possible explanations for full clinical expression of X-linked recessive conditions in females include several mechanisms, such as skewed X inactivation or homozygosity/compound heterozygosity of the causal mutation. Both of these were excluded in our case. Given that the possibility of full expression of SGBS in females is now firmly established, we recommend that GPC3 analysis be performed in all suggestive female cases. © 2016 Wiley Periodicals, Inc