Sex chromosome mosaicism in males carrying Y chromosome long arm deletions

Microdeletions of the long arm of the Y chromosome (Yq) are a common cause of male infertility. Since large structural rearrangements of the Y chromosome are commonly associated with a 45,XO/46,XY chromosomal mosaicism, we studied whether submicroscopic Yq deletions could also be associated with the development of 45,XO cell lines. We studied blood samples from 14 infertile men carrying a Yq microdeletion as revealed by polymerase chain reaction (PCR). Patients were divided into two groups: group 1 (n = 6), in which karyotype analysis demonstrated a 45,X/46,XY mosaicism, and group 2 (n = 8) with apparently a normal 46,XY karyotype. 45,XO cells were identified by fluorescence in-situ hybridization (FISH) using X and Y centromeric probes. Lymphocytes from 11 fertile men were studied as controls. In addition, sperm cells were studied in three oligozoospermic patients in group 2. Our results showed that large and submicroscopic Yq deletions were associated with significantly increased percentages of 45,XO cells in lymphocytes and of sperm cells nullisomic for gonosomes, especially for the Y chromosome. Moreover, two isodicentric Y chromosomes, classified as normal by cytogenetic methods, were detected. Therefore, Yq microdeletions may be associated with Y chromosomal instability leading to the formation of 45,XO cell lines.     

Large Y chromosome (Yq+) and increased risk of abortion

Previous reports have indicated an association between Yq+ in the family and increased risk of abortion. In a chromosome investigations of 11,148 consecutively newborn children, we found 58 boys with a large Y chromosome (Y/F equal to or greater than 1.00). The mothers of these boys had 22% abortions, compared with 13% among 4,895 mothers of children with normal karyotypes and without Yq+ (P less than 0.001).     

COMMENTS

Human spermatogenesis is regulated by a network of genes located on autosomes and on sex chromosomes, but especially on the Y chromosome. Most results concerning the germ cell function of the Y genes were obtained by genomic breakpoint mapping studies of the Y chromosome of infertile patients. Although this approach has the benefit of focussing on those Y regions that contain most likely the Y genes of functional importance, its major drawback is the fact that fertile control samples were often missing. In fertile men, molecular and cytogenetic analyses of the Y chromosome has revealed highly polymorphic chromatin domains especially in the distal euchromatic part (Yq11.23) and in the heterochromatic part (Yq12) of the long arm. In sterile patients cytogenetic analyses mapped microscopically visible Y deletions and rearrangements in the same polymorphic Y regions. The presence of a Y chromosomal spermatogenesis locus was postulated to be located in Yq11.23 and designated as AZoospermia Factor (ZF). More recently, molecular deletion mapping in Yq11 has revealed a series of microdeletions that could be mapped to one of three different AZF loci: AZFa in proximal Yq11 (Yq11.21), AZFb and AZFc in two non‐overlapping Y‐regions in distal Yq11 (Yq11.23). This view was supported by the observation that AZFa and AZFb microdeletions were associated with a specific pathology in the patients' testis tissue. Only AZFc deletions were associated with a variable testicular pathology and in rare cases AZFc deletions were even found inherited from father to son. However, AZFc deletions were found with a frequency of 10–20% only in infertile men and most of them were proved to be “de novo”, i.e. the AZFc deletion was restricted to the patient's Y chromosome. Based mainly on positional cloning experiments of testis cDNA clones and on the Y chromosomal sequence now published in GenBank, a first blueprint for the putative gene content of the AZFc locus can now be given and the gene location compared to the polymorphic DNA domains. This artwork of repetitive sequence blocks called AZFc amplicons raised the question whether the AZFc chromatin is still part of the heterochromatic domain of the Y long arm well known for its polymorphic extensions or is decondensed and part of the Yq11.23 euchromatin? We discuss also the polymorphic DAZ gene family and disclose putative origins of its molecular heterogeneity in fertile and infertile men recently identified by the analyses of Single Nucleotide Variants (SNVs) in this AZFc gene locus.     

