Does the (tg)13(t)6 variant of cftr gene contributes to male infertility?
Article published in Urologia Polska 2008/61/Supl. 1.

authors

Katarzyna Wertheim, Agnieszka Sobczyńska-Tomaszewska, Józef Pawlicki, Jan Karol Wolski, Jerzy Bal

Zakład Genetyki Medycznej, Instytut Matki i Dziecka w Warszawie
Centrum Leczenia Niepłodności GAMETA w Łodzi
Przychodnia Leczenia Niepłodności „Novum” w Warszawie

summary

Introduction.

CBAVD (Congenital Bilateral Absence of the Vas Deferens) is recognized in 6% of obstructive azoospermia cases. The other name of the disease – genital form of CF (Cystic Fibrosis) refers to CFTR gene, mutations of which are responsible for those diseases. Until now more than 1540 mutations and polimorfic variants of the gene have been identified. Each mutation has different effect on the protein production - severe mutations disturb protein production, mild ones influence protein action. In 45-80% of CBAVD patients, two mutations are found (usually – one severe and the other mild, or, less often - both mild). The most frequent genotypes of CBAVD patients in polish population are F508del/R117H+IVS8-7T and F508del/IVS8-5T. IVS8-5T occurs in polythimidine (polyT) sequence in intron 8. There are three common alleles comprising 9T, 7T and 5T (5%) and two extremely rare: 3T and 6T. Lower number of T repeat influences splicing and may cause skipping of exon 9, leading to production

of truncated mRNA, and further non-functional protein. The exact effect of two rare variants is speculative. The 3T is considered as severe, while the clinical consequence of the 6T is less clear, although it has been identified in a few CAVD or CBAVD patients. The level of mRNA is also modulated by the number of TG repeats located at 5’ side of polyT site. According to many reports, longer TG repeats (≥12), enhance alternative splicing, and thus intensify the effect of IVS8-5 T.

Objectives.

In this paper we present a case of fertile man with unusual genotype (TG)13(T)6/(TG)11(T)5. We also focus on technical aspect of IVS8-6T identification by presenting the comparison of three widely-used genetic tools.

Materials and methods.

Patient and his partner were qualified to ART after 12 years of unsuccessful procreation efforts. Andrologic consultation was negative for risk factors. Gynecological consultation revealed that problems with conception may be due to female factor (one miscarriage and uterine myoma removed). CFTR analysis was performed on patient request before ART procedure. IVS8-5T was initially investigated by a standard method (PCR, XmnI digestion, 12% polyacryloamide gel electrophoresis, ethidium bromide visualization), than the results were confirmed by INNOLiPA17 (Innogenetics NV) and by sequencing.

Results.

The genotype of fertile man presented above is (TG)13(T)6/(TG)11(T)5. IVS8-6T variant of CFTR gene can be identified by direct sequencing. INNOLiPA17 does not permit to discriminate between the IVS8-6T and IVS8-5T variant.

Conclusions.

The major finding of our study is that genotype (TG)13(T)6/(TG)11(T)5 does not determine CBAVD, although the exact influence of this variant on CFTR mRNA needs to be determined. IVS8 polyT XmnI digestion analysis with subsequent sequencing of samples showing untypical digestion pattern seem to be rational method of identification of the IVS8-6T variants. As INNOLiPA17 test do not permit to identify other variants than IVS8-5T, 7T, 9T, we would recommend employing restriction analysis or sequencing as an additional confirmation test in case of homozygosity.