The role of Y chromosome deletions in male infertility

Male infertility affects approximately 2-7% of couples around the world. Over one in ten men who seek help at infertility clinics are diagnosed as severely oligospermic or azoospermic. Recent extensive molecular studies have revealed that deletions in the azoospermia factor region of the long arm of the Y chromosome are associated with severe spermatogenic impairment (absent or severely reduced germ cell development). Genetic research into male infertility, in the last 7 years, has resulted in the isolation of a great number of genes or gene families on the Y chromosome, some of which are believed to influence spermatogenesis.     

A high frequency of Y chromosome deletions in males with nonidiopathic infertility.

Microdeletions of the long arm of the human Y chromosome are associated with spermatogenic failure and have been used to define three regions of Yq (AZFa, AZFb, and AZFc) that are recurrently deleted in infertile males. In a blind study we screened 131 infertile males (46 idiopathic and 85 nonidiopathic) for Y chromosome microdeletions. Nineteen percent of idiopathic males, with an apparently normal 46,XY chromosome complement had microdeletions of either the AZFa, AZFb, or AZFc region. There was no strict correlation between the extent or location of the deletion and the phenotype. The AZFb deletions did not include the active RBM gene. Significantly, a high frequency of microdeletions (7%) was found in patients with known causes of infertility and a 46,XY chromosome complement. These included deletions of the AZFb and AZFc regions, with no significant difference in the location or extent of the deletion compared with the former group. It is recommended that all males with reduced or absence sperm counts seeking assisted reproductive technologies be screened for deletions of the Y chromosome.     

Small deletions of the short arm of the Y chromosome in 46,XY females.

Structural anomalies of the sex chromosomes provide a means to study the location of genes responsible for sex determination. Recently, a type of sex reversal in humans, the 46,XX male, was shown to result in some cases from translocation of Y chromosome material to the X chromosome. In the present report, another type of sex reversal, the 46,XY female, is shown to result, in two cases, from small deletions of the short arm of the Y chromosome. Prometaphase chromosome analysis showed a 46,X,Yp- karyotype. Several Y chromosome-specific DNA probes were found to be deleted in the two female patients. DNA analysis showed that the two deletions were different but included a common overlapping region likely to be essential for male determination.     

The pituitary-testicular axis in Klinefelter's syndrome and in oligo-azoospermic patients with and without deletions of the Y chromosome long arm

OBJECTIVE: The most frequent known genetic causes of severe oligospermia (< 5 million sperm/ml) or azoospermia in men are Klinefelter's syndrome (KS), and deletions in the Y chromosome long arm (Yq). We aimed to compare the function of the pituitary-testicular axis in patients with severe oligospermia or azoospermia, idiopathic or associated with Y chromosome deletions or Klinefelter's syndrome (KS) and in control subjects. PATIENTS: We studied 47 men with idiopathic oligo-azoospermia, 42 with Yq deletions (27 AZFc, 13 AZFb and two AZFa) and oligo-azoospermia, 14 with KS and 39 control subjects (total 143). MEASUREMENTS: We analysed levels of FSH, inhibin-B, LH, free testosterone and oestradiol in all subjects, and we calculated indexes based on those hormones. RESULTS: Inhibin-B levels were indistinguishable between patients with idiopathic and Y deletion-associated oligo-azoospermia, lowest in the Klinefelter's patients and highest in controls. FSH levels followed the reverse pattern: indistinguishable between patients with idiopathic and deletion-associated oligo-azoospermia, highest in Klinefelter's patients and lowest in controls. Oestradiol, free testosterone and the derived indeces were not different in subjects with Yq deletions compared to those with idiopathic oligo-azoospermia. Among the Yq-deleted patients, no measured or derived parameter differed between the subjects with AZFc deletion and those with AZFb deletion. When non-KS oligo-azoospermic patients were classified according to histology [Sertoli cell-only (SCO), n = 18 or non-Sertoli cell only (non-SCO), n= 18] and compared to KS patients, the hormonal pattern did not differ between SCO and non-SCO subjects, but levels in KS patients were significantly different for FSH, inhibin-B and the FSH/inhibin-B ratio. KS patients not only had lower inhibin-B than SCO and non-SCO oligo-azoospermic men, but also higher FSH levels for any given inhibin-B concentration. CONCLUSION: Our data show that Y-deleted patients do not have a lesser impairment of Sertoli cell function than patients with idiopathic oligo-azoospermia, and support the concept that the main determinant of inhibin-B production is the germ cell mass. Also, our results suggest that one or more other factors, apart from inhibin-B, may contribute to increased pituitary secretion of FSH in KS patients.     

