Quantitative evaluation of partial deletions of the DAZ gene cluster

Abstract

Partial deletions of the DAZ gene cluster are thought to cause spermatogenesis impairment. The presence of homologous copies of this gene in the Y chromosome does not allow PCR to be used for the identification of this abnormality. Hence, sequence family variants (SFV), following amplification of sY581, sY587 and sY586 and subsequent enzymatic digestion with Sau3A, DraI and TaqI, respectively, and the dual fiber fluorescence in situ hybridization (FISH) have been used to this aim. However, SFV is not always able to identify single DAZ gene copy deletions. We report a quantitative real-time PCR application to evaluate partial deletions of the DAZ gene cluster. To accomplish this, we designed a probe on exon 6 of the DAZ gene which is repeated 3 times in DAZ1, once in DAZ2 and DAZ3 and twice in DAZ4. Five normozoospermic healthy men (C1-C5) having 4 DAZ gene copies by SFV were selected. Fiber-FISH confirmed this outcome in C1-C4, but not in C5 who had an incomplete DAZ gene cluster. The men underwent then quantitative real-time PCR and C1 was arbitrarily selected as calibrator for the calculation of the DAZ gene signals because of the lowest variation in the threshold cycles. Real-time PCR identified 7.2±0.05 signals in C2-C4 and 5.4±0.05 signals in C5. The overall coefficient of variation was 1.4±0.2%. The loss of two signals in this subject may relate to a deletion of both DAZ2 and DAZ3 or of DAZ4 gene. Since SFV showed clearly the presence of DAZ2, it may be hypothesized that C5 lacks DAZ4. In conclusion, these data suggested that quantitative real-time PCR seems to be an effective and reproducible technique that can be used to study the DAZ gene cluster. In addition, the probe chosen for this approach may give indication on the DAZ gene copy deleted.

Discrimination of Deletion and Duplication Subtypes of the Deleted in Azoospermia Gene Family in the Context of Frequent Interloci Gene Conversion

“…Although it is not typical, the results of several studies suggest the existence of deletions eliminating only one DAZ gene [ 3 , 38 ]. While the potential mechanisms that may result in such deletions are unknown, the possibility of their occurrence can’t be excluded.…”

“…To distinguish the members of a gene family from one another would normally require the use of sequence elements that ( i ) are characteristic of the respective members and ( ii ) do not show inter-individual variation. Several studies utilized genetic traits supposed to fulfill the above requirements for the discrimination of the various DAZ deletion or duplication subtypes [ 3 – 5 , 12 , 38 ]. However, little information has been reported on the inter-individual variation regarding either the actual marker-carrying DAZ family members [see requirement c ( S6 Table )] or the copy number [0, 1 or 2 ( S8 File )] of the tested “family member-specific” features in a control population consisting of individuals who carry one copy of each member of the DAZ gene family [ 14 ].…”

Investigation ofDAZandRBMY1Gene Expression in Human Testis by Quantitative Real-Time PCR

“…In contrast, quantitative real-time PCR measures the amount of PCR product generated in the exponential phase of PCR, thus, providing more reproducible data [22]. Two studies used quantitative real-time PCR to examine the effect of DAZ partial deletions and the DAZ downstream substrate CDC25A [1,2]. In this study, quantitative real-time PCR assays for DAZ and RBMY1, using LightCycler TM and 5 0 exonuclease probe technologies, were developed to determine the cDNA copy numbers of these genes in azoospermic male testicular biopsies and normospermic controls.…”