Human genetic maps.

The international Human Genome Project, initiated in the late 1980's, aimed to determine the nucleotide sequence of the entire human genome by the year 2005. The first steps in this process were the production o f high-resolution genetic and physical maps.

1.3.2.1 Restriction fragment length polymorphisms.

The quality and potential value o f a genetic map depends upon the density and heterozygosity o f the polymorphic markers used to create it. Early genetic maps were composed of restriction fragment length polymorphisms (RFLPs), and variable number of tandem repeat polymorphisms (VNTRs). RFLPs, the first DNA polymorphisms to be detected, are differences in the length of the DNA fragments

obtained following digestion by specific restriction endonucleases (Botstein et al.,

1980). These are based upon a variety o f sequence changes at the nucleotide level. VNTRs, also known as minisatellites, are variations in the number of head to tail

Introduction

Southern blotting (Southern et al., 1975), followed by hybridisation with a specific

DNA probe.

The first RFLP map of the human genome was published in 1987, and it

contained 393 RFLP loci with an average spacing of lOcM (Donis-Keller et al., 1987).

Whilst this publication made possible the genetic mapping o f disease traits, the marker density is inadequate for use in gene isolation. Since RFLPs tend to be bi-allelic they often prove uninformative during linkage analysis. VNTRs are multi-allelic, but have an uneven distribution, being more common at the telomeric ends of chromosomes.

1.3.2.2 Short tandem repeat polymorphisms (STRPs or microsatellites).

Microsatellites are tandem repeats o f simple sequence (e.g. A, AC, AAAN, AAN, AG) that occur abundantly and at random throughout the genome. They are at present the most common source of informative DNA markers available. The most frequently employed (CA)n repeats occur on average once every 30kb in the human

genome and have a repeat unit length o f 10-50 copies (Stallings et al., 1991). They

have the great advantage of being both highly polymorphic and rapidly assayed by PGR. The heterozygosity of a specific microsatellite marker, which is related to its mean repeat length, is calculated from the number of observed alleles and their frequency in a given population.

The advent o f microsatellites lead to the publication of a series of genetic maps

of increasing marker density, culminating in the final Généthon map (Dib et al., 1996)

which contains 5,264 (CA)n repeat markers with an average separation of 1.6cM, and an integrated CEPH/Généthon/CHLC map comprising 5,840 loci (mostly

microsatellite markers) with an average spacing o f 0.7cM (Murray et al., 1994).

These high density genetic maps have greatly facilitated genetic linkage analysis and permitted the critical genetic interval for many disease traits to be refined

sufficiently for physical mapping and gene identification methods to be employed. Genetic maps are also being combined with physical maps to provide integrated maps for each chromosome, which are available via electronic databases on the World Wide Web (WWW). In addition other sources of polymorphic markers are being assessed, such as single nucleotide polymorphisms (SNPs) which although they are bi-allelic and may sometimes be uninformative, have the advantages of being highly abundant (spaced approximately every Ikb) and potentially amenable to automated typing

(Kruglyak et al., 1997).

1.3.2.3 Refîning the critical interval: identification of chromosomal

rearrangements.

Positional cloning projects are very labour intensive, and since both the extent of the physical mapping effort required and the number o f potential candidates which will have to be investigated is directly related to the size o f the disease critical region, it is crucial to refine this region as far as is possible at the outset. Linkage analysis of new pedigrees, or additional members of existing families may narrow down the critical interval, but the smaller this interval becomes the less likely it is that studying further meiotic events will reveal informative recombinations.

Only a few eye disease genes have been identified by positional cloning and in many cases the identification of a chromosomal rearrangement, such as a deletion or a translocation has contributed substantially to the success of the project. The identification o f a female choroideremia patient with a t(X;13) translocation was a milestone in the cloning of the choroideremia gene. Further studies on other patients with choroideremia identified small deletions. Analysis o f the translocation breakpoint and the smallest deletion finally localised the disease gene to a 45 kilobase (kb) region

Introduction

The gene responsible for X-linked RP at the RP3 locus, RPGR, was cloned

following the identification in two affected individuals of a 75kb deletion and a 6.4kb

microdeletion respectively (Meindl et ah, 1996). Whilst the RP3 critical interval had

been refined to just 500kb at the time when RPGR was discovered, the other major X-

linked RP locus, RP2, was genetically mapped to a much larger 5cM region, making

positional cloning efforts very difficult. The region was screened for genomic rearrangements by the YAC representation hybridization technique resulting in the

detection of a LINEl insertion in one X-linked RP patient (Schwann et al., 1998).

This insertion was found to have occurred in an intron of a novel gene, and mutations

in this gene, designated RP2, were subsequently identified in six additional patients.