Screening of Y chromosome microdeletions in 46,XY partial gonadal dysgenesis and in patients with a 45,X/46,XY karyotype or its variants

Normal human sex development involves a series of events: sex determination, which is chromosomally established at fertilization (46,XY or 46,XX); differentiation of the gonads into testes or ovaries; and differentiation of internal and external genitalia, which will follow the male pathway in the presence of testicular hormones, or the female pathway in their absence. Factors adversely affecting any stage of these processes can lead to disorders of sex development (DSD), a group of congenital conditions in which the development of the chromosomal, gonadal or anatomical sex is atypical [1].

Partial and mixed gonadal dysgenesis are relatively rare causes of DSD. Individuals with partial gonadal dysgenesis (PGD) are characterized by genital ambiguity due to variable degrees of testicular dysgenesis, absence of syndromic picture and 46,XY karyotype. The histology of dysgenetic testes may vary from gonads with a few tubular structures and predominance of fibrous tissue to those with mild abnormalities, and they may be found bilaterally or associated with streak gonads [2]. The etiology of most cases of PGD is unknown; mutations in SRY (sex determining region Y) gene are rarely seen [3–5], and in recent years mutations in NR5A1 (Nuclear Receptor Subfamily 5, Group A, Member 1) gene were found in a few patients [6–8].

Mixed gonadal dysgenesis (MGD) is the main differential diagnosis for PGD. Individuals with MGD share similar gonadal and genital features, but the karyotype is 45,X/46,XY or its variants (a 45,X cell line and one or more lineages with a normal or structurally abnormal Y chromosome) [9]. The 45,X/46,XY karyotype may also be found in individuals with female phenotype and Turner syndrome (TS), genital ambiguity and ovotesticular DSD (OT DSD, also known as true hermaphroditism), and in male phenotype with infertility. Whatever the phenotype, this mosaic is associated with short stature, dysmorphisms and cardiovascular and renal anomalies, among other congenital and acquired conditions [10].

Structural rearrangements of the Y chromosome, including deletions, ring chromosomes and isochromosomes may lead to different phenotypes. Yp (short arm) deletions including SRY gene directly affect testis differentiation leading to streak gonads and a female phenotype, whereas deletions of the long arm, specially involving the Azoospermia Factor regions AZFa, AZFb and AZFc on Yq11 lead to male infertility [11].

Detection of Y chromosome deletions in routine analysis depends on the size of the missing segment, which may range from a whole chromosome arm, that may be visualized using regular cytogenetic techniques, to a single sequence tagged site (STS) that may only be detected by molecular techniques [12, 13]. In recent years, evidences of an association between Y microdeletions and 45,X/46,XY mosaicism have been reported in individuals with TS, MGD and infertility, and there are also indications that the instability caused by such deletions might be more significant in germ cells [12, 13].

The aim of this work was to investigate the presence of Y chromosome microdeletions in individuals with 45,X/46,XY mosaicism and its variants bearing different phenotypes and also in patients with PGD, in order to verify if the instability of Y chromosome might be one of the causes of such disorders of gonadal differentiation.

The Y chromosome evolved from an autosome, and its evolution has been characterized by massive gene decay, the human MSY retains only three percent of the ancestral autosome genes [14, 15]. Human Y chromosome has a total length of approximately 60 MB and is composed of two pseudoautosomal regions (PAR1 and PAR2), the euchromatic region, which is rich in testis-specific genes, and the heterochromatic region, containing highly repetitive satellite DNA. The euchromatic region and some of the heterochromatic parts of the Y chromosome are called MSY (male-specific region on the Y chromosome), which comprises 95% of the chromosome’s length [16, 17]. MSY contains 496 genes, including 51 coding genes, 326 pseudogenes, and 119 non-coding genes (Ensembl Genome Browser version 69, accessed January 2013) [18].

The present study did not detect Y chromosome microdeletions in the 13 subjects with PGD and normal 46,XY karyotype, therefore the hypothesis that abnormalities at the molecular level leading to loss of the Y chromosome in gonadal cell lines play a role in the etiology of this disorder was not supported. To the best of our knowledge, this is the first study to investigate the association between Y chromosome abnormalities and PGD. However, the hypothesis that mosaicism restricted to testis could be one of the causes of this condition could not be entirely discarded, because the loss of the Y chromosome giving rise to a 45,X cell line can occur in the absence of structural abnormalities of the Y chromosome. Furthermore, microdeletions found on gonadal DNA can be absent on peripheral blood lymphocyte DNA [19].

On the other hand, microdeletions were found in six of 15 patients with a 45,X/46,XY karyotype or its variants, all of them with MGD phenotype. Proximal breakpoints inside the AZF region were defined in four of the six carriers of structural Y chromosome abnormalities which had already been detected in routine karyotype. The molecular study of Y-STS not only allowed to refine the breakpoints, but also made it possible to detect the loss of euchromatic regions of this chromosome.

