Deletion of azoospermia factor a (AZFa) region of human Y chromosome caused by recombination between HERV15 proviruses

Deletion of any of three regions of the human Y chromosome results in spermatogenic failure and infertility. We previously sequenced one of these regions, azoospermia factor a (AZFa) and found that it spanned approximately 800 kb. By sequence-tagged site (STS) content mapping, we roughly defined deletion breakpoints in two unrelated, azoospermic men with AZFa deletions. The positions of proximal and distal breakpoints were similar in the two men. Hypothesizing that the deletions might have been generated by homologous recombination, we searched electronically for DNA sequence similarities between the proximal and distal breakpoint regions. These comparisons revealed the most striking sequence similarities anywhere along or near the AZFa region. In the proximal breakpoint region, we identified a 10 kb provirus of the recently defined HERV15 class of endogenous retroviruses. In the distal breakpoint region, we found a second HERV15 provirus, 94% identical in DNA sequence to the first and in the same orientation. (A partial LINE insertion in this distal provirus proved to be the basis of the previously described DYS11/p12f polymorphism.) The AZFa deletions in the two men differed slightly, but all breakpoints fell within the HERV15 proviruses. Indeed, sequencing of deletion junctions from the two men revealed that homologous recombination had occurred within large domains of absolute sequence identity between the proximal and distal proviruses. When combined with published STS mapping data for other AZFa-deleted men, our findings suggest that recombination between these two HERV15 proviruses could account for most AZFa deletions.     

Divergent outcomes of intrachromosomal recombination on the human Y chromosome: male infertility and recurrent polymorphism

The Y chromosome provides a unique opportunity to study mutational processes within the human genome, decoupled from the confounding effects of interchromosomal recombination. It has been suggested that the increased density of certain dispersed repeats on the Y could account for the high frequency of causative microdeletions relative to single nucleotide mutations in infertile males. Previously we localised breakpoints of an AZFa microdeletion close to two highly homologous complete human endogenous retroviral sequences (HERV), separated by 700 kb. Here we show, by sequencing across the breakpoint, that the microdeletion occurs in register within a highly homologous segment between the HERVs. Furthermore, we show that recurrent double crossovers have occurred between the HERVs, resulting in the loss of a 1.5 kb insertion from one HERV, an event underlying the first ever Y chromosomal polymorphism described, the 12f2 deletion. This event produces a substantially longer segment of absolute homology and as such may result in increased predisposition to further intrachromosomal recombination. Intrachromosomal crosstalk between these two HERV sequences can thus result in either homogenizing sequence conversion or a microdeletion causing male infertility. This represents a major subclass of AZFa deletions.     

Detection of sperm in men with Y chromosome microdeletions of the AZFa,AZFb and AZFc regions

BACKGROUND: Y chromosome microdeletions are associated with severe male factor infertility. In this study, the success rate of testicular sperm retrieval was determined for men with deletions of AZF regions a, b or c. METHODS: AZF deletions were detected by PCR of 30 sequence-tagged sites within Yq emphasizing the AZFa, b and c regions. Semen analysis and diagnostic testis biopsy or testicular sperm extraction (TESE) findings were correlated with the specific AZF region deleted. RESULTS: A total of 78 men with AZF deletions included three with AZFa deletion, 11 with AZFb, 42 with AZFc, 16 with AZFb+c and six with Yq (AZFa+b+c). All men with AZFa, AZFb, AZFb+c and Yq deletions were azoospermic and no sperm were found with TESE or biopsy. Of men with isolated AZFc deletion, sperm were found in 75% (9/12) by TESE and 45% (9/20) on biopsy (56% overall); 62% (26/42) were azoospermic and 38% (16/42) severely oligozoospermic. A total of 7 patients with deletion patterns that included the complete AZFa region and 23 that included the complete AZFb region who underwent TESE or biopsy did not have sperm detected by these surgical measures. CONCLUSIONS: Microdeletion of the entire AZFa or AZFb regions of the Y chromosome portends an exceptionally poor prognosis for sperm retrieval, whereas the majority of men with AZFc deletion have sperm within the semen or testes available for use in IVF/ICSI.     

