Especificaciones de los casos de uso

Several clinical and m olecular studies have dem onstrated a

connection between male infertility and Y chrom osom e deletions and/or m icrodeletions. Such studies have tried to define the position and extent of these deletions and the frequency w ith which they occur.

In order to determ ine w hether deletions o f the m ll3 d lO and m l22a3 TTY 2-like sequences occur in infertile patients, D N A from a panel o f m ale individuals (n=52) experiencing fertility problem s was analysed by PCR (provided by C. Quilter at the Cytogenetics Unit, UGH hospital). These patients were all selected for idiopathic severe oligozoosperm ia (<5x10^ sperm /m l of semen) or azoosperm ia (com plete absence of sperm in semen) and showed no evidence of obstructive azoosperm ia, endocrine deficiencies or any know n cytogenetic defects. In addition another set of 30 DNA

samples from oligo- and azoosperm ie patients w ere provided by Prof. K leim an at the M aternity Hospital, Tel Aviv. These patients had been

previously analysed by Prof. Kleim an for several STS markers on both arms o f the Y chrom osom e (Kleiman et al., 1999).

The quality of the DNA was checked by am plification with m l22d8 prim ers (22d8.F and 22d8.R), which amplify both Chrs Y and 15 (see section 5.2). One of the DNA samples (no. 48) did not am plify with either m ll3 d lO or ml22a3 specific primers or ml22d8 primers and was excluded from further analysis. As a positive control and in order to ensure that the sequence o f the prim er site was not polym orphic in the general population, 40 unrelated CEPH fathers were included in this study. As a negative control for each experim ent, PCRs were set up in the absence of DNA.

PCR amplification using 13dl0.2F and 13dl0.2R prim ers gave products of the correct size (233bp) for all but 2 DNA samples from U CH no.7 and no. 18 (Fig 4.34). PCR products were obtained for these two samples using prim ers for ml22a3 and the ml22d8 control.

The results for patients no.7 and no. 18, could be explained if deletion(s) were present, which eliminate part of the ml 13d 10 TTY 2-like gene. The location of the deletions must include the region corresponding to either/or both m ll3 d lO specific primers. Until further analysis of the region is carried out, it is difficult to be certain that this interpretation is correct. It m ight have been possible to design other pairs o f primers from the ml 13d 10 sequence, but it would be necessary to ensure that these prim ers do not am plify other TTY 2-like gene sequences.

A m plification of ml22a3 in the patient panel showed a correct sized product in all the UCH samples. However, am ongst the samples from Tel- Aviv, these primers failed to amplify DNA from two patients (n o .l and no.2) (Fig 4.35). In contrast, PCR products of correct size were obtained for both, using prim ers specific for ml 13d 10 and for the ml22dS. Kleim an et al

(1999), have reported that analysis of STS markers and expressed sequences in the DNA of patient n o .l (Kleiman no.95, idiopathic azoosperm ia),

indicates that he lacks the AZFc region only, whereas patient 2 (Kleim an no. 102, Sertoli cell-only syndrome) lacks the whole of AZFa-c region. The results presented here, from the PCR am plification of the Tel Aviv patients n o .l and 2 DNA are in agreem ent with the published data.

In summary, results from screening the panel o f 82 oligo- and azoosperm ie patients for the presence of two of the TTY 2-like genes, ml 13d 10 and ml22a3, suggested that deletions of both genes m ay occur at low frequency and m ight contribute to the infertile phenotype. H owever, the infertile phenotype might be the result of deletions or m utations of other genes, which are present in the region where ml 13d 10 and ml22a3 have been localised. PCR screening o f these patients with either STS prim ers around the TTY 2-like location, or for the presence o f m utations in other surrounding genes, would clarify whether TTY2 genes have an im pact in m ale infertility.

M l 2 3 4 5 6 7 8 9 10 11 12 13 14 hi M 500bp- 233hp- SOOhp- 178hp- SOObp- 264bp- mI13dlO mI22a3

i I

Figure 4.34 Screening of D N A from a panel o f oligo- and azoospermie patients with primers specific for m il 3d 10 and ml22a3. Results for UC H patients 1 to

14 are shown in Fig. A and for UC H patients 15 to 28 in Fig. B. Amplification of the same panel of DN A with primers for ml22dS (Chrs Y and 15) is also shown as a control; M: DNA size marker; b l-2 : no DNA

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M 500bp- 233bp- m ll3dl0 5(K)bp- 178bp- M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M ml22a3 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 50(H)p- 264bp- mI22d8

F ig u re 4.35 Screening of DNA from a panel of oligo and azoosperm ie patients (Tel Aviv), with prim ers specific for ml 13d 10 and mI22a3. A m plification of the same panel of DN A with prim ers for m l22d8 (Chrs Y and 15) is also shown as a control; M: DNA size m arker; b l-2 : no DNA

4.10 DISCUSSION

In this section, the TTY 2-like genes m ll3 d lO and ml22a3 and the published TTY2 gene will be discussed further, particularly in the context o f how Y chrom osom e evolutionary process m ay im pact on the characteristics of Y -linked genes like the TTY 2 gene family.

