D ISC U SSIO N

3.7.1 r and susceptibility to NTD

A possible genetic association betw een the hum an T gene and

susceptibility to NTD was investigated using newly identified polym orphic m arkers within the T gene. Three markers were used; a variant site in intron 7,

TIVSyC, and two polym orphism s identified by SSCP in the D N A binding dom ain o f T, w ithin the conserved T-box, C 363T in exon 2 and G530A in exon 3. In man, the Asp 177 variant occurs at a high frequency in different ethnic groups, w ith a frequency as high as 0.59 in the CEPH U tah population. The functional significance of this polym orphism is discussed in more detail in chapters 4 and 5.

Using the TDT, an association was detected between the TIVS7-2 (TlVSy-C) allele and susceptibility to NTD in the fam ilial cases, with a strong contribution from the Dutch families, where the num ber o f transm issions is higher. This result may suggest that the apparent increase in the frequency of the TÎVSy-2 allele in Dutch and British fam ilial cases is real. However, in direct case/control com parisons no statistically significant difference could be detected in the allele or genotype frequencies of affected and control

populations. This discrepancy could possibly be due to the small sample sizes, which would affect the case/control studies m ore dram atically than the TDT.

There is no evidence that the TlVSyC polym orphism affects Tm RN A or protein synthesis or function. It seems more likely that the TIVSj-2 (C) allele is a m arker for another mutation, which lies either within the T gene or at another locus nearby. The T-box G530A variation was o f particular interest from this point of view, because it involves a change from a conserved, neutral glycine residue to a negatively charged aspartic acid residue (G lyl77A sp) and is therefore potentially functional. However, haplotype and linkage

disequilibrium analysis reveals that the Asp 177 allele occurs m ost frequently on a chrom osom e carrying the TIVSy-l allele and furtherm ore, there was no evidence for association betw een the G lyl77A sp variation and NTD.

The TIVSy-2 allele occurs most frequently on a chrom osom e carrying the C363 and G530 alleles (designated the 1.2.2 haplotype). Haplotype TD T analysis showed an association betw een this haplotype and susceptibility to NTD. The proposition that an as yet unidentified aetiologic m utation which im poses susceptibility to NTD lies on the 1.2.2 chrom osom e is being pursued in our laboratory, by determ ining the complete sequence of the T gene of affected individuals hom ozygous for the 1.2.2 haplotype.

To date, there have been two other studies using the TIVSyC

polym orphism as a marker. Trembath et a l (1996) investigated NTD in an A m erican population and detected an unequal transm ission o f alleles from 17 heterozygous parents to their affected offspring (41.2% for allele 1, 58.5% for allele 2). Although this was a relatively small-scale study and the result is not significant, it shows the same trend of higher transm ission o f the TIVSy-2

allele. In a m ore recent study. Shields and colleagues analysed 218 Irish NTD case/parent triads and found an increased frequency of the TIVSy-2 allele in cases, w hich was particularly striking in NTD cases born before 1980; relative risk RR=2.09 (95% C l 1.23-3.55), p=0.006 (Shields et a l , 1999, personal communication).

3.7.2 M T H F R and susceptibility to NTD

Six studies have dem onstrated an association betw een hom ozygosity for the com m on therm olabile variant of the M THFR gene (C677T) and N TD in Dutch, Irish and A m erican populations (Ou et a l , 1995; van der Put et a l ,

1995; W hitehead et a l , 1995; Kirke et a l , 1996; Ram sbottom et a l , 1997; van der Put et a l , 1997b; see section 1.2.6). However, this association could not be detected in this study. Apparent differences in allele or genotype

frequencies w ere not statistically significant and the TD T did not detect a distortion in the transm ission of the C677T alleles. In agreem ent with this result, two other studies have failed to dem onstrate an association in

N orw egian and French populations (Bjorke-M onsen et a l, 1997; M ornet et a l , 1997). Also, although Shaw et a l (1998) report a m odestly increased risk of spina bifida am ong infants who were hom ozygous for the C677T

polym orphism , inspection of their data shows that their result is not statistically significant.

