Conclusion by gyvwpsjkko


									                                                                                      Chapter 6

This comparative study has covered a broad range of VDR genotypes and haplotypes,
to determine the influence of the individual SNPs or BsmI-ApaI-TaqI SNP combination
on VDR protein level and VDR function. Table 6.1 compiles the most statistically
significant findings, in terms of the influence of VDR SNPs or haplotypes on VDR
protein level and biological responsiveness to 1,25(OH)2D3, of the current study.

Table 6.1 A compilation of all the results obtained in the study

Polymorphisms               VDR level             Inhibition of cell proliferation
or SNP                                            Alamar Blue                        Trypan blue
                    Significance    Effect        Significance        Effect         Significance     Effect
                    (p-value)       (Eta)         (p-value)           (Eta)          (p-value)        (Eta)

FokI                *n.s            high-medium   n.s                 small          0.003          high

BsmI                n.s             small         0.071               medium         0.004          high

ApaI                n.s             small         0.024               high-medium    0.002          high

TaqI                n.s             high medium   n.s                 medium         0.008          high


BAt vs baT          n.s             medium        0.042               medium         0.007          high

n.s - not significant

Taken together the following conclusions were drawn from the current study (refer to
Table 6.1):
       a. All VDR SNPs investigated and SNP combinations of the 3’ end that occurred at
          high frequency had no effect on VDR level.
       b. FokI, the functional VDR SNP in exon II, and TaqI, the silent, exon IX SNP, had
          high-medium effects on VDR level although these effects were not significant.
       c. The BsmI-ApaI-TaqI SNP combination had a medium effect on VDR level but
          was not significant.
       d. All VDR SNPs studied, individually and the 3’ end SNPs in combination
          (specifically BAt and baT haplotypes), significantly influenced inhibition of B-
          lymphocyte proliferation, measured by the trypan blue dye exclusion method,
          despite the lack of a significant effect on VDR protein levels. This implies that

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     VDR SNPs, alone or in combination, influences the quality of VDR-mediated
     responses, rather than the levels of VDR protein (quantity). Subtle changes in
     VDR protein architecture effected by the studied SNPs or linked functional
     polymorphisms is most likely responsible for the differential biological
     responsiveness to 1,25(OH)2D3 mirrored by variable inhibition of cell
e. Results observed for cell viability measured by Alamar Blue and cell counts
     done by Trypan blue were not always consistent. This was attributed to the fact
     that the methods are based on different criteria: viability as reflected by
     reduction (Alamar Blue) and counts based on membrane integrity (Trypan blue).
     Although prone to human error, Trypan blue counting is a more direct measure
     of cell proliferation.
f.   Results from the current study underscore the relevance of studying VDR
     polymorphisms in the context of haplotypes rather than individual SNPs. There
     has been much controversy about the biological impact of the VDR
     polymorphisms as individual variants or in haplotypes in recent years. Haplotype
     knowledge may permit a rational choice of polymorphisms to use in
     epidemiology studies or improve our understanding of the significance of VDR
     genetic polymorphisms in infection.
g. The FokI SNP in combination with BAt and baT haplotypes, significantly
     influenced inhibition of B-lymphocyte proliferation. The FBAt SNP combination
     was more responsive to 1,25(OH)2D3 compared to the fbaT SNP combination.
     Leading to the conclusion that different SNPs and SNP combinations influence
     the quality of VDR-mediated responses, rather than the levels of VDR protein
     (quantity; Figure 6.1).

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                             SNP in the coding region   3’-end SNP combinations
         SNPs:               FokI                       BsmI-ApaI-TaqI haplotypes:
                                                        BAt and baT

Scenario:                    Individual A                         Individual B
SNP and haplotype

                  f              BAt                                       F              BAt

                  F              baT                                       f               baT
VDR protein level:

            FbaT FbaT       fBAt fBAt fBAt                     FBAt FBAt FBAt            fbaT fbaT

            Insignificant increased levels of                    Insignificant increase levels of
                 “less active” ‘fbaT’ VDR                          “more active” ‘FBAt’ VDR

Functionality: Inhibition of cell proliferation         Significant inhibition of cell proliferation
                                                        And responsiveness to 1,25(OH)2D3

Figure 6.1 A model illustrating the influence of combinations of functional FokI with the
different VDR haplotypes in two representative individuals. Individual A and B have identical
genotypes however they have different haplotypes. The FokI polymorphism influences the length
of VDR while the 3’end SNP combination is involved in stability and degradation of mRNA. The
combined effects of the FokI SNP and the BsmI-ApaI-TaqI combination insignificantly influence
VDR protein level, although the biological responsiveness to the ligand (1,25(OH)2D3) is
significantly influenced. Leading to the conclusion that different SNPs and SNP combinations
influence the quality of VDR-mediated responses, rather than the levels of VDR protein
(quantity). Adapted from Uitterlinden et al. (2002).

     a. This study was performed as a pilot study to start clarifying the underlying
         mechanism of associations found between VDR polymorphisms and infectious
         diseases (specifically M.tb) in Sub-Saharan Africa. A better understanding of the
         basis of these associations may contribute to the development of new

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   identification and treatment strategies. Similar studies on cells from populations
   of African origin in whom associations were found seem a plausible next step.
b. Future studies might also focus on searching for polymorphisms in nearby
   genes by means of sequencing or genome walking.
c. VDR variants and 1,25(OH)2D3 may influence the Th1/Th2 balance by inhibition
   of Th1 cytokines. Therefore, functional studies based on cytokine analysis using
   immune cells from the T-cell linage might be a valuable future study.
d. Intronic polymorphisms (BsmI and ApaI) possibly influence splicing and can be
   studied by means of RT-PCR, amplifying the regions containing the splice
   junctions. This may shed more light on the influence of VDR polymorphisms,
   located in introns, influencing VDR function.
e. The organization levels at which functionality of VDR polymorphisms can be
   “read out” is diverse (Figure 6.2). In the current study functionality was analyzed
   on cellular level. A number of other “read outs” may be considered in future
   studies. For example, since VDR polymorphism does not seem to influence
   VDR levels, future studies on protein architecture may elucidate the influence of
   VDR genetic variation on VDR ligand binding and protein-protein or protein-DNA

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Figure 6.2 A diagram illustrating the different organizational levels on which
functionality of VDR polymorphisms can be studied. Molecular, cellular and
physiological analysis can be used to “read out” the functional impact of VDR
polymorphism. Cellular analysis quantifying cell growth inhibition was used in the current
study. From Uitterlinden et al. (2002).


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