Natural transmission of USP9Y gene mutations: a new perspective on the role of AZFa genes in male fertility

Deletions of the azoospermia factor (AZF) regions of the Y chromosome are associated with severe spermatogenic failure and represent the most frequent molecular genetic cause of azoospermia and severe oligozoospermia. The exact role of the candidate AZF genes is largely unknown due to both the extreme rarity of naturally occurring AZF gene-specific mutations and the lack of functional assays. Here, we report the fine characterization of two different deletions in the USP9Y gene (one of the two candidate genes in the AZFa region), which have been transmitted through natural conception in two unrelated families. The associated mild testicular phenotype, in both cases, is in sharp contrast with that of the two previously reported infertile patients bearing a mutation of the same gene. In conclusion, to date, the USP9Y gene has been considered as one of the major Y-linked spermatogenesis genes, based on both its position within the AZFa region and previous reports that correlated USP9Y mutation to severe spermatogenic failure and infertility. This view is now substantially changed because our findings clearly demonstrate that during human spermatogenesis, USP9Y is more likely a fine tuner that improves efficiency, rather than a provider of an essential function. More importantly, the observed natural conceptions suggest that the protein is not required for the final sperm maturation process or for the acquisition of sperm fertilizing ability, providing a new perspective on the role played by the USP9Y gene in male fertility.     

无精子因子C区部分缺失与男性不育相关性研究

目的 探讨Y染色体无精子症因子C区(azoospermia factor C,AZFc)部分缺失与男性不育的关系.方法 采用多重PCR技术对实验组453例原发不育患者(无精子症158例,严重少精子症160例,少精子症135例)和对照组236名已正常生育男性进行AZFc区部分缺失检测,并应用限制性片段长度多态性技术,对部分缺失携带者进行DAZ基因拷贝缺失分析.结果 在所发现的两种常见缺失中,少精症组和严重少精症组与正常对照组b2/b3缺失率差异均有统计学意义,而在各组中gr/gr缺失显示相似的频率.结论 Y染色体AZFc区b2/b3缺失可能是该人群生精障碍发生的风险因素,并与男性原发不育相关.     

The human Y chromosome genes BPY2, CDY1 and DAZ are not essential for sustained fertility

Deletions of the AZFc interval of the human Y chromosome are found in >5% of male patients with idiopathic infertility and are associated with a severely reduced sperm count. The most common deletion type is large (>1 Mb) and removes members of the Y-borne testis-specific gene families of BPY2, CDY1, DAZ, PRY, RBMY2 and TTY2, which are candidate AZF genes. Four exceptional individuals who have transmitted a large AZFc deletion naturally to their infertile sons have, however, been described. In three cases, transmission was to an only son, but in the fourth case a Y chromosome, shown to be deleted for all copies of DAZ, was transmitted from a father to his four infertile sons. Here we present a second family of this latter type and demonstrate that an AZFc-deleted Y chromosome lacking not only DAZ, but also BPY2 and CDY1, has been transmitted from a father to his three infertile sons. Polymerase chain reaction (PCR) and Southern blot analyses revealed no difference in the size of the AZFc deletion in the father and his sons. We propose that the father carries rare alleles of autosomal or X-linked loci which suppress the infertility that is frequently associated with the absence of AZFc.     