4 .I0 .I TTY2 gene structure

The ancestral TTY2 gene M apping studies have shown that

m em bers of the TTY2 gene fam ily appear to be Y -specific with no strongly hom ologous sequences elsew here in the hum an and prim ate genome.

However, Southern blot analysis at low stringency (section 4.5) has

dem onstrated that some bands are com m on betw een m ale and fem ale hum an DNA, which suggests that there are TTY2-like sequence(s) elsew here in the genome.

Perhaps this non-Y sequence was the ancestral version, from which via a duplication and transposition event, the first Y-linked TTY 2 copy emerged. Failure of FISH analysis to detect an autosomal or X -linked sequence (section 4.5) suggests that the hom ology betw een the Y-linked copy and its prototype is low and implies that the transfer of a TTY2 progenitor onto the Y

chrom osom e happened early during m am m alian evolution.

The Y-linked RBM Y gene seems to have arisen in a sim ilar way. It seems m ost likely that a single copy of this large genomic fam ily was derived from an autosom al hnRN PG -like ancestor and copied on the X and Y more than 130 m illion years ago, before the radiation of mam m als. This gene accum ulated sequence changes during evolution to evolve from an X-Y housekeeping gene, into a m ale specific gene fam ily that shares very low

hom ology with its X counterpart (Delbridge et al., 1997; 1999, Chai et al.,

1998X

Presence o f m ultiple TTY2 copies The identification o f 34 distinct Y -linked TTY2-cosm ids and their sequence analysis m ade it clear that TTY 2 sequences are present in many copies on the Y chrom osom e. A partial sequence analysis revealed that there is a m inim um of 26 TTY 2-like genes, arranged in 16 subfamilies. W ithin each subfamily, the level of hom ology betw een different copies is high (93-99%), whereas betw een subfam ilies the level of hom ology drops significantly (55-87%). This indicates that during the evolution of the TTY2 gene family, duplications gave rise to several TTY2 copies that each in turn form ed a subfamily. Gene duplications often occur as a result o f unequal crossover events and very frequently create a tandem ly arranged gene cluster (Smith., 1976).

The gene m ultiplicity showed by TTY2, is a com m on characteristic of m ale specific, Y-linked genes that do not appear to have any close

hom ologues on the X chromosome. Other exam ples are the RBM Y, DAZ and TSPY gene families. This characteristic is associated with the Y

chrom osom e’s haploid nature and the evolutionary pathways that led to the form ation of the sex chromosomes.

One school of thought is that in the absence of extensive recom bination on the Chr Y, gene duplication may serve as a m echanism to produce a reservoir o f active copies of the same gene (Nowak et al., 1997, Vogel and Schm idtke., 1998). The resulting gene redundancy w ould be m aintained by selective pressure as such a reserve that would avoid the com plete loss of gene activity due to accum ulation of mutations. W ithout selection, it m ight be assum ed that genetic drift would eliminate gene copies by m utations, leading to loss of function.

The loss of recom bination between the Y and X chrom osom es, is thought to have arisen because early in its evolution, the proto-Y acquired gene(s) that favored male sex determination, whereas absence of this gene(s) led to fem ale differentiation. In stages, alleles of other genes responsible for prim ary or secondary differentiation of the two sexes evolved and becam e located near the sex-determ ining region of the proto-Y (M axson; 1990).

Progressively, a m echanism o f selection and protection o f these genes resulted in suppression of genetic exchange between the sex chrom osom es. This was accom panied by divergence o f the proto-X and -Y sequences to include a non­ recom bining region. Lack o f extensive recom bination, resulted in the

appearance of internal rearrangem ents on the proto-Y, such as gene

duplications, followed by internal structural changes and m utations that were tolerated because of the pressure to maintain sequence hom ology needed for recom bination (M itchel et at, 1998).

The best characterised genes that, like TTY2, are located in the non­ recom bining portion of the Y chrom osom e and exist in multiple copies, are DAZ, TSPY and RBMY. Fam ily members include both functional copies (although exact numbers are not clear) and pseudogenes (Saxena et a t , 1996, Chai et a t , 1997, M anz et a t , 1993). Lahn and Page (1997) added a further 7 genes to this list. These are CDY, B P Y l, BPY2, XKRY, PRY, T T Y l and the TTY2, all of which are expressed in testis.

A rrangem ent o f T T Y 2 genes in distinct clusters In addition to duplications, translocations can occasionally break-up gene clusters and scatter genes, or groups of genes in different places within the genome. This has occurred in the history of the TTY2 genes, which form two clusters, a large group on Yq (AZFc) and an apparently smaller one on Yp. The distance betw een TTY 2-like genes w ithin the cluster (Figs 4.25 and 4.26) varies

betw een 4Kb and 40Kb.