Spermatogenesis pp 187-204 | Cite as
Human Y Chromosome Microdeletion Analysis by PCR Multiplex Protocols Identifying only Clinically Relevant AZF Microdeletions
Abstract
PCR multiplex assays are the method of choice for quickly revealing genomic microdeletions in the large repetitive genomic sequence blocks on the long arm of the human Y chromosome. They harbor the Azoospermia Factor (AZF) genes, which cause male infertility when functionally disrupted. These protein encoding Y genes are expressed exclusively or predominantly during male germ cell development, i.e., at different phases of human spermatogenesis. They are located in three distinct genomic sequence regions designated AZFa, AZFb, and AZFc, respectively. Complete deletion of an AZF region, also called “classical” AZF microdeletion, is always associated with male infertility and a distinct testicular pathology. Partial AZF deletions including single AZF Y genes can cause the same testicular pathology as the corresponding complete deletion (e.g., DDX3Y gene deletions in AZFa), or might not be associated with male infertility at all (e.g., some BPY2, CDY1, DAZ gene deletions in AZFc). We therefore propose that a PCR multiplex assay aimed to reduce only those AZF microdeletions causing a specific testicular pathology—thus relevant for clinical applications. It only includes Sequence Tagged Site (STS) deletion markers inside the exon structures of the Y genes known to be expressed in male germ cells and located in the three AZF regions. They were integrated in a robust standard protocol for four PCR multiplex mixtures which also include the basic principles of quality control according to the strict guidelines of the European Molecular Genetics Quality Network (EMQN: http://www.emqn.org). In case all Y genes of one AZF region are deleted the molecular extension of this AZF microdeletion is diagnosed to be yes or no comparable to that of the “classical” AZF microdeletion by an additional PCR multiplex assay analyzing the putative AZF breakpoint borderlines.
Key words
AZFa, AZFb, AZFc classical microdeletions PCR multiplex assays AZF DNA polymorphisms and border lines AZF gene deletionsNotes
Acknowledgments
We are indebted to the head of our department, Prof. Dr. Thomas Strowitzki, who financially supported the establishment and running of all of the PCR multiplex assays. We also thank our former colleagues Octavian Henegariu (now at Yale University School of Medicine; HTI, Immunobiology department), Angela Edelmann and Peter Hirschmann (now at Biopharm GmbH, Heidelberg), Karin Huellen (University of Heidelberg; Institute of Human Genetics), and Alexandra Schadwinkel who have contributed to earlier setups of the PCR multiplex assays for the detailed analysis of AZF (gene) microdeletions.
References
- 1.Ngo KY et al (1986) A DNA probe detecting multiple haplotypes of the human Y chromosome. Am J Hum Genet 38:407–418PubMedGoogle Scholar
- 2.Torroni A et al (1990) Y chromosome DNA polymorphisms in human populations: differences between Caucasoids and Africans detected by 49a and 49f probes. Ann Hum Genet 54:287–296PubMedCrossRefGoogle Scholar
- 3.Jobling MA (1994) A survey of long-range DNA polymorphisms on the human Y chromosome. Hum Mol Genet 3:107–114PubMedCrossRefGoogle Scholar
- 4.Seielstad MT et al (1994) Construction of human Y-chromosomal haplotypes using a new polymorphic A to G transition. Hum Mol Genet 3:2159–2161PubMedCrossRefGoogle Scholar
- 5.Santos FR et al (1995) PCR haplotypes for the human Y chromosome based on alphoid satellite DNA variants and heteroduplex analysis. Gene 165:191–198PubMedCrossRefGoogle Scholar
- 6.Jobling MA et al (1996) Recurrent duplication and deletion polymorphisms on the long arm of the Y chromosome in normal males. Hum Mol Genet 5:1767–1775PubMedCrossRefGoogle Scholar
- 7.Tiepolo L, Zuffardi O (1976) Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 34:119–124PubMedCrossRefGoogle Scholar
- 8.Vogt P et al (1992) Microdeletions in interval 6 of the Y chromosome of males with idiopathic sterility point to disruption of AZF, a human spermatogenesis gene. Hum Genet 89:491–496PubMedCrossRefGoogle Scholar
- 9.Ma K et al (1992) Towards the molecular localisation of the AZF locus: mapping of microdeletions in azoospermic men within 14 subintervals of interval 6 of the human Y chromosome. Hum Mol Genet 1:29–33PubMedCrossRefGoogle Scholar
- 10.Johnson MD et al (1989) Molecular scanning of Yq11 (interval 6) in men with Sertoli-cell-only syndrome. Am J Obstet Gynecol 161:1732–1737PubMedGoogle Scholar
- 11.Nagafuchi S et al (1993) A minute deletion of the Y chromosome in men with azoospermia. J Urol 150:1155–1157PubMedGoogle Scholar