Rapid generation of nested chromosomal deletions on mouse chromosome 2

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F(1) hybrid embryonic stem cell line, F(1)(129S1xCast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.     

Cytogenetic and molecular diagnosis of chromosome 5 deletions in myelodysplasia

Deletions of chromosome 5, del(5q), are frequently observed in myelodysplasia (MDS). We evaluated molecular detection of loss of heterozygosity (LOH) as a diagnostic method to detect del(5q) in a series of 60 MDS cases at a single institution. LOH was compared to cytogenetics on the same clinical specimen, resolving ambiguous cases by fluorescent in situ hybridization (FISH) and additional LOH. There was poor concordance between molecular and cytogenetic results, but most discrepancies could be resolved by FISH and additional LOH. Molecular analysis was of low sensitivity because most cases contained a relatively high proportion of cells without del(5q), but it was accurate, while cytogenetics overestimated the proportion of cells with del(5q) and failed to detect some cases with complex rearrangements. Minor clones were detected both by FISH and LOH. Overall, we found an incidence of 23% (14 of 60 cases) for del(5q) in MDS. The results also suggest that there is a high degree of genetic heterogeneity in the cellular population of MDS. Although del(5q) is common in MDS, it may not be present in all cells, leading to diagnostic challenges.     

Molecular analysis of chromosome 11 deletions in aniridia-Wilms tumor syndrome.

We describe five individuals who have constitutional deletions of the short arm of one chromosome 11, including all or part of the band p13. All of these individuals suffer from aniridia; two have had a Wilms tumor removed. We have established lymphoblastoid cell lines from these and in three cases constructed somatic cell hybrids containing the deleted chromosome 11. Analysis of DNA from the cell lines and hybrids with a cloned cDNA probe has shown that the catalase gene is deleted in four of five patients. The catalase locus must be proximal to the Wilms and aniridia-related loci. We have not detected a deletion of the beta-globin or calcitonin genes in any of these individuals; we conclude these genes are likely to be outside the region 11p12-11p15.4. In addition, we have used monoclonal antibodies in fluorescence-activated cell sorting analysis to measure expression in the hybrids of two cell surface markers encoded by genes that map to the short arm of chromosome 11. The genes for both of these are deleted in two individuals but are present in the individual with the smallest deletion.     

Molecular characterization of chromosome 7 long arm deletions in myeloid disorders

Partial deletion of the long arm of chromosome 7 is a common abnormality in the bone marrow cells of patients with myelodysplastic syndrome (MDS) or acute nonlymphocytic leukemia (ANLL). This study was undertaken to characterize the chromosome breakpoints in molecular terms and to determine if hemizygosity or submicroscopic deletions occur in patients without any cytogenetically detectable abnormality of chromosome 7. We studied restriction fragment length polymorphisms with 10 chromosome 7-specific DNA probes in separated WBC fractions. No molecular abnormalities occurred in lymphocyte-derived DNA. Several probes located in band 7q22 or distally thereof detected deletion of one allele in granulocyte-derived DNA from all four patients with chromosome 7 long arm deletion. In the granulocytes of one patient heterozygosity for the T cell receptor beta chain gene (in band 7q35) indicated that the deletion was interstitial. NJ-3, a proalpha2(I)collagen gene probe (in band 7q21-22) detected heterozygosity in the granulocytes of one patient. No hemizygosity or deletions were found in four patients with two normal chromosomes 7. These results confirm that mature granulocytes but not lymphocytes are derived from the abnormal clone. Interstitial deletions exist, and the extent of deleted genomic material varies among patients.     

Determination of sex and Y chromosome]

Years of speculations about the nature of the elusive testis determining factor (TDF) of the Y chromosome have ended last year. A gene named SRY satisfies many criteria expected of the testis determining gene, and gives us a basis to understand molecular mechanisms of the testis differentiation. The different steps which gave rise to SRY cloning are described.     

Y chromosome microdeletions and alterations of spermatogenesis

Three different spermatogenesis loci have been mapped on the Y chromosome and named "azoospermia factors" (AZFa, b, and c). Deletions in these regions remove one or more of the candidate genes (DAZ, RBMY, USP9Y, and DBY) and cause severe testiculopathy leading to male infertility. We have reviewed the literature and the most recent advances in Y chromosome mapping, focusing our attention on the correlation between Y chromosome microdeletions and alterations of spermatogenesis. More than 4,800 infertile patients were screened for Y microdeletions and published. Such deletions determine azoospermia more frequently than severe oligozoospermia and involve especially the AZFc region including the DAZ gene family. Overall, the prevalence of Y chromosome microdeletions is 4% in oligozoospermic patients, 14% in idiopathic severely oligozoospermic men, 11% in azoospermic men, and 18% in idiopathic azoospermic subjects. Patient selection criteria appear to substantially influence the prevalence of microdeletions. No clear correlation exists between the size and localization of the deletions and the testicular phenotype. However, it is clear that larger deletions are associated with the most severe testicular damage. Patients with Y chromosome deletions frequently have sperm either in the ejaculate or within the testis and are therefore suitable candidates for assisted reproduction techniques. This possibility raises a number of medical and ethical concerns, since the use of spermatozoa carrying Y chromosome deletions may produce pregnancies, but in such cases the genetic anomaly will invariably be passed on to male offspring.     

