The degree of eIF2B dysfunction, which is involved in the regulation of protein synthesis during cellular stress, may play a role in the clinical expression of eIF2B-related disorders.

The integrase from the Streptomyces phage (phi)C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, (phi)C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site ((Delta)attB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB(+) and (Delta)attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.

Key Points• Targeted NGS of relapsed/ refractory CLL reveals a high incidence of concurrent mutations that mostly affect the TP53, ATM, and SF3B1 genes.• Concurrent mutations of the TP53, ATM, and/or SF3B1 genes confer short survival in patients with relapsed/ refractory CLL.Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrent gene mutations. The number of genes affected by mutation was ‡2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ‡2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiplehit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P 5 .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome. (Blood. 2015;126(18):2110-2117 IntroductionChronic lymphocytic leukemia (CLL) is characterized by its unique immunophenotype of CD19sIg dim expressing clonal mature B cells and also by a highly variable clinical course. With the emergence of new classes of drugs such as the inhibitor of phosphatidylinositol 3-kinase, idelalisib, 1 and the irreversible inhibitor of Bruton tyrosine kinase, ibrutinib, 2 available treatment options have increased significantly and allow us to begin to develop models of treatment stratification. Over the last 3 years, the CLL genome has been thoroughly characterized by next-generation sequencing (NGS).3 Pioneer reports using this approach unraveled somatic mutations recurrently targeting multiple genes, among which TP53, SF3B1, NOTCH1, MYD88, and ATM were the most frequent. [4][5][6][7][8][9] Large retrospective studies in untreated patients from historical cohorts have recently shown the adverse prognostic impact of the TP53, NOTCH1, and SF3B1 mutations on time to treatment and overall survival (OS). [10][11][12] In addition, mutations in these genes may also be associated with poor progression-free survival (PFS) in frontline patients treated in prospective trials. 13,14 Conversely, the pejorative impact of TP53, SF3B1, and NOTCH1 mutations on the clinical outcome of patients with relapsed/ refractory (R/R) CLL is controversial. [15][16][17] Compared with untreated CLL, relapse, as advanced disease, may be associated with a high level of genomic diversification. 9,18 This process might result in For personal use only. on May 11, 2018. by guest www.bloodjournal.org From accumulation and co-occurrence of these or other genomic events leading to interactions that could be of prognostic relevance. Here, w...

Mutations in each of the five eucaryotic initiation factor 2B (eIF2B) subunits have been found in leukodystrophies of various severity: Cree leukoencephalopathy, childhood ataxia with central hypomyelination/leukodystrophy with vanishing white matter and ovarioleukodystrophy. A continuum was observed from fatal infantile forms to adult forms without neurological deterioration. Disease severity was found to correlate with the age at disease onset and the specific amino-acid substitution. In order to analyze the functional consequences of eIF2B mutations, we measured the guanine nucleotide exchange factor (GEF) activity of eIF2B in transformed lymphocytes from 30 affected patients carrying mutations in eIF2B compared to 10 unaffected heterozygotes and 22 controls without eIF2B mutations. A significant decrease of 20 -70% in GEF activity was observed in all mutated cells. The severity of this decrement of GEF activity correlated with age at onset of the disease. These results suggest that a deficiency in GEF activity underlies the encephalopathy associated eIF2B-related disease. Our study demonstrates that the evaluation of the GEF activity in transformed lymphocytes represents an interesting alternative test to the systematic screening of the five EIF2B genes. This relevant cellular model may also be used to test the functional impact of different molecules on the GEF activity for future therapeutic strategies.

Only 16 patients (8.3%) from 14 families (7.7%) carry GJA12 mutations including five families where we detected only one mutated allele. Among those, we identified 11 novel alterations. Thus, GJA12 mutations are a rather rare cause for Pelizaeus-Merzbacher-like disease. The clinical phenotype of patients with a GJA12 mutation was evaluated and is overall comparable to the clinical features seen in mild forms of proteolipid protein 1 (PLP1) related disorder but with better cognition and earlier signs of axonal degeneration.

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These results highlight the important role of the RCC1-like domain in ALS2 stability and function that is essential for upper motor neuron maintenance.

Upregulation of the telomerase reverse transcriptase (TERT) gene in human cancers leads to telomerase activation, which contributes to the growth advantage and survival of tumor cells. Molecular mechanisms of TERT upregulation are complex, tumor-specific and can be clinically relevant. To investigate these mechanisms in breast cancer, we sequenced the TERT promoter, evaluated TERT copy number changes and assessed the expression of the MYC oncogene, a known transcriptional TERT regulator, in two breast cancer cohorts comprising a total of 122 patients. No activating TERT promoter mutations were found, suggesting that this mutational mechanism is not likely to be involved in TERT upregulation in breast cancer. The T349C promoter polymorphism found in up to 50% of cases was not correlated with TERT expression, but T349C carriers had significantly shorter disease-free survival. TERT gains (15-25% of cases) were strongly correlated with increased TERT mRNA expression and worse patient prognosis in terms of disease-free and overall survival. Particularly aggressive breast cancers were characterized by an association of TERT gains with MYC overexpression. These results evidence a significant effect of gene copy number gain on the level of TERT expression and provide a new insight into the clinical significance of TERT and MYC upregulation in breast cancer.