Are expanded alleles of the FMR1 gene related to unexplained recurrent miscarriages?

BACKGROUND: In women with recurrent miscarriages, up to 50 % of those cases remain unexplained. In this study, we evaluated the impact of Cytosine/Guanine/Guanine (CGG)  trinucleotide expansions of the fragile-X mental retardation 1 (FMR1) gene in women with unexplained recurrent miscarriages. METHODS: This is a prospective case-control pilot study involving 49 women with unexplained recurrent miscarriages and 49 age-matched controls with documented fertility. The case group consisted of women with a history of two or more consecutive miscarriages, in whom no known factor could be identified. The maximum age of recruitment was 40 years. We obtained blood samples that were checked, using polymerase chain reaction with electrophoresis, for the presence of expanded alleles of the FMR1 gene. We further evaluated using sequencing analysis, those women marked as positive. We set the limit at more than 40 repeats. RESULTS: The repeat sizes of CGG expansion in the FMR1 gene differ significantly in the two population groups (p =0.027). We found four women in the miscarriage group and one in the control group positive for carrying premutation alleles (Odds ratio: 4.267, confidence interval: 0.459-39.629). All the positive cases involved intermediate zone carriers. We found no association between the number of abortions each woman had, and her respective CGG repeat number (p =0.255). CONCLUSIONS: Many couples are desperately looking for the cause of their recurrent miscarriage suffering. The CGG expanded allele of the FMR1 gene is possibly to be blamed in some of these cases. More studies are needed to support the results of this prototype study. HIPPOKRATIA 2018, 22(3): 132-136.

Clarithromycin induced reversible sensorineural hearing loss.

OBJECTIVE: We present a rare case of reversible sensorineural hearing loss caused by clarithromycin. METHODS: We present a case report of hearing loss following clarithromycin administration; we also review the current literature and case reports concerning macrolides and, in particular, clarithromycin induced hearing loss. RESULTS: A young pregnant woman presented with sensorineural hearing loss after clarithromycin intake. The subject's hearing returned to normal limits after drug discontinuation and short-term treatment with low dose steroids. CONCLUSION: Newer macrolides are considered to be safer regarding ototoxic effects, and a few cases have been previously described. The present case adds to the body of knowledge concerning clarithromycin ototoxicity. Clinicians should be aware of this rare complication and a thorough otologic history should be established prior to macrolide administration. A baseline audiogram and close observation for patients at higher risk is suggested to identify patients with prior hearing loss and serve as baseline for future reference.

Use of the hereditary persistence of fetal hemoglobin 2 enhancer to increase the expression of oncoretrovirus vectors for human gamma-globin.

The development of oncoretrovirus vectors for human gamma-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of gamma-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the beta-globin locus control region to enhance the expression of an Agamma-globin gene with a truncated -382 bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for Agamma-globin linked to various combinations of the HPFH2 enhancer, the alpha-globin HS40 enhancer, and several versions of the promoter from Agamma-globin or beta-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoter-autonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a -201 bp Agamma-globin gene promoter with the Greek HPFH -117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248+/-99% per copy of mouse alpha-globin (62% of total alpha-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for Agamma-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the Agamma-globin promoter in the context of a gene transfer vector.

An araC-controlled bacterial cre expression system to produce DNA minicircle vectors for nuclear and mitochondrial gene therapy.

The presence of CpG motifs and their associated sequences in bacterial DNA causes an immunotoxic response following the delivery of these plasmid vectors into mammalian hosts. We describe a biotechnological approach to the elimination of this problem by the creation of a bacterial cre recombinase expression system, tightly controlled by the arabinose regulon. This permits the Cre-mediated and -directed excision of the entire bacterial vector sequences from plasmid constructs to create supercoiled gene expression minicircles for gene therapy. Minicircle yields using standard culture volumes are sufficient for most in vitro and in vivo applications whereas minicircle expression in vitro is significantly increased over standard plasmid transfection. By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should also reduce DNA-induced inflammatory responses in a dose-dependent manner. We further describe the generation of minicircle expression vectors for mammalian mitochondrial gene therapy, for which no other vector systems currently exist. The removal of bacterial vector sequences should permit appropriate transcription and correct transcriptional cleavage from the mitochondrial minicircle constructs in a mitochondrial environment and brings the realization of mitochondrial gene therapy a step closer.