A molecular and FISH analysis of structurally abnormal Y chromosomes in patients with Turner syndrome

Fourteen patients with Turner syndrome and a structurally abnormal Y chromosome were analysed by PCR amplification and fluorescence in situ hybridisation for the presence of sequences specific to defined regions of the Y chromosome. Thirteen patients had a mosaic karyotype including a 45,X cell line and one case was non-mosaic in cultured lymphocytes. Ten patients had a pseudodicentric Yp chromosome, two an isodicentric Yq, one a pseudodicentric Yq, and one a derived Y chromosome. Two of the patients with a psu dic(Yp) chromosome had complex karyotypes with more than two cell lines, one of which exhibited five morphologically distinct mar(Y) chromosomes, presumably derived from a progenitor psu dic(Yp). Nine of the ten psu dic(Yp) chromosomes were positive for all Yp and Yq probes used except DYZ1 which maps to Yq12, suggesting a common breakpoint near the Yq euchromatin/heterochromatin boundary. In the three patients with a dicentric Yq chromosome two different breakpoints were observed; in two it was between PABY and the subtelomeric repeat sequence and in one it was between DYZ5 and AMGY in proximal Yp. Our results suggest that the great majority of structurally abnormal Y chromosomes found in Turner syndrome mosaics contain two copies of virtually all of the functional Y chromosome euchromatin.     

Polymorphic DAZ gene family in polymorphic structure of AZFc locus: Artwork or functional for human spermatogenesis?

Human spermatogenesis is regulated by a network of genes located on autosomes and on sex chromosomes, but especially on the Y chromosome. Most results concerning the germ cell function of the Y genes were obtained by genomic breakpoint mapping studies of the Y chromosome of infertile patients. Although this approach has the benefit of focussing on those Y regions that contain most likely the Y genes of functional importance, its major drawback is the fact that fertile control samples were often missing. In fertile men, molecular and cytogenetic analyses of the Y chromosome has revealed highly polymorphic chromatin domains especially in the distal euchromatic part (Yq11.23) and in the heterochromatic part (Yq12) of the long arm. In sterile patients cytogenetic analyses mapped microscopically visible Y deletions and rearrangements in the same polymorphic Y regions. The presence of a Y chromosomal spermatogenesis locus was postulated to be located in Yq11.23 and designated as AZoospermia Factor (ZF). More recently, molecular deletion mapping in Yq11 has revealed a series of microdeletions that could be mapped to one of three different AZF loci: AZFa in proximal Yq11 (Yq11.21), AZFb and AZFc in two non-overlapping Y-regions in distal Yq11 (Yq11.23). This view was supported by the observation that AZFa and AZFb microdeletions were associated with a specific pathology in the patients' testis tissue. Only AZFc deletions were associated with a variable testicular pathology and in rare cases AZFc deletions were even found inherited from father to son. However, AZFc deletions were found with a frequency of 10-20% only in infertile men and most of them were proved to be "de novo", i.e. the AZFc deletion was restricted to the patient's Y chromosome. Based mainly on positional cloning experiments of testis cDNA clones and on the Y chromosomal sequence now published in GenBank, a first blueprint for the putative gene content of the AZFc locus can now be given and the gene location compared to the polymorphic DNA domains. This artwork of repetitive sequence blocks called AZFc amplicons raised the question whether the AZFc chromatin is still part of the heterochromatic domain of the Y long arm well known for its polymorphic extensions or is decondensed and part of the Yq11.23 euchromatin? We discuss also the polymorphic DAZ gene family and disclose putative origins of its molecular heterogeneity in fertile and infertile men recently identified by the analyses of Single Nucleotide Variants (SNVs) in this AZFc gene locus.     