A fiber-FISH contig spanning the non-recombining region of the human Y chromosome

Using fluorescence in-situ hybridization on interphase chromatin fibers (fiber-FISH), we have constructed an overlapping fiber-FISH contig spanning the non-recombining region of the human Y chromosome (NRY). We first established a standard FISH-signal pattern for a distinct panel of DNA clones on prometaphase Y chromosomes in six healthy fertile men. Clones in the panel were selected from all R-bands as well as deletion intervals 1 through 7 plus PAR1 and PAR2 of the human Y chromosome. We next used signals of these marker clones to build a fiber-FISH contig for the multicopy gene families, CDY, DAZ, RBMY, TSPY and XKRY, along the NRY. Our fiber-FISH contig of human NRY may help to close the four gaps that still exist in the current physical map of the human Y chromosome. Furthermore, it provides a more complete picture with respect to the positions and arrangements of the multicopy gene families along the human NRY.     

Class switch from mu to delta is mediated by homologous recombination between sigma mu and sigma mu sequences in human immunoglobulin gene loci

Class switch of immunoglobulin from mu to gamma occurs by recombination between two repetitive switch sequences: S mu and S gamma. However, there are no such sequences in the mu-delta introns of human and mouse genomes. Although the frequency of IgD-secreting cells is extremely low in mouse about 1% of patients with myeloma produce IgD in human. In a previous report (Nucleic Acids Res. 1988. 16: 9497) we reported that a 442-bp DNA sequence located in the JH-mu intron (defined as sigma mu) was inserted into the mu-delta intron (defined as sigma mu) in human genome. There is no such insertion in mouse. We analyzed Ig H chain gene loci of two human IgD myelomas: one was analyzed by cloning and sequencing and the other by Southern hybridization. We found that recombination had occurred between these two homologous DNA sequences, resulting in loss of the DNA segment from sigma mu to sigma mu. On the other hand, in a Burkitt lymphoma, Daudi, the DNA fragment from sigma mu to sigma mu was duplicated. These results suggest that homologous recombination between sigma mu and sigma mu sequences mediates class switch from mu to delta in human and that it occurs via unequal crossing-over between sister chromatids or daughter chromosomes.     

Independent intrachromosomal recombination events underlie the pericentric inversions of chimpanzee and gorilla chromosomes homologous to human chromosome 16

Analyses of chromosomal rearrangements that have occurred during the evolution of the hominoids can reveal much about the mutational mechanisms underlying primate chromosome evolution. We characterized the breakpoints of the pericentric inversion of chimpanzee chromosome 18 (PTR XVI), which is homologous to human chromosome 16 (HSA 16). A conserved 23-kb inverted repeat composed of satellites, LINE and Alu elements was identified near the breakpoints and could have mediated the inversion by bringing the chromosomal arms into close proximity with each other, thereby facilitating intrachromosomal recombination. The exact positions of the breakpoints may then have been determined by local DNA sequence homologies between the inversion breakpoints, including a 22-base pair direct repeat. The similarly located pericentric inversion of gorilla (GGO) chromosome XVI, was studied by FISH and PCR analysis. The p- and q-arm breakpoints of the inversions in PTR XVI and GGO XVI were found to occur at slightly different locations, consistent with their independent origin. Further, FISH studies of the homologous chromosomal regions in macaque and orangutan revealed that the region represented by HSA BAC RP11-696P19, which spans the inversion breakpoint on HSA 16q11-12, was derived from the ancestral primate chromosome homologous to HSA 1. After the divergence of orangutan from the other great apes approximately 12 million years ago (Mya), a duplication of the corresponding region occurred followed by its interchromosomal transposition to the ancestral chromosome 16q. Thus, the most parsimonious interpretation is that the gorilla and chimpanzee homologs exhibit similar but nonidentical derived pericentric inversions, whereas HSA 16 represents the ancestral form among hominoids.     

