The human X chromosome

The X chromosome is the most extensively studied of all human chromosomes due to a wide interest in X-linked diseases and the phenomenon of X chromosome inactivation (Lyon

1988). Both of these features are related to the different dosage of X in males and females; because males have only a single X, recessive diseases tend to be revealed, which accounts for the large numbers of X-linked diseases. As a result, the X chromosome was the first to have a genetic map based on RFLPs and systematic approaches to physical coverage were undertaken and expanded as part of the Human Genome Initiative. By 1993, 200 structural genes had been assigned to the X chromosome, together with 400 anonymous DNA segments (Sclessinger et al 1993). Genes for two X-linked diseases (chronic granulomatous disease and Duchenne muscular dystrophy) were the first to be isolated by positional cloning (Royer- Pokora et al 1986; Monaco et al 1986) and it was by elucidating the mutation mechanism o f two X-linked diseases, fragile X syndrome and spinobulbar muscular atrophy (Sutherland and Richards 1995) that expanded trinucleotide repeats were discovered.

1.8.1 Mapping X-linked diseases

X-linked diseases have features that facilitate positional cloning. Chromosomal assignment is obvious because of the inheritance pattern, reducing mapping on the X chromosome to regional mapping. For several diseases, rare affected females have been found with balanced X-autosome translocations. In these patients, the normal X chromosome is generally inactive and the translocated X active, because of selection in early embryogenesis in order to maintain expression of genes on both autosomes. Translocations that have a breakpoint in a gene will lead to expression of the corresponding disease, as the uninterrupted copy on the normal X is inactive. Such translocations have been instrumental in the cloning o f several X-linked disease genes (section 1.1.1).

Rare male patients exist with deletions (often detected cytogenetically) encompassing several megabases of DNA. In most cases the lack of function for genes in the deleted region results in a contiguous gene syndrome. In other words, several diseases are associated in a single patient, allowing very accurate mapping of the relevant genes if a series of overlapping deletions are available for analysis. This was first observed in the the case of the BB deletion

encompassing part of DMD and genes for CGD {CYBB), McLeod syndrome (XK) and retinitis

pigmentosa-3 (RP3) in Xp21 (Francke et al 1985). The BB deletion was instrumental for the cloning of the DMD and CYBB in 1986, and XK more recently (Ho et al 1994). It is likely that such regions have relatively low gene densities or the deletions would be lethal. In the Xp21.2 region this can be accounted for in part by the huge size of the DMD gene (~2Mb), but more generally it illustrates the great heterogeneity in gene density throughout the genome, with gene-poor regions (in general AT-rich, Giemsa dark bands) and gene-rich regions (GC-rich, Giemsa light bands) (Bickmore and Sumner 1989).

1.8.2 Mutations in X-linked genes

For X-linked diseases that severely decrease reproductive fitness in affected males, the number of mutations in each generation decreases by one-third (since males have one-third of the X chromosomes in the population) and the particular mutation becomes extinct after a few generations. The spectrum of mutations and incidence of severe X-linked diseases is due to the constant input of new mutations and directly reflects the mutational sensitivity of the gene. It is now clear that there is a striking difference in the deletion frequency for various diseases (reviewed in Mandel et al 1992); in X-linked ichthyosis, 80-90% of the mutations are large deletions encompassing the entire gene (many due to unequal recombination between flanking low-copy repetitive elements) while DMD has a high frequency of partial deletions (60-70%) and a considerable level of partial duplications (6-7%) (due in part to the large size of the dystrophin gene). For most other diseases analysed, the frequency of deletions or rearrangements detectable by Southern blot is about 5-15%. Analysis of the factor IV gene in cases of hemophilia B has revealed about 400 different point mutations and one case where the gene was disrupted by de novo insertion of an ALU repeat. The fragile X syndrome is is caused by an unstable expansion of a CGG repeat in the 5’ exon of the FMR-1 gene beyond a normal threshold, which is correlated in patients with abnormal méthylation of the adjacent CpG island leading to loss of expression of the gene transcript. In SMA, the mutation is a more moderate expansion of a GAG repeat in the NHz-terminal coding region of the androgen receptor gene (AR) while other heterogeneous mutations in AR result in the completely different phenotype of testicular féminisation.

The above examples illustrate the mutational diversity associated with most X-linked disorders as a result of the high proportion of families with new mutations, which can severely hamper precise localisation by linkage analysis. Even with a dense genetic map composed of highly polymorphic markers, most rare diseases will not be mapped to intervals smaller than 2- 5cM (potentially containing 100 genes!), and precise localisation by linkage disequilibrium

studies is only advisable in homogeneous populations capable of showing a founder effect for the disease (section 1.2.7).

1.8.3 The genetic and physical map of the human X chromosome

A unified genetic, physical and transcriptional map of the human X chromosome is being built through a concerted, international effort. Two large groups (Nagaraja et al 1996; Crollius et al 1996) have recently published integrated maps specifically for the X chromosome, owing to its poor representation compared with the autosomes in the whole- genome physical maps (Chumakov et al 1995; Hudson et al 1995). This is prinicipally due to the low representation of the X in the CEPH Y AC library used to construct these maps, and the fact that they are based on microsatellite STSs which are lower in frequency on the X chromosome (Dietrich et al 1996). Y AC contigs with an average clone depth of at least four­ fold coverage now span more than 80% (125Mb) of this 160Mb chromosome (Crollius et al

1996), with an average inter-STS resolution of 85kb and a current total of around 2000 STSs in 5200 YACs (Nagaraja et al 1996).

Numerous YAC contigs have also been established in smaller regions surrounding specific disease genes in positional cloning strategies, and contigs are rapidly being linked by common STSs and clones such that the ‘consensus’ physical map is virtually at the point of completion (Nelson et al 1995). The gaps that remain suffer a variety of contigging problems: regions that are unstable or unrecovered in cloned DNA; sequence elements that are repeated along the X; clones that are chimaeric or contain large internal deletions. Closure will most likely accrue from efforts to construct higher resolution maps in bacterial cloning systems (section 1.3.2.3).

There are 6 major genetic maps for the X chromosome (Murray et al 1994, Matise et al 1994, Donnelly et al 1994, Wang et al 1994, Fain et al 1995, Dib et al 1996) consisting o f RFLP’s and microsatellite markers. Basic features of these maps are summarised in Table 1.2. The maps are described as ‘framework’ (F) or ‘comprehensive’ (C) depending on the statistical support for marker order; 1000:1 (lod 3) is required for a framework map (detailed in section 5.1.2.1).

These genetic maps show a high degree of overall consistency and the genetic lengths are in good agreement with that postulated by Morton in 1991 (220cM). Differences in the resolution and genetic length among maps reflect differences in error screening, number o f markers analysed, ordering criteria and map format. The higher resolutions of the 2D map (3.2cM; Fain et al 1995) and the map of Wang et al (1994) reflect the high marker density provided by map integration, and make them extremely valuable for positional cloning projects where it is more useful to know and refine the probable marker order in a dense map, both to refine disease gene intervals and to assist in locus assignment for X-linked disorders that display genetic heterogeneity (e.g. retinitis pigmentosa, Teague et al 1994; mental retardation; Lubs et al 1996). The high-confidence framework maps serve as a guide to low resolution linkage mapping.