Analysis of Yq microdeletions in infertile males by PCR and DNA hybridization techniques

Defects in spermatogenesis have been found associated with deletions of different portions of Y chromosome long arm (Yq), suggesting the presence of the azoospermia factor in the control of spermatogenesis. We studied 67 men with idiopathic azoospermia and severe oligozoospermia, cytogenetically normal, for the presence of microdeletions on Yq chromosome. By using polymerase chain reaction (PCR) and Southern blotting techniques we analysed the AZFa, AZFb and AZFc loci on Yq, where deletions have been associated with defects in spermatogenesis. Deletions of a portion of the Y chromosome were detected in five patients. Four of these patients shared deletions in distal Yq11 interval 6, including the DAZ gene, while one patient lacked loci in the proximal Yq11. Testicular histology of two patients bearing distal Yq11 deletions showed two different spermatogenic defects including Sertoli cell-only (SCO) syndrome and maturation arrest, while the patient with microdeletions in the proximal Yq11 showed a SCO phenotype.      

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.     

The impact of large Y chromosome on pregnancy, foetus and birth

In a current investigation of children born in the Arhus area (Denmark) chromosome examinations were made in 6,691 newborns. Of these children, 170 boys had a large Y chromosome (2.6%). The present material was examined using a bi-variate stratified analysis to eliminate social and simple biological factors that could act as confounders. No increased frequency of malformations was found, and birth weight and length was nearly equal in the probands and the controls. A significantly increased frequency of prostaglandin stimulation of labour was found for the mothers of the Yq+ boys. Differences in the frequency of mechanical disproportion or abnormal presentation could not explain this. The Yq+ boys suffered more frequently from intrauterine asphyxia leading to acute Caesarean section. This finding cannot be explained by long-standing placenta problems alone. A possible mechanism which could link these findings together is suggested, and it is concluded that the boys with Yq+ most probably should be regarded as being at a certain risk at the time of birth.     

Molecular characterization of an unusual variant of the short arm of chromosome 15 by FISH-technique

Summary One of the most frequent translocations involving the long arm of chromosome Y with autosomes is with the short arm of chromosome 15. The regions which are involved in this translocation fluoresce brightly, are highly heteromorphic and thus escape detection. Therefore, these abnormalities could not be fully characterized, especially in cases where parents are not available or paternity is disputed. Results from the employment of the selective staining techniques DA/DAPI and Q-banding have been inconclusive. FISH-technique using whole chromosome painting (WCP) probes should be used to decipher such translocations. We present a case where, even after using a battery of probes, the origin of extra material on chromosome 15p could not be identified though it was not a part of Yq.     

Lectin staining of carbohydrates of haemic cells. III. The cells of Hodgkin''s disease and other lymphomas

When stained for reactive sialyl groups with fluorescein-labelled Aprotinin (FLA), lymphocytes of three diffuse lymphomas were uniformly faintly fluorescent. The nodules of a nodular lymphocytic lymphoma showed dimly fluorescing lymphocytes surrounded by brightly fluorescing, apparently normal cells. The spleens of eight patients with Hodgkin's disease showed involvement in six cases. With FLA, the two uninvolved spleens contained only brightly fluorescing lymphocytes, whereas the foci of Hodgkin's lesions in the six spleens and in eight involved lymph nodes from a further eight patients contained varying proportions of brightly and dimly fluorescing lymphoid cells. Mononuclear Hodgkin's cells and bi- or multinucleated Reed-Sternberg cells fluoresced faintly. Fluorescein-labelled Ricinus communis agglutinin (FL-RCA) for galactose, and Concanavalin A (FL-Con A) for mannose or glucose, showed eosinophils, reticulin and collagen fibres especially in nodular sclerosing Hodgkin's disease, whereas all lymphocytes, Hodgkin and Reed-Sternberg cells stained faintly with either lectin. The reduction of reactive sialyl groups in malignant lymphocytes of lymphomas and Hodgkin's lesions is similar to that in lymphocytic leukaemias. It is suggested that in Hodgkin's disease these lymphocytes together with the Hodgkin and Reed-Sternberg cells represent the malignant component, whereas the brightly fluorescent normal lymphocytes, together with histiocytes, eosinophils (and neutrophils) represent a reactive component in the lesions. Similarly, the reactive lymphocytes in sarcoid lesions and sinus histiocytosis were brightly fluorescing.     

