In this study an attempt was made to understand the mechanism o f association o f the Xbal R FLP with differences in serum lipid and lipoprotein levels and some haplotypes which may account for these differences have been detected. The haplotype carrying the Ag(x) and Xbal X- alleles is associated with a small mean lowering effect on LDL-cholesterol and apoB, acting in a recessive manner. By contrast haplotypes, uniquely defined by the A g (a) and Xbal X + alleles, are associated with mean raised levels o f apoB with a mild but dominant effect. It will be of interest to see if these associations are also confirmed in other population samples that demonstrated the Xbal association, and to examine their effect at the molecular level.
During the course o f this study, aspects o f the evolutionary structure o f the apoB gene that account for the reported strong linkage disequilibria and allelic associations have been revealed. It is the very existence o f this high degree o f linkage disequilibrium that has enabled haplotypes to be deduced without family studies. In general, the evidence indicates that novel mutation, rather than recombination, is the driving force for change, particularly in the 3’ end o f the apoB gene. This poses the question o f whether this lack of recombination is due to chance or selection. In order for selection to be the cause, it would require that the forms of apoB created by recombination would be deleterious.
This study has also revealed the existence o f more complex apoB haplotypes, the existence o f which cannot be explained by simple novel mutation. There are several possible mechanisms by which these complex haplotypes could have occurred. Recombination events between two different apoB alleles is the most likely possibility, but it is also possible that some of the polymorphisms considered here are at hyperm utable sites and that either the same mutation has
occurred twice on two different haplotypes, or that a mutation having occurred once, has subsequently reverted back to its original form. If recombination is the mechanism by which these unusual haplotypes are generated, it is notable that in all except one o f the complex haplotypes seen, the site o f recombination would appear to be 5 ’ to the Xbal site. It is notable that more than 99% o f the intron sequences within the apoB gene are also located 5 ’ to the Xbal site and exon 26 (Blackhart et al. 1986) This implies that apparent recombination events may correlate with the presence o f intron sequences, as suggested previously by Gilbert (1985).
The higher frequency o f the complex haplotypes among the Swedish patient group, as compared to the controls, raises the possibility that recombination events within the apoB gene may be deleterious. This would be o f obvious clinical importance and, if confirmed, these individuals should be investigated for dislipidaemia or other clinical risk factors. However, it is hard to imagine that increased risk of cardiovascular disease could affect Darwinian fitness, leading to a reduced overall frequency o f complex haplotypes since this type o f late-onset disease seldom affects people before they have reproduced. If selection is to be invoked it is perhaps more likely to have been mediated though differential lipid metabolism and resistance to famine or m alnutrition during human evolution. Alternatively, there is evidence that apoB-containing lipoproteins have a role in neutralising endotoxin relaeased by gram-negative bacteria (Liaow and Floren 1992). It is possible that some variants o f apoB that are better at neutralising endotoxin, enhance resistance to certain bacterial infections, which would be another mechanism through which natural selection could act. It is not inconceivable that the apoB alleles that confer resitance to famine or bacterial infection, are the same ones that now predispose to hyperlipidaemia under dietary excess.
If haplotypes 7 and 8 do have a functional effect on serum apoB levels, the question is raised as to what is the mechanism o f this effect. It could be due to the presence o f another
mutation, also carried on haplotypes 7 and 8, that either alone or in combination with the Ala^pi to Val change, alters the metabolism o f the LD L particle or affects the production o f apoB protein. This could be one o f the apoB variants listed in Table 1.5.2 or may not yet have been discovered. Alternatively, if haplotypes 7 and 8 arose by recombination between other haplotypes, the functional effect might be due to the new combinations o f existing amino acid
yv
polymorphisms in the apoB protein. Evidence from a pig model for hyperlidiaemia supports this hypothesis. A group o f pigs with spontaneous hyperlidiaemia and reduced FCR o f LD L have been examined (Checovitch et al. 1988, Lowe et al. 1988). Although this phenotype is associated with one allele of apoB, termed Lpb5, which has been extensively sequenced, no single amino acid change is unique to this allele (Purtell et al. 1993). Instead, the hyperlipidaemia appears to be due to the unique combination of Asp3 , 6 4 and Ala^^^? within this allele. This hypothesis
assumes that the apoB protein has a significant tertiary structure within the lipoprotein particle, for which there is growing evidence (Walsh et al. 1990, Chatterton et al. 1991, Koduri et al. 1991).
Chapter 6
ARG^.^^ t o THR - A NEW VARIANT IN EXON 2 9 .
Summary
A new variant has been found in a search o f exon 29 o f the apoB gene. A G to C base- change at nucleotide 12937 results in the substitution o f threonine for arginine at residue 4243. Protein secondary structure modelling predicts that the Thr^^w variant would have reduced local net positive charge and increased hydrophobicity. The carrier frequency o f Thr^^^^ is estimated to be 0.035 - no homozygotes have yet been detected. There is evidence that carriers o f Thr^^^s may be m ore frequent among CAD patients than among controls and that carriers may have 4- 20% higher serum apoB levels than Arg4 2 4 3 homozygotes, but these differences do not reach
statistical significance in the samples presented here. Twelve o f the thirteen Thr4 2 4 3 alleles
examined to date share a common apoB haplotype and thus may be identical by descent.
6.1 B ackground
W hilst working in this laboratory, Dr. Helmut Renges sought new apoB variants in individuals with high or low binding of LDL to the LDL-receptor as measured by the U937 cell growth assay (Frostegârd et al. (1990). Twelve individuals were investigated: seven with reduced receptor-binding and five with increased - binding and biochemical details for these individuals are given in Table 2.1.1. Dr. Renges used a combination o f chemical cleavage mismatch analysis and (SSCP) to investigate the gene region encoding the prim ary receptor binding domain (using the system described in Chapter 3) and the whole o f exon 29. No variants were found in exon 26 in these individuals. Only one new variant was discovered in exon 29 - an extra band in the hydroxylam ine track o f the mismatch analysis o f PCR fragments c/d (Figure 4.1.1) in individual 15 (Table 2.1.1). Shown in Figure 6.1.3, this band corresponds to a mis-paired C in the "probe" DNA approximately 350bp from one end o f the amplified DNA fragment.
HO I HO
VC"'
— SOObp
350bp
230bp
Figure 6.1.3 Chemical Cleavage Mismatch Analysis .
T h e DNA fro m in d iv id u als 15 and 16 is sho w n . T h e re a re tw o tracks p e r perso n from the h y d ro x y la m in e reactio n (H) and the o sm iu m te tro x id e re a ctio n (O). T h e u n cleav ed full-length PCR g iv e s a band o f SOObp. T h e 230bp b an d co rre sp o n d s to the A sp,,,, to S e r c h a n g e sh o w n in F ig u re 4 .1 .2 . T h e new 350bp b a n d , c o rresp o n d in g to a m ism atched C. is in th e h y d ro x y lam in e track o f in d iv id u a l 15. A u t o r a d i o g r a p h c o u r t e s y o f D r . H R e n g e s .