EVALUACIÓN FINAL DEL PROYECTO

1.8.1 General and historical aspects

In humans, as in other mammals, the sex chromosomes are heteromorphic, dififering substantially in size. The X chromosome is submetacentric, euchromatic and contains approximately 165 Mb of DNA whereas the much smaller, acrocentric Y chromosome is heterochromatic and contains only about 60 Mb of DNA. During meiosis in human females, the two equivalent X chromosomes can pair and recombine anywhere along their entire length. In contrast during male meiosis, recombination is restricted to two small areas of sequence identity known as the pseudoautosomal regions (reviewed by Rappold 1993).

Pairing between the short arms of the X and Y chromosomes was first observed in humans in 1970 by Pearson and Bobrow, who examined spermatocytes during meiotic prophase (Pearson and Bobrow 1970). Crossing over events in the region were then observed cytogenetically. Later it was proposed that the distal short arms of the X and Y chromosomes were the site of a single obligatory crossover during male meiosis, and that this X-Y pairing was a prerequisite for successful male meiosis (Burgoyne 1982). It was argued that this region would behave like an autosomal segment and therefore the term ‘pseudoautosomal’ was proposed. The occurrence of crossing over was confirmed by the identification of molecular markers that recombine between the sex chromosomes. In

1992, Frege Qt al. identified the existence of a second pseudoautosomal region by

demonstrating crossover events between Xq and Yq (Frege et al. 1992).

The major pseudoautosomal region (PARI) is a 2.6 Mb region of homology between the X and Y chromosomes located at Xp22.3 and Y pll.3. The second, smaller pseudoautosomal region (PAR2) is a 0.4 Mb region located at the tip of Xq/Yq. The pseudoautosomal boundary (PABX or PABY) marks the interface between sex-specific and pseudoautosomal sequences, and is defined as the proximal limit of recombination of the PAR. Sequences proximal to the pseudoautosomal boundary are non-homologous and sex-specific. The distal boundary of each PAR is the telomere.

1.8.2 Recombination within PARI

Telomeres in general are known to have a higher recombination rate per physical length compared to other chromosomal regions, but due to the obligatory cross over in male meiosis, PARI has an exceptionally high overall recombination frequency, at least

10-fold higher than the genome average (Rouyer et al. 1986). The frequency of this

recombination shows a gradient through the PARI such that loci at the telomere have almost a 50 % chance of crossover between the X and the Y, reducing to 0 % at the pseudoautosomal boundary. Thus pseudoautosomal genes are inherited in an apparently autosomal fashion with a varying degree of sex linkage depending on their physical location within PAR. Double recombinants are detected in approximately 1 % of meioses

(Lien et al. 2000). There is a large sex difference in recombination frequency Avithin the

PAR, since in female meiosis the recombination frequency in PARI is comparable to the X chromosome average.

Deletion of PARI in humans results in failure of pairing and male sterility, supporting the hypothesis that the cross over within PARI is obligatory for successful

male meiosis (Mohandas et al. 1992). Although recombination has been observed in

PAR2, it is neither obligatory nor sufficient for male meiosis.

Occasionally an ‘illegitimate recombination’ can occur outside the PAR and may

result in the testis determining factor SRY, located 5 kb proximal to the pseudoautosomal

boundary o f the Y, being transferred to the X chromosome. Thus a 46,XX individual with

a male phenotype may arise (Page, Brown, and de la Chapelle 1987; Petit et al. 1987).

The reciprocal product of this crossover is a Y chromosome deleted for SRY, which may

result in a 46,XY female with gonadal dysgenesis.

1.8.3 Evolutionary aspects: origin of the FARs

Human X and Y chromosomes show several regions of homology on both the long and short arms in addition to the two pseudoautosomal regions, which provides support for the generally accepted theory that the heteromorphic sex chromosomes evolved from a homomorphic sex chromosome pair. This is thought to have occurred by gradual reduction of the Y, in combination vrith a series of rearrangements (Ohno 1967).

The human PARI is proposed to represent a remnant of part of an autosomal region added to both the X and Y chromosomes between 80 and 150 million years ago. Evidence supporting this hypothesis comes from interspecies comparison of the PARs, which has shown that no PAR is present in marsupials (a group of mammals that diverged from placental mammals about 130 million years ago), but that the homologues of human PARI genes are autosomally located in marsupials, and co-localise with human Xp22 genes (Toder and Graves 1998). The PAR o f placental mammals is very variable, with human and mouse PARs being completely non-homologous (Blaschke and Rappold 1997). There is even variation in the gene content of the PARs within primates, observations which argue for the rapid evolution of the human PARI (reviewed by Toder and Graves 1998).