Intrachromosomal insertion translocation resulting in duplication of chromosome band Yq11.2 in two fertile brothers

Chromosome analysis in a couple referred because of two spontaneous abortions showed a normal 46,XX karyotype in the 28-year-old female and an aberrant Y chromosome with an enlarged short arm in the 30-year-old male. Subsequent chromosome analysis showed that his 33-year-old brother was carrier of the same Y chromosome aberration. Further characterization of the aberrant Y chromosome with FISH using probes specific for chromosome bands Yp11.32, Yq11.2, the centromere and the subtelomeric region of the p-arm of the Y chromosome showed that chromosome band Yq11.2 was duplicated and inserted in the p-arm of the Y chromosome. Combining the results of the analysis of GTG-banded chromosomes and of the FISH analysis we conclude that both patients have a 46,X,ins dup(Y)(pter --> p11.23::q12 --> q11.1::p11.23 -->) karyotype. The clinical and cytogenetical findings are reported and discussed.     

Polymorphic variants in human chromosome 15

We found eight polymorphic variants in human chromosome 15 using Q, C, Q-C and Ag-NOR staining methods. These variants included brightly or dully fluorescent pericentric segments and satellites, giant satellites, increased amounts of short arm heterochromatin (ph+) and darkly (C band-positive) or lightly (C band-negative) Giemsa-stained pericentric Q-negative segments. These staining properties indicated that the entire short arm of 15 contained at least four distinct chromatin segments: Q-negative centrometric heterochromatin, a Q-vriable distal segment, a Q-negative satellite stalk, and Q-variable satellites, in that order, from proximal to distal ends. The Brd U-Hoechst 33258-stained R bands (RBH) and high resolution G subbands were also studied for karyologic characterization of chromosome 15. Most of these variants were reported also in 13, but insufficiently documented in other D and G chromosomes. Together with polymorphic pericentric fluorochromes seen in 3 and 4, Yq, and nonpathogenic t(D;Yq), the pattern of these variants can be used as karyologic fingerprints for identification of each individual and his or her cell explants both in vivo and in vitro.     

Y染色体末端缺失患者的细胞遗传学及分子生物学分析

目的确定1例少弱精子患者G显带和C发现Yq末端缺失病例的核型,探讨YYq12缺失与表型关系。方法应用实验室常规染色体标本制备方法进行G显带和C显带,并应用Yq12区DYZ1探针和Yp11.1-q11.1区DYZ3探针与病例的中期分裂相进行荧光原位杂交(fluorescence in situ hybridization,FISH),同时应用PCR技术对患者进行了Y染色体微缺失的检测。结果G显带、C显带和FISH检测结果一致,均显示为Yq12区的缺失;Yq11区生精基因微缺失检测未发现该患者存在缺失。结论FISH结合细胞遗传学检测可以明确诊断染色体微小结构异常,Yq12区缺失可能是导致男性不育的原因之一。     

Deleted Yq in the sterile son of a man with a satellited Y chromosome (Yqs).

Disturbed spermatogenesis and azoospermia are reported in a man with a deleted Y chromosome. The anomalous Y chromosome appears in the karyotype as a small metacentric marker. In situ hybridisation using three different Y specific DNA probes shows that deletion at Yq11 has resulted in loss of all distal heterochromatin. The sterility of the patient indicates loss also of the azoospermia factor (AZF) located at the Yq distal euchromatic/heterochromatic interface. Microspread and air dried meiotic preparations show a severe impairment of spermatogenesis but rare cells are seen to be progressing to the late prophase stage. The testicular histology shows most of the seminiferous tubules to be completely hyalinised. The father and a fertile brother of the proband show a satellited Y chromosome (Yqs) in their karyotypes. The case appears to be the first of its kind reported in which a father with a satellited Y chromosome has produced a son carrying a different Y chromosome anomaly. The possible derivation of the one from the other is discussed.     

Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion

Submicroscopic deletions of the Y chromosome and polymorphisms of the androgen receptor (AR) gene in the X chromosome have been observed in men with defective spermatogenesis. To further define the subregions/genes in the Y chromosome causing male infertility and its relationship to polymorphisms of the AR polyglutamine tract, we screened the genomic DNA of 202 subfertile males and 101 healthy fertile controls of predominantly Chinese ethnic origin. Y microdeletions were examined with 16 sequence-tagged site (STS) probes, including the RBM and DAZ genes, spanning the AZFb and AZFc subregions of Yq11, and related to the size of trinucleotide repeat encoding the AR polyglutamine tract. Y microdeletions were detected and confirmed in three out of 44 (6.8%) of azoospermic and three out of 86 (3.5%) severely oligozoospermic patients. No deletions were detected in any of the patients with sperm counts of >0.5 x 10(6)/ml, nor in any of the 101 fertile controls. All six affected patients had almost contiguous Y microdeletions spanning the entire AZFc region including the DAZ gene. The AZFb region, containing the RBM1 gene, was intact in five of the six subjects. Y deletions were not found in those with long AR polyglutamine tracts. Our study, the first in a Chinese population, suggest a cause and effect relationship between Y microdeletions in the AZFc region (possibly DAZ), and azoospermia or near-azoospermia. Y microdeletions and long AR polyglutamine tracts appear to be independent contributors to male infertility.      

Microdeletions of a Y-specific marker,Yfm1, and implications for a role in spermatogenesis

We have detected deletions of a Y-specific microsatellite marker, Yfm1, located on the Y chromosome (Yq) within interval 6 and near the DAZ (deleted in azoospermia) genes, in 9/89 oligospermic and 17/68 azoospermic Japanese men. No Yfm1 deletions were detected in the 150 normal fertile males examined as controls. Yfm1 deletions in the oligo- and azoospermic males were associated with other deletions that removed entire DAZ genes in those infertile men. These deletions indicated that all Yfm1 loci are located within azoospermia factor c (AZFc) in interval 6 on the long arm of the Y chromosome. Mapping Yfm1 on the Y chromosome using the draft sequence of the human genome revealed that at least three Yfm1 loci are located within about 25–30 kbp of the DAZ genes. Moreover, the Yfm1 marker showed the least number of copies in Japanese males derived from a Y chromosomal lineage called haplotype II, defined by having the Y Alu polymorphism (YAP) insertion. Males from this haplotype II lineage are known from our previous studies to have lower spermatogenic abilities, with higher rates of oligo- and azoospermia than other haplotypes. The least number of Yfm1 loci, whose copy number may correspond to that of the DAZ genes, may be a risk factor predisposing an individual to azoospermia or oligospermia. Received: October 26, 2001 / Accepted: February 12, 2002     

Dicentric chromosome Y associated with Leydig cell agenesis and sex reversal

The nature of a non-mosaic marker Y chromosome observed in a pseudohermaphrodite patient with Leydig cell agenesis was investigated by high-resolution chromosome analysis and molecular probes from the Y chromosome. Cytogenetically, the marker chromosome appeared to be an isodicentric, with breakage in Yq11.21. Double copies of all Yp-specific loci tested, including SRY, were present. The most distal Yq portion detected in patient DNA was DXS278-C, which maps to interval D in the chromosome Yq deletion map. Fragment DXS278-B, which maps to deletion interval E, was absent. The possible relationship between this cytogenetic abnormality and Leydig cell agenesis, a finding never reported in association with Y chromosome rearrangements, is discussed.     

