Compound heterozygous variants in MAPK8IP3 were detected in severe congenital hypotonia mimicking lethal spinal muscular atrophy.

Mitogen-activated protein kinase 8-interacting protein 3 gene (MAPK8IP3) encodes the c-Jun-amino-terminal kinase-interacting protein 3 (JIP3) and is involved in retrograde axonal transport. Heterozygous de novo pathogenic variants in MAPK8IP3 result in a neurodevelopmental disorder with or without brain abnormalities and possible axonal peripheral neuropathy. Whole-exome sequencing was performed on an individual presenting with severe congenital muscle hypotonia of neuronal origin mimicking lethal spinal muscular atrophy. Compound heterozygous rare variants (a splice and a missense) were detected in MAPK8IP3, inherited from the healthy parents. Western blot analysis in a muscle biopsy sample showed a more than 60% decrease in JIP3 expression. Here, we suggest a novel autosomal recessive phenotype of a lower motor neuron disease caused by JIP3 deficiency.

Multiple splice variants of EWSR1-ETS fusion transcripts co-existing in the Ewing sarcoma family of tumors.

PURPOSE: The Ewing sarcoma family of tumors (EFT) is characterized by fusions of the EWSR1 gene on chromosome 22q12 with either one of the genes encoding members of the ETS family of transcription factors, in the majority of cases FLI1 or ERG. Many alternative EWSR1-ETS gene fusions have been encountered, due to variations in the locations of the EWSR1 and ETS genomic breakpoints. The resulting heterogeneity in EWSR1-ETS fusion transcripts may further be increased by the occurrence of multiple splice variants within the same tumor. Here we present a retrospective study designed to detect all of the EWSR1-FLI1 and EWSR1-ERG fusion transcripts in a series of 23 fresh frozen EFT tissues. METHODS: RT-PCR and nested fluorescent multiplex PCR were used to amplify EWSR1-FLI1 and EWSR1-ERG transcripts from EFT tissues. Fusion transcripts were identified by laser-induced fluorescent capillary electrophoresis and confirmed by sequence analysis. RESULTS: Nine different EWSR1-FLI1 fusion transcripts and one EWSR1-ERG fusion transcript were identified in 21 out of 23 fresh frozen EFT tissue samples. In five cases multiple fusion transcripts were found to coexist in the same tumor sample. We additionally reviewed previous reports on twelve cases with multiple EWSR1-ETS fusion transcripts. CONCLUSIONS: Alternative splicing may frequently affect the process of EFT-associated fusion gene transcription and, as such, may significantly contribute to the pathogenic role of EFT-associated chromosome translocations. In a considerable number of cases this may result in multiple splice variants within the same tumor.

Utilisation of fluorescent multiplex PCR and laser-induced capillary electrophoresis for the diagnosis of Ewing family of tumours in formalin-fixed paraffin-embedded tissues.

AIMS: The localisation of the translocation breakpoint of the Ewing sarcoma family of tumours shows significant variability on relatively large regions of fusion partner genes. As a consequence, many alternative forms of EWSR1-ETS translocation exist which make the RNA-based molecular diagnostics of Ewing sarcoma family of tumours complicated. In addition to the heterogeneity of fusion transcripts, the degradation of RNA also presents a significant difficulty in the molecular analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Our aim was to establish a sensitive method which is able to identify all combinatorially possible EWSR1-FLI1 and EWSR1-ERG translocation transcripts in FFPE tissue samples despite significant RNA-degradation. METHODS: The combination of fluorescent multiplex PCR with laser-induced capillary electrophoresis was used to detect and identify EWSR1-FLI1 and EWSR1-ERG chimeric transcripts on the basis of amplicon size, and forward primers labelled by distinct fluorophores. RESULTS: Using this method, we processed 60 FFPE samples of Ewing sarcoma family of tumours, and identified six types EWSR1-FLI1 and one type EWSR1-ERG chimeric transcripts acceptable for RT-PCR analysis in 27 out of 45 samples. This result shows 60% sensitivity for detecting the most frequent Ewing family of tumour (EFT)-related fusion transcripts. CONCLUSIONS: The utilisation of fluorescent multiplex PCR and laser-induced fluorescent capillary electrophoresis is effective for the diagnosis of EFT in FFPE tissue, and after the defined modifications it can offer a sensitive method to overcome the diagnostic difficulties connected with heterogeneity of the variant translocations in EFT.