Interpretación y explicación categorial (franja horizontal)

Mutation analysis of the PMM2 gene was carried out on all of the patients with CDG-Ia by various methods, including SSCP analysis, direct sequencing and restriction enzyme digestion. Nine different mutations were found in the CDG-Ia patients, 8 of which were missense mutations. One novel deletion, 284delT in exon 4 of the PMM2 gene was the only other change found. Therefore, the detection of mutations in the CDG-Ia patients in our study confirms the enzymic diagnosis. A summary of the missense mutations found is shown in Table 4.7:

Exon cDNA nucleotide number Mutation Codon Change Amino Acid Change Number of Occurrences 5 357 C^A TTC-^TTA F119L 3

5 395 T-^A ATT^AAT I132N* 1

5 422 G^A CGC^CAC R141H 11 5 442 G^A CGA^CAA D148N* 1 7 548 T-^C TTT—TCT F183S* 1 7 623 G-^C GGA^GCA G208A 2 8 691 G^A GTG-^ATG V231M 5 8 710 C^T ACG^ATG T237M 1

Table 4.7: Summary of missense mutations found in PMM2 gene (*) denotes novel mutation

All of the patients were compound heterozygotes. Two mutations were found in 11 of the 14 CDG-Ia families (Table 4.7), but only one mutation was found in two patients DC, JR and in the siblings JS and TS, where DNA only from the father was available. In the cases of DC and JR, all of the complete coding sequence and intron/exon boundaries of the PMM2 gene were sequenced in the forward and reverse directions. The detection of the normal sequence ruled out the complete deletion of the gene in the

Chapter 4 Discussion CDG-Ia

coding regions of the PMM2 gene, which may affect normal splicing, or in the promoter region, which would affect transcription. In order to verify these possibilities, further investigation using mRNA analysis of the PMM2 gene would need to be carried out.

Patient Genotype Mutation 1 Mutation 2 PMM activity (nmol/min per mg of protein) normal control: mean=2,7±0.7; range: 1.9-3.8 Clinical phenotype (survival <2yrs=severe; >2yrs=mild) CB R141H V231M 0.18 severe JB R141H G208A 0.05 severe LB D148N F183S 0.05 mild TB R141H V231M 0.24 mild VB R141H V231M 0.02 mild DC - V231M 0.13 severe

MC 284 del T G208A 0.07 severe

JD R141H F119L 0.00 mild KF R141H F119L 0.10 mild LM R141H F119L 0.25 severe JR I132N - 0.23 severe JS R141H - N.D severe TS R141H - N.D severe KT R141H V231M 0.13 severe EW R141H T237M 0.05 mild

Table 4.8: Genotypes of CDG-Ia patients

(N.D) denotes that the enzyme assay was not performed due to lack of patient sample Clinical phenotype was classified as either ‘severe’, where the patient died before the age of 2 years, or ‘mild’ if the patient survived over 2 years of age. (For a detailed clinical description of patients see Appendix Table A.1)

All of the missense mutations were found in exons 5, 7 and 8 of the PMM2 gene. These exons contain regions that are highly conserved in human PMM2, human PM M l, yeast PMM (SEC53) and Candida albicans PM M genes (Matthijs et a l, 19997b; Schollen et a l, 1998; Imtiaz et al., 2000). All of the mutations caused an alteration to a conserved or semi-conserved arnino acid, therefore supporting the idea that they are disease- causing. Furthermore, none of novel mutations was found in 100 normal chromosomes

Chapter 4________________________Discussion__________________________CDG-Ia

screened by SSCP analysis (Matthijs et al., 2000), indicating that they are not polymorphisms.

The R141H mutation was the most common mutation found (35% of alleles) where 9 of the 13 CDG-Ia families were heterozygous for this mutation. A high incidence of R141H has been observed worldwide and it accounts for 37% of all reported mutant chromosomes (Matthijs et al., 2000). R141H has an incidence of approximately 1 in 80 in the normal Western European population (Matthijs et al., 1998b), but as yet, has not been found in the homozygous state (Matthijs et al., 1997a; 1998b; Schollen et al.,

1998; Carchon et al., 1999; Imtiaz et al., 2000; Matthijs et al., 2000). The residual activity of the R141H recombinant protein when expressed in vitro is almost zero (Pirard et ai, 1999). This finding supports the hypothesis by Matthijs and colleagues (1998a) that R141H is deleterious in the homozygous state, leading to fetal wastage, miscarriage or early death. Homozygosity for R141H may also give rise to a different phenotype. However, in view of the fundamental role of PMM2 in the normal functioning of the cell, it was hypothesized that the R141H/R141H genotype is lethal soon after conception. R141H is associated with a specific haplotype and it has been suggested that the mutation is the result of a single mutational event (Schollen et al.,

1998). R141H is caused by a CGC to CAC transition at a CpG dinucleotide, a hotspot for mutations.

The second most common mutation was V231M, found in 5 patients. V231M has been reported as the most common mutation found in exon 8 of the PMM2 gene and its presence has been reported worldwide (For review, see Matthijs et al., 2000).

Chapter 4________________________Discussion__________________________CDG-Ia

The third most common mutation was F119L, found in 3 patients. F119L has been reported to be the second most common CDG-Ia mutation found worldwide (Matthijs et al., 2000), accounting for 16% of all mutant chromosomes. Bjursell et al, (1997; 1998) have documented a clear founder effect for F119L in the Southern Scandinavian population, as in Denmark where the mutation accounts for 48% of the disease alleles (Matthijs et al., 1999a; 2000). The proportion gradually decreases from north to south and the incidence varies between 17% in the Netherlands and 11% in Germany but has not yet been reported in Spain, Portugal or Italy. A patient homozygous for F119L has been reported (Matthijs et al., 1998; 2000) which is in accordance with the Hardy- Weinburg equilibrium, whereas the lack of a patient homozygous for R141H is not. As the F119L/R141H genotype has been found in this and other investigations (Matthijs et al., 1998; 2000), it can therefore be concluded that the combination of the two most frequent disease mutations is not lethal.