The interpretation of these conflicting results is difficult. The incidence of NTD varies in populations of different ethnic origin, as does the frequency of the M TH FR therm olabile variant. However, the differences do not

correlate. For exam ple, the frequency of hom ozygosity for the therm olabile M TH FR allele is approxim ately 8% in Ireland, w here the prevalence o f NTD is high and greater than 16% in Italy, where NTD is low (Fletcher and

Kessling, 1998). One explanation could be that differences in dietary habits that affect folate levels counteract the m odest negative effect o f the

therm olabile variant. Interestingly, in the Spanish population, an association has been proposed betw een an increase in the num ber of babies born

hom ozygous for the therm olabile variant and the start of the use o f early folate supplem entation in 1977 (M unoz-M oran et a l, 1998). A lthough it is difficult to envisage how diet could influence genotype frequencies in such a relatively short time-span, for the future, it would seem im portant to take into account dietary factors and the use o f folate supplem entation when carrying out the type o f genetic studies described here. Only a single published study o f the

role o f M TH FR in NTD has been able to subdivide cases/m others by these characteristics (Shaw et a l , 1998). In the study reported here, there was no inform ation about the folate status of the affected children or their m others, or w hether folate supplements were taken periconceptionally. Since only 70% of NTD pregnancies can be prevented by folic acid supplem entation, the

rem aining 30% are thought to be unresponsive to folate. It would be useful to be able to exclude this latter group from genetic studies, w hich look at

association betw een enzymes of the folate m etabolism pathw ay and NTD. In this context, studies in m ice have shown that in the case o f curly-tail and axial defects m ice, administration of folate during pregnancy has no effect on either the frequency or severity of the defects (Seller, 1994). However, studies in

curly-tail m ice have shown that inositol can reduce the incidence o f spina bifida, possibly by upregulating the expression o f retinoic acid receptor beta in the caudal region of the embryo (Greene and Copp, 1997). D espite the

considerable emphasis placed by researchers on the im portance o f folate, it should be born in mind that there is no relationship betw een frank deficiencies of enzym es involved in folate metabolism and NTD.

Recently, another com m on variant of the M TH FR gene (A1298C), that changes a glutam ate residue to alanine, was analysed in NTD patients (van der Put et a l , 1998). The C1298 allele is not a risk factor for NTD and is never found on the same chrom osom e as the therm olabile T677 allele. Individuals hom ozygous or heterozygous for the A 1298C variant show reduced M TH FR activity, w ith a more significantly decreased activity in individuals

heterozygous for both A 1298C and C677T variants. Double heterozygotes have increased hom ocysteine levels, although the A1298C variant alone does not affect hom ocysteine levels and CdZZTheterozygotes have norm al levels. Van der P ut et al. suggest that com bined heterozygosity for the two M TH FR

3.7.3 Interactions betw een T and M T H F R

Com plex interactions betw een genes and betw een genes and

environm ent could explain the clinical heterogeneity and variable penetrance o f N TD (sections 1.2.2 and 1.2.3). The possibility that the TIVSyC or G530A

(G ly l7 7 A sp ) T variants and the therm olabile MTHFR variant act together to increase susceptibility to NTD was investigated. Only one positive

association was detected. The TIVSy-2 allele in children occurs in com bination with the m aternal M TH FR therm olabile T677 variant m ore frequently than expected. However, exam ining two markers sim ultaneously reduces the num ber of informative individuals and thus, this result should be treated w ith caution.

Studies on allele interactions and NTD have been reported elsewhere. In a recent study of 218 Irish NTD cases. Shields and colleagues (Shields et a l , 1999) did not detect an increased hom ozygosity for the therm olabile M TH FR variant in NTD cases carrying a TIVSy-2 allele, de Franchis et a i

(1997) found an association betw een NTD and a com bined genotype consisting o f the M THFR thermolabile variant and a com m on 68 bp

insertion/deletion polym orphism in cystathionine-(3-synthase, in Italian and Irish N TD populations (see section 1.2.6). In Dutch and British mothers of N TD cases (n=64), M orrison et al. (1998) found that the m ethionine synthase Gly919 variant (see section 1.2.6) occurs in com bination w ith the M TH FR therm olabile variant more frequently than expected.