PRODUCTOS

In total eleven different mutations of y^ were identified in this study. Four of these cause amino acid substitutions. The remaining seven are disruptive mutations, that is they could be expected to cause an structurally aberrant message or protein to be produced. The disruptive mutations are made up of two stop mutations, three

deletions, one insertion and one splice site mutation. Thus a wide range of mutagenic mechanisms must have been involved in the generation of these mutations suggesting that this gene demonstrates no bias with respect to a particular mechanism of

mutagenesis.

4.4.2.2 Distribution of mutations identified in

Mutations were identified in five of the eight exons of y^. No mutations were found in exons 1, 3 or 8. However, other studies report mutations in these exons (Noguchi et a l, 1993c; Puck et al, 1993b; DiSanto et al, 1994a and b; Ishii et al, 1994;

Markiewicz et al, 1994; Puck, 1994) suggesting that the failure to find mutations in these exons may be a consequence of the small sample size or may reflect the

influence of the sequence context of a mutation on the sensitivity of SSCP (Sheffield

et a l, 1993). Considering all of the published mutations (see Appendix VI and Puck, 1994) together with those reported here, y^ mutations seem reasonably evenly

distributed throughout the gene. However, closer analysis reveals a possible cluster in the 3' half of exon 5. This region is less than lOObp and yet ten of the 61 mutations identified in the y^ gene have been found here (4/11 from this study, 5/23 published as described in Appendix VI and 1/27 reported in a review by Puck, 1994 but precise location and nature of mutations not given). This observation suggests that this region may be particularly prone to mutation. With respect to this it is interesting to note that five of the 18 mutation prone CpG dinucleotides found in the y^ gene (discussed in Section 4.4.2.3) lie in this region of exon 5, two of which are sites of reported mutations (patient 9, DiSanto et al, 1994a and Pepper et al, 1995). However, it is important to note that other mutagenic mechanisms are at work in this region, as at least three of the ten mutations in this region are deletions and one is an insertion.

this region includes the "WSXWS" motif which is conserved between all members of the cytokine receptor superfamily and is disrupted by a number of the mutations in this region.

4.4.2.3 Mechanisms of mutagenesis in y,.

It has been reported that deletions and insertions often occur at positions in a gene flanked by direct or inverted repeats, monotonie runs of bases and other sequences which could form secondary structures or permit slippage of DNA polymerases during DNA replication (Cooper and Krawczak, 1991 ; Krawczak and Cooper, 1991). For two of the deletions and the insertion described here there are no obvious sequences of this kind. However, the single base deletion identified in patient 2 lies at the centre of a region of sequence with some inverted repeat elements as indicated in Figure 4.21 which may be involved in causing this mutation, for example by facilitating the formation of a hairpin structure.

Figure 4.21 : Sequence surrounding the single base pair deletion found in exon 5 in patient 2

TG G G CAG AAACGCTACACGTTTCSTGTTCGGAGCCGCTTTAACCCA TGGG* * * A A A * * * * * * *C G * * * * Q* * * * CG* * * * * * * T T T * *C C C A

Upper sequence shows complete sequence surrounding the deleted base Lower sequence highlights inverted repeat elements

G indicates deleted base

* indicates sequences which do not show any inverted repeat elements

Seven of the eleven mutations described here are single base substitutions. Of these, four are C to T transitions in the context of a CpG dinucleotide on the coding or non-coding strand, the latter causing a G to A transition on the coding strand (see Table 4.1 for details of mutations). In the human genome the dinucleotide CpG is particularly prone to mutation because méthylation of the cytosine makes it vulnerable to deamination to thymine. Consequently it has been observed that C to T and G to A transitions are 42 times more likely to occur than the frequency predicted by random mutation (Cooper and Youssoufian, 1988). It is therefore unsurprising that transitions of this kind account for more than half of the single base substitutions found in this study.

The remaining three single base pair substitutions identified may be caused by a range of other mechanisms. For example, single base alterations can result from damage caused by environmental agents such as ionising radiation, UV light and mutagenic chemicals if it is not faithfully repaired (reviewed in Weeda et al, 1993). In addition, spontaneous errors during DNA replication or repair account for some base alterations. The demands of the DNA polymerases for the precise geometry of the Watson-Crick base pair are rigorous but it has been estimated that the error rate of DNA polymerases is

approximately 10’^ (Lewin, 1993). This rate is improved by the proofreading action of the 3 '^ 5 ' exonuclease activity of the DNA polymerases. Thus, the compound effect of the polymerase fidelity and the proofreading activity gives an estimated mutation

frequency of 10'^ to 10'^°. These estimates are based on prokaryotic model systems but it is assumed that such mechanisms exist in eukaryotic cells (Echols and Goodman, 1991).

4.4.2 4 Mutation "hotspots"

Three mutations have been identified more than once during this study, and another described here has also been reported elsewhere (Markiewicz et al, 1994). In two of these cases it has been possible to show that the pairs of families involved are very unlikely to be related by analysis of a chromosome 7 VNTR locus. Significantly, three of these four "hotspots" are caused by a C to T transition on the coding or non-coding strand in the context of a CpG dinucleotide (see Table 4.1). As discussed in the previous section these dinucleotides are particularly vulnerable to this transition and this may account for multiple mutations occuring independently at these positions. Another mutation "hotspot" in the gene has been reported (Pepper et a l, 1995) which lies in exon 5 at genomic position 2233 and is found in four unrelated

individuals in the study by Pepper et a/. (1995) and also in one individual in a separate study by Disanto et al (1994a). Interestingly, this "hotspot" is also reported to be at a CpG dinucleotide (Pepper et al., 1995; DiSanto et al, 1994a).