The average L:M cone ratio in 27 males of African descent who have normal color vision was statistically lower than the average ratio for males of Caucasian descent with normal color vision (). The two primary sources of error in estimates of the L:M cone ratio from FP-ERG are contributed by variation in the wavelength of peak absorption (λmax
) of the L pigment and variation in the optical density of the lens (Bieber, Kraft, & Werner, 1998
) The L:M cone ratios for both the Africans and Caucasians were corrected for both of these sources of error. To account for variation in the λmax
of L pigments, the L cone spectrum used to determine the weighted sum of an L and an M cone spectral sensitivity that best fit the FP-ERG derived spectral sensitivity function for each subject was the one predicted for the L pigment encoded by the gene in the first position in the array for each subject. We previously catalogued the L opsin gene sequences and λmax
values for dichromats who had a single X-chromosome opsin gene (Carroll, McMahon, Neitz, & Neitz, 2000
). Fourteen of the subjects in the present study had an L opsin gene that encoded a pigment corresponding to one for which the spectrum had been measured in a dichromat. The remaining 13 subjects differed from catalogued pigments only by polymorphisms that have been previously demonstrated not to influence the λmax
of L pigments. To account for variation in the optical density of the lens, the spectral sensitivity curves used to calculate the L:M cone ratios were determined using an age-specific correction for lens absorption (Pokorny, Smith, & Lutze, 1987
It is possible that there is a correlation between high skin pigmentation and increased lens density (c.f. Said & Weale, 1959
). We considered the possibility that the downward shift in L:M cone ratio in African subjects compared to Caucasian subjects is due to an underestimated lens density for the former. However, increasing the lens density values for a given set of spectral sensitivity data decreases the L:M ratio further. Thus we conclude that the FP-ERG-derived L:M ratio estimates reflect a real difference in the average L:M ratio between males of African versus Caucasian descent.
According to the model proposed by Nathans and colleagues, a difference in the distance between the LCR and M opsin gene promoter in Caucasians versus Africans could account for the dissimilarity in L:M cone ratio, however, molecular genetic results ruled out all of the obvious possibilities. Both Africans and Caucasians have arrays in which an L gene is closest to the LCR, so it is not due to a difference in gene order. The absence of a correlation between L:M cone ratio and the short versus long variant of the L opsin gene rules out the possibility previously suggested by Mollon (1999)
that the DNA insert in the long variant has a measurable affect on L:M cone ratio. An alternative hypothesis proposed by Smallwood et al. (2002)
that promoter sequence polymorphisms can account for cone ratio variation can be eliminated here by the almost complete absence of promoter sequence differences both between and among groups.
In the model illustrated in the DNA is envisioned to bend to allow the LCR to form a complex with the opsin gene promoter (Smallwood, Wang, & Nathans, 2002
). In the absence of other constraints, the relative probability of spatial coincidence between the LCR and a promoter should decrease in proportion to the volume of the sphere, or in proportion to the cube of the distance from the LCR, so that a cone photoreceptor is 1000 times more likely to express the first gene (43
) versus the second gene (403
) making the expected L:M ratio approximately 1000:1 (). There must be constraints on the ability of the LCR to choose to complex with the L versus M gene promoter that are not accounted for in the Nathans model that prevent such a disparate ratio as predicted when only linear distance is considered.
Figure 4 Relative accessibility of the L or M opsin gene promoters to the LCR. (A) The L gene enjoys a huge advantage compared to the M genes if linear distance is the determining factor. The L gene is only 4 kb away from the LCR whereas the distance between the (more ...)
It has long been known that eukaryotic DNA is packaged into complex higher order structures, and there is emerging evidence that both the structure and physical position of DNA within the nucleus play key roles in regulating gene expression (Cai, Han, & Kohwi-Shigematus, 2003
). Actively transcribed regions of chromosomes are organized into chromatin loop domains, and although there is considerable variability in the size of the loops, the average in humans has been estimated to be about 40 kb or about the size of the repeat unit of the L and M opsin genes (Vollrath, Nathans, & Davis, 1988
). The precise organization of the chromatin loop domains for the X-chromosome opsin gene locus remains unknown; however, as illustrated in , packaging into chromatin loops can bring the LCR into nearly equal proximity to the promoters of both the first and second genes in the array account for the observation that the relative numbers of L and M cones is much more nearly equal than might be predicted by the extreme difference in distance between the LCR and the two promoters respectively.
In summary, the three dimensional chromatin organization of the L and M opsin gene locus must certainly play a key role in determining the probability of whether a photoreceptor cell will express an L versus an M pigment gene. The artificial L/M opsin gene array described by Wang et al. (1999)
and Smallwood et al. (2002)
might not mimic the normal chromatin context of the X-chromosome opsin gene locus in humans and would not provide the ideal model of the native locus in experiments aimed at understanding how variation in L:M cone ratio is regulated in humans if chromatin context is important. The challenge for the future will be to characterize the chromatin loop organization of the X-chromosome opsin gene locus, and to determine whether there are sequence differences that influence the L:M cone ratio by altering the chromatin organization.