Quantitative Fluorescent-PCR Detection of Sex Chromosome Aneuploidies and AZF Deletions/Duplications

Abstract

The most common genetic causes of spermatogenic failure are sex chromosomal abnormalities (most frequently Klinefelter's syndrome) and deletions of the azoospermia factor (AZF) regions (AZFa, AZFb, and AZFc) of the Y chromosome. Several studies have proposed that partial AZFc deletions/duplications may be a risk factor for spermatogenic impairment. We describe a multiplex quantitative fluorescent-polymerase chain reaction (QF-PCR) method that allows simultaneous detection of these genetic causes and risk factors of male infertility. The 11-plex QF-PCR permitted the amplification of the amelogenin gene, four polymorphic X-specific short tandem repeat (STR) markers (XHPRT, DXS6803, DXS981, and exon 1 of the androgen receptor gene), nonpolymorphic Y-specific marker (SRY gene), polymorphic Y-specific STR marker (DYS448), and coamplification of DAZ/DAZL, MYPT2Y/MYPT2, and two CDY2/CDY1 fragments that allow for determination of the DAZ, MYPT2Y, and CDY gene copy number. A total of 357 DNA samples from infertile/subfertile men (n = 205) and fertile controls (n = 152) was studied. We detected 14 infertile males with sex chromosome aneuploidy (10 with Klinefelter's syndrome, 2 XX, and 2 XYY males). All previously detected AZF deletions, that is, AZFc (n8), AZFb (n1), AZFb + c (n1), gr/gr (n11), gr/gr with b2/b4 duplication (n3), and b2/b3 (n5), gave a specific pattern with the 11-plex QF-PCR. In addition, 32 DNA samples showed a pattern consistent with presence of gr/gr or b2/b4 and 4 with b2/b3 duplication. We conclude that multiplex QF-PCR is a rapid, simple, reliable, and inexpensive method that can be used as a first-step genetic analysis in infertile/subfertile patients.

Male infertility in Northeast China: molecular detection of Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome

“…The fact that Northeast Chinese/Asian KFS patients generally lack AZF microdeletions, as was shown in this study, might suggest that the occurrence of these two defects most likely represents two separate independent genetic events that can occur in the same individual by chance. This observation has also been illustrated in other studies (Lee et al, 2000;Plaseski et al, 2008;Behulova et al, 2011;Rajpert-De et al, 2011). Furthermore, it was proposed that Y chromosome deletions do not facilitate non-disjunction events in paternal sex chromosomes, and do not cause a gain of X chromosomes (Rajpert-De et al, 2011).…”

Detection of the Most Common Genetic Causes of Male Infertility by Quantitative Fluorescent (QF)-PCR Analysis

“…During our previous work we have detected eight different AZF deletions, including the two partial AZFc deletions, gr/gr and b2/b3 deletions (Plaseski et al, 2006, Plaseski et al, 2008. Schematic presentation of the deletions is shown in Figure 2.…”

“…Thus, our initial results indicated a possible association between CAG repeat length and mild oligozoospermia. (Plaseski et al, 2008). This 11-plex QF-PCR analysis was shown as a rapid, simple, reliable and inexpensive method that can be used as a first-step genetic analysis in infertile patients.…”

“…Y microdeletions are found almost exclusively in patients with azoospermia or severe oligozoospermia (Simoni et al, 1998). The prevalence of Y microdeletions among the infertile males from the Republic of Macedonia is 6.4%, among patients with azoospermia 16.7% and among those with severe oligozoospermia 2.8% (Plaseski et al, 2003 (Plaseski et al, 2006;Plaseski et al, 2008 Distant homologous recombination between specific palindromic sequences is believed to be the mechanism for majority Yq deletions ( Figure 1C) (Kamp et al, 2000;Repping et al, 2002;Repping et al, 2003), although deletions based on mechanism of nonhomologous recombination were also identified (Costa et al, 2008). The AZFa deletions are located in proximal Yq and are caused by recombination that take place between retroviral homologous sequences.…”

Quadruplex real-time polymerase chain reaction assay for molecular diagnosis of Y-chromosomal microdeletions

“…In the past few years, several approaches has been developed to constitute the important adjuncts to the EAA/EMON guidelines method for the rapid diagnosis of YCMD (18)(19)(20), but the performance or throughput limitations of these methods constrain their use in routine testing. For example, Zhu et al (18) reported a novel YCMD detection method based on multianalyte suspension array technology, but the method could not be applied widely in a short time owing to the high cost and relatively complicated operation.…”