1.8.4 X-inactivation

In mammalian females, one of each pair of X chromosomes undergoes the process of X-inactivation, resulting in the transcriptional silencing of most genes on the inactive X chromosome. X-inactivation allows gene dosage equivalence to be maintained between males and females for the vast majority of X chromosome genes which do not have a homologue on the Y chromosome. The process of X-inactivation occurs early in development and is random, but once an X chromosome is inactivated in a cell, the same X chromosome tends to remain inactivated in all descendant cells, making females mosaic for cell lines in which either the maternally or paternally derived X chromosome is inactivated. The mechanism by which X-inactivation occurs involves inhibition of

transcription thought to be initiated by expression of the XIST gene from the inactivated

X chromosome (Brown et al. 1992).

Genes in the pseudoautosomal region, however, escape X-inactivation and are expressed whether or not the chromosome is subject to X- inactivation, so that males and females both have two active copies. An increasing number of genes are known to escape X-inactivation and most of these genes have a homologue on the Y chromosome, which may be actively expressed or a pseudogene. For example, the region of the human X chromosome proximal to PARI contains several genes known to escape X-inactivation,

including XG (Xg blood group), STS (the steroid sulphatase gene), KALI (Kallmann

syndrome), ARSE (aryl sulphatase E) and one or more loci for X-linked mental

retardation (XLMR).

1.8.5 The short stature critical region of PARI

In 1989, Ballabio et al. studied a group of male and female Xp deletion patients,

and found that they almost invariably showed short stature (Ballabio et al. 1989b). The

authors proposed that this observation was consistent with haploinsufiSciency for a gene affecting height located within PARI, two copies of which are required for normal

growth. Ogata et al. performed further similar genotype-phenotype correlations to refine

the region and concluded that haploinsufficiency for a dosage sensitive gene(s) within the

distal 700 kb o f PARI results in short stature (Ogata et al. 1992; Ogata et al. 1995a). The

distal 700 kb of PARI became known as the short stature critical region since individuals who are hemizygous for this region are short, whereas those with deletions elsewhere within PARI are of normal stature. Hemizygosity for this putative gene was also suggested to contribute to the short stature of Turner syndrome (Ogata and Matsuo 1995).

Rao et al. constructed a cosmid contig across the short stature critical region and,

using FISH to map precisely the breakpoints of Xp deletion patients, performed

genotype-phenotype correlations to narrow the critical region to 270 kb (Rao et al.

1997a). This group went on to fijrther refine the interval to 170 kb by analysis of three additional informative Xp deletion patients with and without short stature, and fi’om this

region the 5hort stature HOmQohoX gene SHOX was identified using a combination of

cDNA selection, exon amplification and 3’ and 5’ RACE (Fig. 1.7) (Rao et al. 1997b). A separate group also independently identified the gene by positional cloning and named it

PHOG, for Pseudoautosomal //bmeobox-containing Osteogenic Gene (Ellison et al.

1997). The Nomenclature Commission of the Human Genome Database assigned the

name SHOX. Both groups proposed that the novel gene plays a role in the short stature of

Turner syndrome and in idiopathic short stature, but in addition Rao et al. identified a

p 22.3 \ 1 7 0 kb critical r e g io n NJ

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Y

m

Schem atic representation o f the cloning o f SHOX from the short stature critical region o f PA R I. The location o f the 2.6 Mb PARI at Xp22.3 and Y p l l . 3 is illustrated, show ing the 700 kb 'short stature critical region’ w ithin it. A cosm id contig across this 700 kb region was constructed by Rao et a i , and deletion m apping refined the critical interval to 170 kb. From this region, the short stature hom eobox gene SHOX was identified by cDNA selection and exon am plification, approxim ately 500 kb from the Xp and Yp telom eres. (A dapted from Zinn, 1997).

n

□ R egion en c o d in g p roline-rich stre tc h □ R egion en c o d in g OAR do m ain □ Alu r e p e a t 5 89 bp 6a 243 bp 6b 42 bp H Figure 1.8

The structure o f SHOX. T he gene com prises 7 exons, the first being non-coding. Two transcripts, SHOXa and SHOXh, are produced by alternative splicing o f exons 6a and 6b at the 3 ’ end o f the gene. SHOXa has an open reading fram e o f 876 bp, encoding a protein o f 292 am ino acids. SHOXh has an open reading fram e o f 675 bp, encoding a protein o f 225 am ino acids. T he start codon is m arked in exon 2, with the coding region shown in black, and the position o f the hom eobox m arked in red, situated partly in exon 3 and partly in exon 4. The stop codons in exons 6a and 6b are indicated. A region in exon 6a encoding a short proline-rich stretch w ith sim ilarities to an SH 3-binding dom ain, is show n in yellow. The region encoding the OAR dom ain is show n in blue. An Alu repeat elem ent located in the 3 ’ untranslated region o f SHOXa is shown in green. The non-coding 5 ’ and 3 ’ untranslated regions are show n unfilled.