Usher syndrome (USH), the most prevalent cause of hereditary deafness–blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1–3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.
BackgroundIn spite of significant improvement after multi-modality treatment, prognosis of most patients with glioblastoma remains poor. Standard clinical prognostic factors (age, gender, extent of surgery and performance status) do not clearly predict long-term survival. The aim of this case-control study was to evaluate immuno-histochemical and genetic characteristics of the tumour as additional prognostic factors in glioblastoma.Patients and methodsLong-term survivor group were 40 patients with glioblastoma with survival longer than 30 months. Control group were 40 patients with shorter survival and matched to the long-term survivor group according to the clinical prognostic factors. All patients underwent multimodality treatment with surgery, postoperative conformal radiotherapy and temozolomide during and after radiotherapy. Biopsy samples were tested for the methylation of MGMT promoter (with methylation specific polymerase chain reaction), IDH1 (with immunohistochemistry), IDH2, CDKN2A and CDKN2B (with multiplex ligation-dependent probe amplification), and 1p and 19q mutations (with fluorescent in situ hybridization).ResultsMethylation of MGMT promoter was found in 95% and in 36% in the long-term survivor and control groups, respectively (p < 0.001). IDH1 R132H mutated patients had a non-significant lower risk of dying from glioblastoma (p = 0.437), in comparison to patients without this mutation. Other mutations were rare, with no significant difference between the two groups.ConclusionsMolecular and genetic testing offers additional prognostic and predictive information for patients with glioblastoma. The most important finding of our analysis is that in the absence of MGMT promoter methylation, longterm survival is very rare. For patients without this mutation, alternative treatments should be explored.
A simple bone cyst (SBC) is a benign bone lesion of unknown etiology. It can be differentiated from an aneurysmal bone cyst (ABC) by radiologic and histopathologic features, as well as by the absence of fusions of the USP6 gene characteristic of an ABC. In an attempt to differentiate between ABC and SBC in a recurrent bone cyst, we performed targeted RNA sequencing and found an EWSR1-NFATC2 fusion and no fusion of the USP6 gene. We subsequently analyzed additional 10 cysts, consistent with SBCs after radiologic-pathologic correlation, for the presence of an NFATC2 gene fusion, by targeted RNA sequencing, reverse-transcription polymerase chain reaction (RT-PCR) and Sanger sequencing, and fluorescent in situ hybridization. Targeted RNA sequencing showed a FUS-NFATC2 fusion in 4 of 11 SBCs and an EWSR1-NFATC2 fusion in 2 of 11 SBCs. No fusion was identified in 3 SBCs and the analysis was not successful in 2 SBCs because of the low quantity or poor quality of isolated RNA. All the 6 fusions detected by targeted RNA sequencing were confirmed by RT-PCR and Sanger sequencing, and 5 of the 6 fusions by fluorescent in situ hybridization. An additional FUS-NFATC2 fusion was identified by RT-PCR, Sanger sequencing, and fluorescent in situ hybridization in 1 of the 3 cases negative for fusions by targeted RNA sequencing. At least a large subset of SBCs represents cystic neoplasms characterized by FUS-NFATC2 or EWSR1-NFATC2 fusions, which also define a group of distinct, rare “Ewing-like” sarcomas that predominantly arise in long bones. Our results provide additional evidence of the existence of benign lesions with FUS-NFATC2 or EWSR1-NFATC2 fusions. Although they can recur locally in a nondestructive manner, their clinical course and possible relation to sarcoma with EWSR1-NFATC2 or FUS-NFATC2 fusion remains to be elucidated.
Aneurysmal bone cyst (ABC) is a benign but locally aggressive neoplasm, with a tendency for local recurrence. In contrast to other bone tumors with secondary cystic change, ABC is characterized by USP6 gene rearrangement. There is a growing list of known USP6 fusion partners, characterization of which has been enabled with the advent of next‐generation sequencing (NGS). The list of known fusion partners includes CDH11, CNBP, COL1A1, CTNNB1, EIF1, FOSL2, OMD, PAFAH1B1, RUNX2, SEC31A, SPARC, STAT3, THRAP3, and USP9X. Using NGS, we analyzed a series of 11 consecutive ABCs and identified USP6 fusions in all cases, providing further evidence that USP6 fusions are universally present in primary ABCs. We identified four novel fusion partners in five ABCs and confirmed them by RT‐PCR and Sanger sequencing, ASAP1, FAT1, SAR1A, and TNC (in two cases). Because of high sensitivity and specificity, detection of a USP6 fusion by NGS may assist in differentiating between ABC and its mimics, especially in small biopsy samples when a definite diagnosis cannot be achieved on morphological grounds alone. Further studies with a large number of cases and follow‐up are needed to determine whether different fusion partners are associated with specific clinical and pathologic features of ABCs.
Analysis of Y-chromosomal markers in five Slovenian regions revealed a diverse genetic landscape. Slovenian population display close genetic affiliations with West Slavic populations. The homogenous genetic strata of the West Slavic populations and the Slovenian population suggest the existence of a common ancestral Slavic population in central European region.
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