- 12.Kobayashi K et al (1994) PCR analysis of the Y chromosome long arm in azoospermic patients: evidence for a second locus required for spermatogenesis. Hum Mol Genet 3:1965–1967PubMedCrossRefGoogle Scholar
- 13.Pryor JL et al (1997) Microdeletions in the Y chromosome of infertile men. N Engl J Med 336:534–539PubMedCrossRefGoogle Scholar
- 14.Vogt P et al (1991) Towards the molecular localization of AZF, a male fertility gene on the human Y chromosome by comparative mapping of microdeletions in the Y chromosome of men with idiopathic infertility. Am J Hum Genet 49:258Google Scholar
- 15.Vogt PH et al (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 5:933–943PubMedCrossRefGoogle Scholar
- 16.Simoni M et al (1999) Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int J Androl 22:292–299PubMedCrossRefGoogle Scholar
- 17.Rodovalho RG et al (2008) Tracking microdeletions of the AZF region in a patrilineal line of infertile men. Genet Mol Res 7:614–622PubMedCrossRefGoogle Scholar
- 18.Repping S et al (2003) Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 35:247–251PubMedCrossRefGoogle Scholar
- 19.Vogt PH (2005) AZF deletions and Y chromosomal haplogroups: history and update based on sequence. Hum Reprod Update 11:319–336PubMedCrossRefGoogle Scholar
- 20.Repping S et al (2006) High mutation rates have driven extensive structural polymorphism among human Y chromosomes. Nat Genet 38:463–467PubMedCrossRefGoogle Scholar
- 21.Quintana-Murci L et al (2001) The relationship between Y chromosome DBA haplotypes and Y chromosome deletions leading to male infertility. Hum Genet 108:55–58PubMedCrossRefGoogle Scholar
- 22.Fernandes S et al (2004) A large AZFc deletion removes DAZ3/DAZ4 and nearby genes from men in Y haplogroup N. Am J Hum Genet 74:180–187PubMedCrossRefGoogle Scholar
- 23.Krausz C et al (2011) The Y chromosome-linked copy number variations and male fertility. J Endocrinol Invest 34:376–382PubMedGoogle Scholar
- 24.Stouffs K et al (2011) What about gr/gr deletions and male infertility? Systematic review and meta-analysis. Hum Reprod Update 17:197–209PubMedCrossRefGoogle Scholar
- 25.Simoni M et al (2004) EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State of the art 2004. Int J Androl 27:240–249PubMedCrossRefGoogle Scholar
- 26.Lahn BT, Page DC (1997) Functional coherence of the human Y chromosome. Science 278:675–680PubMedCrossRefGoogle Scholar
- 27.Skaletsky H et al (2003) The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423:825–837PubMedCrossRefGoogle Scholar
- 28.Vogt PH (2005) Azoospermia factor (AZF) in Yq11: towards a molecular understanding of its function for human male fertility and spermatogenesis. Reprod Biomed Online 10:81–93PubMedCrossRefGoogle Scholar
- 29.Nakahori Y et al (1994) A locus of the candidate gene family for azoospermia factor (YRRM2) is polymorphic with a null allele in Japanese males. Hum Mol Genet 3:1709PubMedCrossRefGoogle Scholar
- 30.Girardi SK et al (1997) Submicroscopic deletions in the Y chromosome of infertile men. Hum Reprod 12:1635–1641PubMedCrossRefGoogle Scholar
- 31.Stuppia L et al (1996) Widening of a Y-chromosome interval-6 deletion transmitted from a father to his infertile son accounts for an oligozoospermia critical region distal to the RBM1 and DAZ genes. Am J Hum Genet 59:1393–1395PubMedGoogle Scholar
- 32.Najmabadi H et al (1996) Substantial prevalence of microdeletions of the Y-chromosome in infertile men with idiopathic azoospermia and oligozoospermia detected using a sequence-tagged site-based mapping strategy. J Clin Endocrinol Metab 81:1347–1352PubMedCrossRefGoogle Scholar
- 33.Foresta C et al (1997) Y-chromosome deletions in idiopathic severe testiculopathies. J Clin Endocrinol Metab 82:1075–1080PubMedCrossRefGoogle Scholar
- 34.[Laboratory manual of the WHO for the examination of human semen and sperm-cervical mucus interaction]. Ann Ist Super Sanita 37: I–XII, 1–123 (2001)Google Scholar
- 35.Kamp C et al (2001) High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome. Mol Hum Reprod 7:987–994PubMedCrossRefGoogle Scholar
- 36.Mirfakhraie R et al (2010) High prevalence of AZFb microdeletion in Iranian patients with idiopathic non-obstructive azoospermia. Indian J Med Res 132:265–270PubMedGoogle Scholar
- 37.Siffroi JP et al (2000) Sex chromosome mosaicism in males carrying Y chromosome long arm deletions. Hum Reprod 15:2559–2562PubMedCrossRefGoogle Scholar
- 38.Patsalis PC et al (2002) Effects of transmission of Y chromosome AZFc deletions. Lancet 360:1222–1224PubMedCrossRefGoogle Scholar
- 39.Kuroda-Kawaguchi T et al (2001) The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 29:279–286PubMedCrossRefGoogle Scholar
- 40.Vogt PH (2004) Genomic heterogeneity and instability of the AZF locus on the human Y chromosome. Mol Cell Endocrinol 224:1–9PubMedCrossRefGoogle Scholar