A Combination of M50I and V151I Polymorphic Mutations in HIV-1 Subtype B Integrase Results in Defects in Autoprocessing

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus.

Surveying Mutagenic and Anti-mutagenic Effects of Citrus Limon

Background and objective: Cancer is one of the main causes of mortality in the world and countless mutagenic chemical compound kill millions of cancer patients every year. Scientists are searching for natural foodstuff to prevent cancer. The present paper examines anti-mutagenic and anti-carcinogenic effects of citrus lemon.Methodology: Human astrocytoma cancerous cell line was cultured in DMEM (Gibco) with FBS10% (fetal bovine serum), L-glutamine, penicillin, and streptomycin at 37ºC for two days. Then the samples were treated with citrus lemon juice and biotic potential of cells was examined through the MTT method. Anti-mutagenic and anti-carcinogenic effects of citrus lemon were studied through back mutation assessment standard method (Ames test). The test was done using Salmonella typhimurium strain (TA100) with the mutation in histidine operon that needs external histidine source. The strain creates the reverse colony in presence of carcinogenic agents (sodium azide). Findings: Based on the MTT method, human astrocytoma cancerous cell line demonstrated significant cellular death compared with the control groups (p<0.01). Ames test showed that the juice prevented back mutation and rate of inhibition, based on the anti-mutagenic effect, of half-ripen and ripen citrus lemon was 71.7% and 34.3% respectively. In addition, the anti-carcinogenic effect of half-ripen and ripen citrus lemon were 83.3% and 50% respectively. Conclusion: Anti-mutagenic and anti-carcinogenic effects of citrus lemon were examined for the first time. The results showed that in this regard half-ripen citrus lemon was more effective than ripening citrus lemon.

Clonal Reversion in Genetic Syndromes: Gene Therapy at Its Best

Many genetic syndromes are characterized by a wide spectrum of clinical severity. Even within one family clinical presentations can show extreme variation. Mosaic tissue distribution caused by spontaneous correction of a germline pathogenic allele is one of multiple explanations for variety in phenotypic expression of an inherited mutation. This phenomenon, called somatic reversion, is infrequently observed and can be easily overlooked. Reversion needs to be considered if a person presents with a milder than expected clinical course or with a mixture of phenotypically normal and abnormal cells.1 Mechanisms that may explain reversion include mitotic gene conversion, back mutation, intragenic mitotic recombination and the occurrence of compensatory mutations. A mosaic pattern of somatic reversion only becomes apparent if several criteria are met. The non-mutant cells need to have a selective growth advantage over surrounding mutant cells. Furthermore, to facilitate expansion of the revertant clone the affected genes need to be expressed in regenerating organ systems like skin and blood.1 The chance of spontaneous correction of a pathogenic allele is likely increased in diseases with an underlying mechanism resulting in genomic instability or high mutation rates, like Bloom syndrome and Fanconi anemia, which are both caused by gene defects in DNA repair pathways.2,3 In skin disorders an evolving mosaic revertant pattern is easily visible. Ichthyosis with confetti, caused by mutations in KRT10, is an example of a skin disorder displaying multiple events of reversion.4 In this condition, normal skin spots appear early in life and increase in number and size over time. Each normal spot results from a separate event of loss of heterozygosity on chromosome 17q, which harbors KRT10, via mitotic recombination. Also in the genetic skin fragility disorder epidermolysis bullosa revertant mosaicism has been described repeatedly.5,6 We have observed reversion, caused by mitotic recombination of mutant TE RC (telomerase RNA component) alleles in a family affected by dyskeratosis congenita (DC).7 DC is a multisystem disorder characterized amongst others by bone marrow failure and lung fibrosis. The observation of mosaic stretches of uniparental disomy (UPD) of chromosome 3q as an indication of revertant mosaicism encouraged us to develop a highly sensitive method for detecting genomic regions with low mosaic UPD in SNP array data. Indeed this tool supported us in identifying additional cases of DC and a mosaic reversion pattern in blood cells. Revertant mosaicism being a recurrent event in DC related conditions was recently confirmed by others.8 Awareness of revertant mosaicism is important for improving diagnostic testing. In DC for instance it is common practice that analysis of the DC genes is performed on DNA isolated from peripheral blood cells. In case no pathogenic mutation is found, an obvious conclusion can be that the phenotype in the family is caused by an aberration in an as yet to be identified DC gene. Based on our findings, we recommend sequence analysis on DNA extracted from other cells, such as skin fibroblasts, particularly in individuals without bone marrow failure. The observation of reversion in hematological conditions is also of importance for the development of future therapies: Isolation of autologous reverted stem cells can probably circumvent more toxic and harmful therapies, like allogeneic stem cell transplantation, in a subset of individuals. 1. Hirschhorn R. In vivo reversion to normal of inherited mutations in humans. J Med Genet 2003;40 (10):721-728. 2 Ellis NA, Ciocci S, German J. Back mutation can produce phenotype reversion in Bloom syndrome somatic cells. Hum Genet 2001;108 (2):167-173. 3 Waisfisz Q, Morgan NV, Savino M, et al. Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism. Nat Genet 1999;22 (4):379-383. 4 Choate KA, Lu Y, Zhou J, et al. Mitotic recombination in patients with ichthyosis causes reversion of dominant mutations in KRT10. Science 2010;330 (6000):94-97. 5 Jonkman MF, Scheffer H, Stulp R, et al. Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion. Cell 1997;88 (4):543-51. 6 Kiritsi D, Garcia M, Brander R, et al. Mechanisms of natural gene therapy in dystrophic epidermolysis bullosa. J Invest Dermatol 2014;134 (8):2097-104. 7 Jongmans MC, Verwiel ET, Heijdra Y, et al. Revertant somatic mosaicism by mitotic recombination in dyskeratosis congenita. Am J Hum Genet 2012;90 (3):426-33. 8 Alder JK, Stanley SE, Wagner CL, et al. Exome Sequencing Identifies Mutant TINF2 in a Family With Pulmonary Fibrosis. Chest 2015;147 (5):1361-8. Disclosures No relevant conflicts of interest to declare.