COMPENSACIONES

A total o f 10 genetic alterations were identified by SSCP analysis in seven different exons o f the RPGR gene (from exons 1 to 19 plus exon 15a) in 27 XLRP families segregating with the RP3 locus (Zito et al. 1999). Five o f the ten changes were classified as polymorphisms (Table 4.3) either because they were previously described as polymorphisms

or because they were also present in unaffected control chromosomes. The remaining five changes were classified as potential novel mutations found in four different exons. Each mutation is described as a correspondent change at the nucleotide and/or the amino acid level (Table 4.2a, see also Figure 1.11).

In families RP106 and RPl 120 a nucleotide change was found in the donor splice site respectively at the first and at the fifth nucleotide downstream of the exon/intron boundary o f exon 1 (Figure 4.3a and 4.3b). Exon 1 was sequenced in 80 control chromosomes and these changes were not detected. In addition for both families the segregation o f the nucleotide change with disease was demonstrated by sequencing 3 affected and 3 unaffected males (data not shown) and analysis o f the entire gene in both families did not reveal any other sequence alterations. Biological proof that these nucleotide changes are causing the disease in these families requires further experimentation initially by reverse transcription PCR. Unfortunately fresh patient blood was unavailable. Nevertheless all intron splice acceptor and donor sequences o f the human RPGR gene follow the GT-AG rule and the first nucleotide o f a donor splice sequence is 100% conserved (Horowitz and Krainer 1994), hence a G->A transition at this site is very likely to affect RNA splicing. The fifth nucleotide is 80% conserved, therefore a G->A transition at this position is also likely to affect RNA splicing.

Direct automated sequencing of DNA from another 3 patients that presented a shift on an SSCP gel led to the identification o f 3 different frameshift mutations. Family F75 (Figure 4.3c) has a 2 base pair deletion in exon 7 corresponding to nucleotides 807 and 808 in the normal sequence. In family XRP90 (Figure 4.3d) 2 base pairs have been inserted in exon 10 between nucleotides 1297 and 1298 o f the normal sequence. Family RP87 (Figure 4.3e) has a 2 base pair deletion in exon 11 corresponding to nucleotides 1435 and 1436 in the normal sequence. In all three cases, the resulting frameshift gives rise to a considerably truncated protein predicted to lead to lack of function. Segregation o f the mutations with disease was demonstrated in each family by SSCP analysis, examples o f which are seen in Figure 4.4a-b (families F75 and XRP90 respectively).

Figure 4.3:

Electropherograms depicting patient mutations and normal sequences for the RPGR gene. Arrows indicate the sites of mutations. a) A G>A change at the first nt downstream o f the exon 1/intron 1 boundary

in an affected male from family RPl 06 and normal sequence o f the corrisponding region.

b)

A G>A change at the fifth nt downstream o f the exon 1/intron 1 boundary in an affected male from family RPl 120 and normal sequence o f the corrisponding region.

c) A 2-bp deletion (GT) in exon 7 in an affected male from family F75 and normal sequence o f the corrisponding region.

d)

A 2-bp insertion (AG) in exon 10 in an affected male from family XRP90 and normal sequence from the corresponding region.

e) A 2-bp deletion (TC) in exon 11 in an affected male from family RP87 and normal sequence o f the corresponding region.

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a C T G A T G C C CG A T Û AG T T Q C Q C | ) C C C CG G T G A A T T G C G C C C G C

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C T G A T G C C C G G T G A G T T G C G C G C C C G G T G A G T T G C G C C C G C n n n . A A A m i Y ' i c C fl fl G Tfl G C C T G G TG G fl G R G C fl T d T G C G G C 6 fl fl G fl G fl G fl G fl G G G T fl C C f l f l G T R G C C T G T G G T G G R G R G C f l 'A T G C G G C G R R G f l G R G R G G G T R C f l e C C f l f l G R G R G T G T T R T C T G R R C f l G C C R f l G R G R G T G T C T T R T C T G f l f l C f l

Figure 4.4:

Segregation analysis o f two novel RPGR mutations. Arrows on the right depict the informative bands. Pedigrees are aligned to correspond to the respective gel lane.

a) Segregation o f the exon 7 frameshift mutation in family F75.

b)

Segregation of the exon 10 frameshift mutation in family XRP90.

m

4.3.2 Additional RPGR mutations and polymorphisms identified by

In document Propuesta de modelo de gestión de recursos humanos, centrado en competencias, aplicado al personal de una empresa productora del medio televisivo (página 43-71)