In addition, in two of nine individuals with an apparently normal Y chromosome (cases 8 and 11) the abnormalities were detected only by the molecular techniques. This frequency (2/9) was similar to that observed by Alvarez-Nava et al. (2008) (3/11) [19] and lower than that found by Patsalis et al. (2005) (4/7) [12]. Taken together, these findings suggest that 9/27 or one in three individuals with 45,X/46,XY and apparently normal Y chromosome may have Y microdeletions.

All alterations identified in the six individuals were located on the Y chromosome long arm at the beginning of Yq11.221 region, where the AZFb region is located, and extending to AZFc. Only in case 8 the deletion was practically limited to the AZFc region. In the four cases in which an abnormal Y chromosome had already been detected, Y-STS testing were not useful to define distal breakpoints, which might be located between the q11-q12 boundary and terminal Yq. In case 11 the analysis of Y-STS showed that the lost fragment was not confined to the heterochromatic region.

The same fragment in the euchromatic region was lost in cases 6 and 15; the deletion starts at STS sY127, located in Yq11.222, and includes 11 STS. In the karyotype of case 6, only a Yq12 deletion was detectable, while in case 15 there were four cell lines containing markers with different Yq sizes. In case 8, two STS were missing (sY157 and sY3168), indicating a breakpoint within Yq11.223. Upon karyotyping, there was an apparently normal Y chromosome. The deletion in case 10 started at sY149 (Yq11.223), with absence of seven STS.

Deletions found in cases 11 and 13 led to loss of 13 and 17 STS, respectively. In patient 11, whose karyotype was 45,X/46,XY, the missing fragment started in sY117 (Yq11.221) and included all subsequent STS. In case 13 the deletion started at STS sY105, also located in Yq11.221; the karyotype had already detected a structural chromosome abnormality with loss of the heterochromatic region Yq12.

In cases 10, 13 and 15, in which there were Y-derived markers, STS study detected the presence of a terminal deletion of the euchromatic region; deletion in case 10 was the smallest, encompassing AZFc and the overlapping region of AZFb and AZFc.

No deletions (including those of Yp) were found in the two subjects with isodicentric Y chromosomes (cases 5 and 14); these chromosomes are often unstable during cell division, resulting in mosaicism with a 45,X line [20]. As the STS analyzed in this study included all the euchromatic region of Yp from the centromere to p11.31, the break in the short arm that originated these isodicentrics may have occurred in the PAR1 region. It has been suggested that a hot spot located in the PAR1 is prone to chromosomal breakage and reunion with generation of isodicentric Y chromosomes [20].

Yq microdeletions with loss of genes specifically expressed in the testis directly contribute to male infertility. AZFc is most often involved, followed by AZFb, and more rarely AZFa [21–23]. In addition, deletions involving the entire AZFb region or AZFb-c remove a large stretch of Yq chromatin, which may result in more severe disturbance of X-Y chromosome pairing during meiosis than isolated AZFc deletion leading to meiotic breakdown [24, 25]. All microdeletions found in our patients with mosaicism were located in AZFb and AZFc regions; their effect on Y chromosome instability is not mediated by the loss of function of genes related to spermatogenesis, but one could speculate that the loss of these large stretch of Yq could also be more dangerous in mitosis than AZFc deletions alone in increasing the risk of mosaicism due to loss of the abnormal Y chromosome.

The mechanism of Y chromosome deletions is accidental homologous recombination between highly similar or identical sequences, which are found in great abundance on this chromosome [11]. Three of our patients (cases 8, 10 and 11) had a breakpoint within regions consisting of palindromes [26, 27], which may have contributed to the emergence of these deletions [26, 27].

Most genes involved in Y chromosome deletions are expressed specifically in the testes during spermatogenesis, but do not appear to be essential for fertilization or embryogenesis; thus, these deletions does not seem to adversely affect the fertilization results in men whose sperm was obtained by testicular sperm extraction (TESE). Thus, subjects with MGD may also benefit from procedures such as TESE followed by in vitro fertilization with intracytoplasmic sperm injection (ICSI) [11].

Thereby, patients with a 45,X/46,XY karyotype or its variants reared as males who have Y microdeletions may, by means of assisted reproductive technologies, generate not only male offspring with sterility, but also individuals who are carriers of an unstable Y chromosome that may originate mosaicism with a 45,X cell line in the early stages of embryonic development leading to various anomalous phenotypes (TS, MGD and OT DSD) [13]. This is the case of the three patients with microdeletions from this sample reared as males, two with an apparently normal Y chromosome (cases 8 and 11) and one with a deletion apparently limited to Yq12 (case 6). In these three cases, as well as in case 13, in whom the structural alteration of the Y was more evident on karyotype, the presence of these abnormalities in euchromatic region should be taken into account in counseling regarding assisted reproduction.

Absence of deletions in individuals with PGD suggests that the Y chromosome is intact, indicating that it is not likely that its instability in the gonads is one of the causes of this condition; further studies that aim to identify the etiology of this condition are needed. However, deletions identified in the second group indicate that mosaicism may be associated with Y chromosome abnormalities detectable only at the molecular level. If patients with mosaicism and Y microdeletions reared as males decide to undergo in vitro fertilization, Y chromosomes which tend to be unstable during cell division may be transmitted to offspring.