Lack of association between Y chromosome haplogroups and male infertility in Japanese men

The Y chromosome carries several genes involved in spermatogenesis, which are distributed in three regions in the euchromatic part of the long arm, called AZFa (azoospermia factor a), AZFb, and AZFc. Microdeletions in these regions have been seen in 10-15% of sterile males with azoospermia or severe oligozoospermia. The relatively high de novo occurrence of these microdeletion events might be due to particular chromosome arrangements associated with certain Y chromosome haplogroups. To test whether there is any association between Y chromosome types and male infertility, we studied a sample of 84 Japanese oligozoospermic or azoospermic males. The patients were analyzed for the presence of Yq microdeletions and also typed with a battery of unique event polymorphisms (UEPs) to define their Y haplogroups. Six of the infertile patients presented likely pathological microdeletions detectable with the sequence tagged sites (STS) markers used. There was no significant association between Y chromosome haplogroups and the microdeletions. We also compared the Y haplogroup frequencies in our subset sample of 51 idiopathic azoospermia patients with 57 fertile control Japanese males, and did not observe any significant differences. Contrary to previous reports, our data suggest that Y microdeletions and other molecular events causally associated with male infertility in Japan occur independently of the Y chromosome background.     

Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.     

178例男性不育患者Y染色体AZF微缺失筛查

目的探讨男性不育症患者Y染色体微缺失分子检测临床意义。方法应用多重PCR对178例不育症患者进行Y染色体AZFa、AZFb和AZFe基因微缺失检测。结果41例特发性无精症患者中有10例缺失,占24．3％;34例严重少精子症患者中有4例缺失,占11．7％;其余103例少弱精子症患者中没有检出缺失。结论在男性不育症患者中,Y染色体AZF基因微缺失是特发性无精子或严重少精子症发生的重要原因,基因检测可为正确诊断和合理治疗提供科学依据。     

人Y染色体类热休克蛋白因子编码特征和男性生精障碍

精子生成障碍是男性不育的主要原因之一,而无精症和少精症在男性不育症患者中,90％是由于生精功能障碍引起,其中特发性生精障碍占60％。近年来通过人类基因组计划的实施,发现不少Y染色体上诱发不育症的候选基因,包括类热休克蛋白因子HSFY、LW-1和mHSFYL。本文通过HSFY、LW-1和mHSFYL的编码特征,阐述它们在精子生成过程中的作用和诱发生精障碍、导致无精症和少精症的遗传学机理。     

Y chromosome gene copy number and lack of autism phenotype in a male with an isodicentric Y chromosome and absent NLGN4Y expression

We describe a unique male with a dicentric Y chromosome whose phenotype was compared to that of males with 47,XYY (XYY). The male Y‐chromosome aneuploidy XYY is associated with physical, behavioral/cognitive phenotypes, and autism spectrum disorders. We hypothesize that increased risk for these phenotypes is caused by increased copy number/overexpression of Y‐encoded genes. Specifically, an extra copy of the neuroligin gene NLGN4Y might elevate the risk of autism in boys with XYY. We present a unique male with the karyotype 46,X,idic(Y)(q11.22), which includes duplication of the Y short arm and proximal long arm and deletion of the distal long arm, evaluated his physical, behavioral/cognitive, and neuroimaging/magnetoencephalography (MEG) phenotypes, and measured blood RNA expression of Y genes. The proband had tall stature and cognitive function within the typical range, without autism features. His blood RNA showed twofold increase in expression of Yp genes versus XY controls, and absent expression of deleted Yq genes, including NLGN4Y. The M100 latencies were similar to findings in typically developing males. In summary, the proband had overexpression of a subset of Yp genes, absent NLGN4Y expression, without ASD findings or XYY‐MEG latency findings. These results are consistent with a role for NLGN4Y overexpression in the etiology of behavioral phenotypes associated with XYY. Further investigation of NLGN4Y as an ASD risk gene in XYY is warranted. The genotype and phenotype(s) of this subject may also provide insight into how Y chromosome genes contribute to normal male development and the male predominance in ASD.     

Deletion of the long arms of the Y chromosome with normal male development and intelligence

Normal male development was found in a man with a proven deletion of the long arms of the Y chromosome. The only phenotypic effect was on his build. This study offers additional proof that all, or most of the long arms of the Y chromosome are not primarily concerned with the determination of male sexual characteristics.