Coincidence of synteny breakpoints with malignancy-related deletions on human chromosome 3

We have found previously that during tumor growth intact human chromosome 3 transferred into tumor cells regularly looses certain 3p regions, among them the approximately 1.4-Mb common eliminated region 1 (CER1) at 3p21.3. Fluorescence in situ hybridization analysis of 12 mouse orthologous loci revealed that CER1 splits into two segments in mouse and therefore contains a murine/human conservation breakpoint region (CBR). Several breaks occurred in tumors within the region surrounding the CBR, and this sequence has features that characterize unstable chromosomal regions: deletions in yeast artificial chromosome clones, late replication, gene and segment duplications, and pseudogene insertions. Sequence analysis of the entire 3p12-22 revealed that other cancer-associated deletions (regions eliminated from monochromosomal hybrids carrying an intact chromosome 3 during tumor growth and homozygous deletions found in human tumors) colocalized nonrandomly with murine/human CBRs and were characterized by an increased number of local gene duplications and murine/human conservation mismatches (single genes that do not match into the conserved chromosomal segment). The CBR within CER1 contains a simple tandem TATAGA repeat capable of forming a 40-bp-long secondary hairpin-like structure. This repeat is nonrandomly localized within the other tumor-associated deletions and in the vicinity of 3p12-22 CBRs.     

Y chromosome microdeletion screening in infertile men

Molecular analysis of Y-chromosomal microdeletions is routinely performed in the work-up of male infertility, in order to establish a diagnosis and for genetic counseling of the couple, since such microdeletions are transmitted to the male offspring. The review of published data shows that microdeletions are relatively common in patients with azoospermia or severe oligozoospermia, with wide variations in the reported deletion frequency depending mainly on the selection criteria. In general, patients with proximal deletions, involving the AZFa and/or the AZFb region show severe defects of spermatogenesis with a high prevalence of Sertoli cell only syndrome, while deletions of the distal AZFb and of the AZFc region can be compatible with residual spermatogenesis. Microdeletions have been only sporadically found in normozoospermic patients. For the time being the molecular analysis of microdeletions of the Y chromosome is indicated in infertile patients with sperm concentration <5 x 10(6)/ml and in men undergoing assisted reproduction techniques, since the genetic defect and, most probably, the related infertility problem will be transmitted to the sons.     

Familial absent pulmonary valve syndrome without deletions of chromosome 22q11

Deletions of chromosome 22q11 are common in patients with tetralogy of Fallot, and in those with absent pulmonary valve syndrome. In this report, we describe a pair of siblings with absent pulmonary valve syndrome, neither of whom had deletions of chromosome 22q11. The finding of familial absent pulmonary valve syndrome without deletion of 22q11 in our patients suggests an alternative genetic basis for this rare condition.     

衍生9号染色体缺失对慢性髓细胞白血病的预后价值

目的研究慢性髓细胞白血病（CML）中衍生9号染色体[der（9）]缺失的发生率及预后意义。方法随机选取48例CML-急变期（BC）患者,以BCR-ABL双色双融合荧光原位杂交（FISH）技术检测其染色体标本。对于FISH技术检测到的间期细胞中只有单个融合信号的标本,则观察其中期细胞,以明确是否为der（9）缺失。对于der（9）缺失患者,以相同方法检测其初诊时的染色体标本,以明确der（9）缺失是否和Ph易位同时发生。患者慢性期以羟基脲治疗。结果48例CML-BC患者中有8例（16．7％）der（9）缺失,其初诊时的染色体标本der（9）也缺失。der（9）缺失组慢性期及总生存期分别平均为20．9和36．4个月,明显短于未缺失组（55．1和70．3个月）（P〈0．05）。CML-BC急淋变与急非淋变患者中der（9）缺失概率无统计学意义（P〉0．05）。结论FISH技术可有效检测der（9）缺失。der（9）缺失与Ph易位同时发生。具有der（9）缺失的CML疾病进展较快,预后较差。羟基脲不具有逆转CML中der（9）缺失的不良预后的作用。der（9）缺失不导致cML向某一特定类型转化。