CÁLCULO DE MEDIDAS DE TENDENCIAS CENTRAL Y DISPERSIÓN DE

Exon

-2

-1

Consensus Sequence

A G

CO

CO

Intron

g t a/g a g t

g t a a g t

a t a a g t

Likelihood

score

86

68

Figure 5.3.4.2.2 : Schematic representation of the 5' donor splice site of a consensus sequence compared to the normal intron 5 of a-fucosidase and patient 2.

deficiency of functional enzyme. It is probable that the mutated splice site would not be recognised by the Ul snRNP and therefore splicing of the 5' donor splice site would not occur as in the normal splice site. There are two possible consequences of this mutation on RNA splicing. Firstly the mutant splice site will not be recognised and therefore the exon will not be recognised and spliced out as an intronic sequence. This would result in an infi*ame deletion. Secondly the splice site will not be recognised but a different sequence may be used as a cryptic splice site located in either exon 5 or intron 5. This would result in either some of the exon being spliced out or some of the intron included as an exonic sequence and therefore translated. Analysis of the sequence of exon 5 revealed no sections of sequence that would be predicted to act as a cryptic splice site as they all had a probability of usage lower than the mutated splice site. Therefore if a cryptic site is used it must be located in intron 5.

Further evidence that this g-a transition is the putative disease-causing mutation is obtained from earlier studies that were performed on patient 2 and her family (Williamson et al., 1993). Original analysis of genomic DNA of patient 2 and her family by Southern blotting revealed that DNA digested with the restriction enzyme Taq\ and probed with a FUCAl probe A+B (Figure 2.3.9.2.1) produced an altered pattern. The 5kb band that was observed in all the controls disappeared and an extra l.Skb band was observed. Patient 2 was homozygous for this alteration and her parents heterozygous. Family studies revealed that this novel 1.8kb band obtained during Taql restriction cosegregated with the mutated fucosidosis allele, indicating strongly that this is the disease-causing mutation in this family. The homozygosity of the mutation can be accounted for by the parents being distantly related. The clinical symptoms of patient 2 were detected

between 1 and 3 years of age and were characteristic of flicosidosis. Negligible a-fucosidase activity was found in leukocytes. Further analysis of mRNA was not performed because no material was available therefore the effect of this splice site mutation on the mRNA remains unknown.

There are several published examples of the different effects of a mutation at the +1 position of the 5' splice site in lysosomal enzymes. For example Sakuraba et al., (1992) describes a patient with Fabry disease in which a g - t transversion at the invariant +1 position of the 5' donor splice site of intron 6 of the a-galactosidase gene constantly resulted in exon 6 being skipped out during

mRNA processing. This effect has also been found in a non-Finnish patient suffering from aspartylglucosaminuria, who had a point mutation in the same + 1 position of the donor splice site

(Mononen et al., 1992). In Tay Sachs disease Alki et al., 1990 reported that exon 2 is skipped in a patient showing a point mutation in the invariant +1 position of the 5’ donor splice site of intron 2 of the hexosaminidase gene. An interesting situation was seen in 3 different Ehlers Danlos patients in the type m procollagen gene in which the same mutation g -a mutation was identified in the same position of the 5' donor splice site, +1, in 3 different exons (Kuivaniemi et al., 1990). These 3 identical mutations in introns 16, 42 and 20 induced very different effects on splicing. The mutation in intron 16 caused skipping of the preceding exon in 71% of transcripts; in intron 42 it resulted in the invariant use of a cryptic splice site and the insertion of 30 nucleotides in jframe into the mature mRNA and finally the same mutation in intron 20 resulted in the use of a cryptic splice site in 53% of transcripts and retention of all the intron sequence in the mature mRNA in 34% of transcripts. This demonstrates the lack of understanding there is of the splicing mechanism for RNA and also the inability to predict with a high degree of certainty the outcome of a mutation in a splice site. It is worth noting that the previously described mutations identified in

splice sites were the disease-causing mutations.

The second identified splice site mutation, a Ibp deletion, S216fs (TGA), in patient 4 was also located in a 5' donor splice site, at position -2 of intron 3. This mutation is predicted to have two possible effects. Firstly it may affect splicing or alternatively, it may alter the reading fi-ame and produce a stop codon upstream from the deletion. As the base at position -2 is deleted it would change the splice site sequence at positions -2 and -3. The base that was at position -3 would be located at position -2 and there is a new base at the -3 position (Figure 5.3.4.2.3). This change would decrease the likelihood score from 94 to 87 ,which would not be expected to affect splicing. Therefore the most probable effect of the S216fs mutation would be to alter the reading frame. The effect of this type of mutations will be discussed in the next section.

Control