Faculty Opinions recommendation of Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.

In recent years, molecular characterization and management of patients with systemic mastocytosis (SM) have greatly benefited from the application of advanced technologies. Highly sensitive and accurate assays for KIT D816V mutation detection and quantification have allowed the switch to non-invasive peripheral blood testing for patient screening; allele burden has prognostic implications and may be used to monitor therapeutic efficacy. Progress in genetic profiling of KIT, together with the use of next-generation sequencing panels for the characterization of associated gene mutations, have allowed the stratification of patients into three subgroups differing in terms of pathogenesis and prognosis: (i) patients with mast cell-restricted KIT D816V; (ii) patients with multilineage KIT D816V-involvement; (iii) patients with “multi-mutated disease”. Thanks to these findings, new prognostic scoring systems combining clinical and molecular data have been developed. Finally, non-genetic SETD2 histone methyltransferase loss of function has recently been identified in advanced SM. Assessment of SETD2 protein levels and activity might provide prognostic information and has opened new research avenues exploring alternative targeted therapeutic strategies. This review discusses how progress in recent years has rapidly complemented previous knowledge improving the molecular characterization of SM, and how this has the potential to impact on patient diagnosis and management.

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Molecular Characterization of eutF Mutants ofSalmonella typhimurium LT2 Identifies eutFLesions as Partial-Loss-of-Function tonB Alleles

Journal of Bacteriology ◽

10.1128/jb.181.2.368-374.1999 ◽

1999 ◽

Vol 181 (2) ◽

pp. 368-374 ◽

Cited By ~ 4

Author(s):

Michael G. Thomas ◽

George A. O’Toole ◽

Jorge C. Escalante-Semerena

Keyword(s):

Amino Acid ◽

Molecular Characterization ◽

Immunoblot Analysis ◽

Phenotypic Characterization ◽

Loss Of Function ◽

Wild Type ◽

Partial Loss ◽

Acid Addition ◽

Fusion Site

ABSTRACT The eutF locus of Salmonella typhimuriumLT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source. Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter. Phenotypic characterization of eutF mutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization. Here we provide evidence that two alleles defining the eutFlocus, Δ903 and eutF1115, are partial-loss-of-function tonB alleles. Both mutations were complemented by plasmids containing a wild-type allele of theEscherichia coli tonB gene. Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carrying eutF alleles. Molecular analysis of the Δ903 allele identified a deletion that resulted in the fusion of the 3′ end of tonB with the 3′ end oftrpA. In-frame translation of the tonB-trpAfusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition. We isolated a derivative of a strain carrying allele Δ903 that regained the ability to grow on ethanolamine as a carbon and energy source. The molecular characterization of the mutation that corrected the Eut−phenotype caused by allele Δ903 showed that the new mutation was a deletion of two nucleotides at the tonB-trpAfusion site. This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition. This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine and eut operon expression to nearly wild-type levels. It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, the eutF locus does not exist.

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Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/854737 ◽

2010 ◽

Vol 2010 ◽

pp. 1-7 ◽

Cited By ~ 4

Author(s):

Lucia Anna Muscarella ◽

Vito Guarnieri ◽

Michelina Coco ◽

Serena Belli ◽

Paola Parrella ◽

...

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Quantitative Polymerase Chain Reaction ◽

Genetic Diseases ◽

Complete Loss ◽

Loss Of Function ◽

Autosomal Dominant Disorder ◽

Cavernous Malformations ◽

Genomic Deletions

Cerebral cavernous malformations (CCMs) represent a common autosomal dominant disorder that predisposes patients to haemorrhagic strokes and focal neurological signs. About 56% of the hereditary forms of CCMs have been so far associated with mutations in theKRIT1(Krev Interaction Trapped 1) gene, located at 7q21.2 (CCM1 locus). We described the complete loss of 7q21.2 locus encompassing theKRIT1gene and 4 flanking genes in a CCM family by using a dense set of 12 microsatellite markers. The complete loss of the maternal copy ofKRIT1gene region was confirmed by Real-Time Quantitative Polymerase Chain Reaction (RT-QPCR) and the same approach was used for expression analysis. Additional RT-QPCR analysis showed the extension of the deletion, for a total of 700 kb, to the adjacent downstream and upstream-located genes,MTERF, AKAP9, CYP51A1, as well as a partial loss of theANKIB1gene. Here we report the molecular characterization of an interstitial small genomic deletion of the 7q21.2 region in a CCMs affected family, encompassing theKRIT1gene. Our findings confirm the loss of function mechanism for the already known CCM1 locus, without any evident involvement of the other deleted genes. Moreover, our investigations highlight the usefulness of the RT-QPCR to the molecular characterization of the breakpoints genomic deletions and to the identification of internal deleted genes involved in the human genetic diseases.

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