High frequencies of Non Allelic Homologous Recombination (NAHR) events at the AZF loci and male infertility risk in Indian men

Abstract

Deletions in the AZoospermia Factor (AZF) regions (spermatogenesis loci) on the human Y chromosome are reported as one of the most common causes of severe testiculopathy and spermatogenic defects leading to male infertility, yet not much data is available for Indian infertile men. Therefore, we screened for AZF region deletions in 973 infertile men consisting of 771 azoospermia, 105 oligozoospermia and 97 oligoteratozoospermia cases, along with 587 fertile normozoospermic men. The deletion screening was carried out using AZF-specific markers: STSs (Sequence Tagged Sites), SNVs (Single Nucleotide Variations), PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) analysis of STS amplicons, DNA sequencing and Southern hybridization techniques. Our study revealed deletion events in a total of 29.4% of infertile Indian men. Of these, non-allelic homologous recombination (NAHR) events accounted for 25.8%, which included 3.5% AZFb deletions, 2.3% AZFbc deletions, 6.9% complete AZFc deletions, and 13.1% partial AZFc deletions. We observed 3.2% AZFa deletions and a rare long AZFabc region deletion in 0.5% azoospermic men. This study illustrates how the ethnicity, endogamy and long-time geographical isolation of Indian populations might have played a major role in the high frequencies of deletion events.

Figure 1

(A) Schematic representation of the human Y chromosome structure. (B) Schematic diagram showing NAHR events between the P5/proximal P1, P4/distal P1, P5/distal P1 and b2/b4 amplicons. (C) Schematic diagram showing the location of direct and inverted repeat sequences, transcription units and STSs markers of the AZFc region, containing the genes and transcription units BPY2, CDY1, CSPG4LY, DAZ, GOLGA2LY, TTY3 and TTY4 in variable numbers of copies. (D) Schematic picture showing the complete deletion of AZFc results from recombination between the b2 and b4 amplicons, which removes 3.5 Mb of DNA. (E) Schematic diagram showing the b1/b3 deletion removes a ~1.6 Mb segment of the AZFc region. (F) Schematic diagram showing the gr/gr deletion, it is the most common deletion type caused by the recombination events between the amplicons g1-r1-r2 and g2-r3-r4. (G) Schematic diagram showing the b2/b3 inversion followed by gr/gr deletion or vice versa remove 1.8 Mb segment of AZFc region. (H) Schematic diagram showing the b3/b4 inversion followed by gr/gr deletion, or vice versa, removes a ~1.6 Mb segment of the AZFc region. Deleted regions are shown as faded, and the possible combinations of inversion and recombination events are given.

Figure 2

Top: Y-chromosomal phylogenetic tree showing the markers tested and the haplogroups they define. Bottom: Distributions and frequencies of haplogroups in fertile and infertile men with deletion and without deletions.

Figure 3

The DAZ and CDY1 genes copies deletions. (A) Schematic diagram showing the organization of DAZ gene copies. The 3 SNV markers sY587, sY581 and sY586 allow the differentiation of DAZ gene copies. (B1) The sequence electropherogram shows allele ‘T’ in the left panel, allele ‘C’ in the right panel and both the alleles in the middle panel. (B2) Schematic representation of the sY587 amplicon digested with restriction enzyme DraI. (B3) The first lane of both panels is the digested amplicon of fertile controls. The second lane of the first panel shows the absence of the 73 bp and 122 bp fragments, suggesting the deletion of DAZ1/DAZ2. The second lane of the second panel shows the absence of the 195 bp fragment, suggesting the deletion of DAZ3/DAZ4. (C1) The sequence electropherogram shows allele ‘C’ in the left panel, allele ‘T’ in the right panel and both the alleles in the middle panel. (C2) Schematic representation of the sY581 amplicon digested with restriction enzyme Sau3A. (C3) The first lane of both panels is the digested amplicon of fertile men. The second lane of the first panel shows the absence of the 189 bp fragment, which suggests the deletion of DAZ1/DAZ4. The second lane of the second panel shows the absence of the 130 bp fragment, suggesting the deletion of DAZ2/DAZ3. (D1) The sequence electropherogram shows allele ‘C’ in the right panel and both ‘C’ and ‘T’ alleles in the left panel. (D2) Schematic representation of the amplicon of sY586 digested with TaqI. (D3) The first lane of the panel is the digested amplicon of fertile man. The second lane shows the absence of the 301 bp fragment, suggesting the deletion of DAZ2. (E) PCR-RFLP of CDY1-specific SNV; CDY1-7750 was amplified and digested with PvuII and size-fractionated in a 2.0% agarose gel. CDY1b has a PvuII restriction site, but CDY1a does not have a PvuII restriction site. Lanes 1, 2, 3 and 14 – showing the intact CDY1a and the digested CDY1b. Lanes 4, 7, 8 and 12 – showing the presence of only CDY1a. Lanes 5, 6, 9, 10, 11, 13 and 15 – showing the presence of only CDY1b. Lane 16 undigested DNA and lane M marker.

Figure 4

DAZ copy deletions confirmed by Southern hybridization analysis. (A) The first lane of the panel is the EcoRV-digested amplicon of fertile man. The second lane shows the EcoRV-digested DNA from an infertile man with a DAZ1 deletion confirmed by hybridized with the 49f probe. (B) The first lane of the panel is the EcoRV-digested amplicon of fertile man. The second lane shows the infertile EcoRV-digested DNA from an infertile man with a DAZ4 deletion confirmed by hybridized with the 49f probe. (C) The first lane of the panel is the EcoRV-digested amplicon of fertile man. The second lane shows the TaqI-digested DNA from an infertile man with a DAZ3 deletion confirmed by hybridized with 49f probe. Although DNA of both fertile and infertile men were run in the same gel, transferred, and hybridized, they were not in the adjacent lanes. Therefore, we have cropped the images and placed the relevant adjacent for better comparison. We have provided the unedited autoradiogram as a Supplementary File.