Different phenotypes in two cases of an apparently identical familial (Yq;13p) translocation.

A chromosome 13 with extra material on the short arm was observed in a 17-year-old boy showing defects in skeletal growth, an altered hormone profile and asthenoteratozoospermia, and in a 46 XX fetus subjected to prenatal diagnosis. The abnormal chromosome 13 had been transmitted from phenotypically normal parents who were the mother (case 1) and the father (case 2). The extra material on the abnormal chromosome 13 was brightly fluorescent after Q-banding, and positive in C-banding (CBG) and distamycin A-Dapi (DA-Dapi) banding. Staining of the nucleolus organizer region indicated its retention. In-situ hybridization of a Yq-specific repetitive DNA probe to chromosomal spreads from both cases demonstrated that the der(13) chromosome contains sequences of the Yq heterochromatic region. However, the apparently identical unbalanced (Y;13) translocation may either interfere (case 1) or not (father of case 2) with meiotic or postmeiotic sperm cell development.     

FISH characterization of a dicentric Yq (p11.32) isochromosome in an azoospermic male

The most common structural rearrangements of the Y chromosome result in the production of dicentrics. In this work, we analyze an abnormal Y chromosome, detected as a mosaic in an azoospermic male ascertained for infertility. FISH with seven different DNA probes specific for Y chromosome sequences (Y alpha-satellite, Y alpha-satellite III, non-alpha-satellite centromeric Y, SRY gene, subtelomeric Yp, subtelomeric Yq, and PNA-tel) and CGH analysis were performed. FISH results showed that the abnormal Y chromosome was a dicentric Yq isochromosome and that the breakpoint was distally in band Yp11.32. Lymphocyte chromosomes showed a mosaicism with 46,X,idicY(qter-->p11.32::p11.32-->qter) (51.7%), 46,XY (45.6%), and other cell lines (2.7%). In oral interphase cells, the mosaicism was 46,XidicY (62.8%), 46,XY (25.7%), 45,X (6.6%), and others (4.9%). The possible origin of this dicentric Yq isochromosome is discussed. Finally, we compare differences in mosaicism and phenotype among three reported cases with the breakpoint at Yp11.32 Copyright 2004 Wiley-Liss, Inc.     

Unique t(Y;1)(q12;q12) reciprocal translocation with loss of the heterochromatic region of chromosome 1 in a male with azoospermia due to meiotic arrest: a case report

A de novo reciprocal translocation 46,X,t(Y;1)(q12;q12) was found in an azoospermic male with meiotic arrest. Cytogenetics and fluorescent in situ hybridization (FISH) were used to define the karyotype, translocation breakpoints and homologue pairing. SRY (Yp), Yq11.2-AZF regions, DAZ gene copies and the distal Yq12 heterochromatin were studied by PCR and restriction analysis using sequence-tagged sites and single nucleotide variants. High resolution GTL, CBL and DA-DAPI staining revealed a (Y;1) translocation in all metaphases and a normal karyotype in the patient's father. FISH showed the presence of the distal Yq12 heterochromatic region in der(1) and loss of the heterochromatic region of chromosome 1. PCR demonstrated the intactness of the Y chromosome, including the SRY locus, AZF regions, DAZ genes and distal heterochromatin. A significant decrease (P = 0.005) of Xp/Yp pairing (18.6%), as compared with controls (65.7%), was found in arrested primary spermatocytes, and cell culture and mRNA expression studies confirmed an irreversible arrest at meiosis I, with induction of apoptosis and removal of germ cells by Sertoli cells. We characterized a de novo t(Y;1)(q12;q12) balanced reciprocal translocation with loss of the heterochromatic region of chromosome 1, that caused unpairing of sex chromosomes followed by meiosis I arrest, apoptotic degeneration of germ cells and azoospermia.