The X-linked high-grade myopia phenotypes investigated in this study are complex, with characteristics that include features such as amblyopia, optic nerve hypoplasia, cone photoreceptor dysfunction, and red-green or green-red color vision deficiencies. In this work, we evaluated and addressed an aspect of X-linked high-grade myopia at the genomic level by performing screening studies on a potential candidate gene.
Segmental duplications have been implicated in disorders that result from inappropriate gene dosage.
13 14 Segmental duplications can mediate cytogenetic alterations such as deletions, duplications, translocations, and inversions, which can give rise to an altered number of copies at the genomic level leading to altered dosage.
15 Examples of genetic disorders mediated by altered gene dosage include Charcot-Marie-Tooth disease type 1A (CMT1A),
16 17 Smith-Magenis syndrome,
18 neurofibromatosis type 1,
19 X-linked ichthyosis,
20 and hemophilia A.
21 Complex disorders such as Parkinson disease,
22 early-onset Alzheimer disease,
23 and hereditary pancreatitis
24 have also been associated with CNV.
In recent times, CNV has been speculatively involved in susceptibility to and etiology of various conditions, including complex multifactorial traits.
25 26 The results obtained in our study are in agreement with this line of thought. The array-CGH data revealed CNVs in DNA samples of affected male subjects of the BED and Minnesota pedigrees in the Xq28 region involving the opsin gene array. Both pedigrees showed predicted duplications in this region, which includes opsin cone pigment and TEX28 genes. CNVs in this region can occur because of the close homology of the genes, making them susceptible to unequal crossing over during meiosis. The real-time assay targeted on the opsin genes confirmed previous predicted findings of opsin gene numbers in the Minnesota pedigree.
4 Only three copies of TEX28 have been reported within the opsin gene array,
9 11 though the real-time assay targeted on the TEX28 gene indicated that the affected male subjects had up to four copies of TEX28 in the BED and five copies in the Minnesota phenotypes in contrast to the 3 copies in the unaffected males. Two families from the United Kingdom showed either fewer (one) or greater (four) number of copies in the affected samples when compared with the unaffected samples.
Triplication of the α-synuclein locus (OMIM 163890) is causal for Parkinson disease,
22 and duplication of the amyloid beta A4 precursor protein locus (OMIM 104760) has been reported to cause early-onset Alzheimer disease and cerebral amyloid angiopathy,
23 all of which are considered complex diseases. Similarly, in CMT1A (OMIM 601097), duplication involving the peripheral myelin protein 22 (
PMP22) gene on chromosome 17p12 has been reported, and it is speculated that the decreased nerve conduction velocities observed are a consequence of gene dosage effect.
17 We speculate that the CNVs we observed in the opsin gene array region may play a role in the X-linked high-grade myopia phenotypes evaluated. The relative higher/lower number of opsin genes in affected male subject DNA may not play a role in color vision deficiencies because the red-green and green-red hybrid genes reported previously
4 explain the color vision associations in the tested Minnesota and BED pedigrees. Moreover, it is known that only the first two opsin genes are expressed in the retina.
27 The relative change in the number of TEX28 copies could be involved in phenotypes with opsin gene arrays containing red-green/green-red hybrid genes such as the ones investigated in this study. No substantive evidence demonstrates which TEX28 copies are functional because the entire protein coding sequence of TEX28 exists in all the copies. Hence, the exact manner by which TEX28 gene regulation might play a role is unknown and requires further investigation. It is also likely that the TEX28 CNVs observed are an indication of a true causal variation in linkage disequilibrium (LD) with these CNVs. The fewer copies observed in United Kingdom Family 2 and Family 3 prompted us to ask whether their phenotypes were different from those in BED, Minnesota, and United Kingdom Family 1. All five families investigated had myopia, cone photoreceptor dysfunction, and red-green or green-red color vision deficiencies as clinical features. To the best of our knowledge, United Kingdom Family 2 and Family 3 do not have any unique features. It is possible that TEX28 variations may not explain all the phenotypes of X-linked myopia examined. The inconsistent genotype-phenotype correlation observed in Family 2 and Family 3 may also suggest the involvement of a variant in LD with TEX28 CNVs.
There were differences in the TEX28 copy gain or loss among the Minnesota, BED, and United Kingdom pedigrees. If one assumes that TEX28 is a nested gene and always follows an opsin gene within the array, the findings in the subject DNA of affected males in the Minnesota pedigree point otherwise (five TEX28 copies, four opsins), indicating an extra copy gain of TEX28 in relation to the opsin copy gain. Similarly, the affected males in the United Kingdom pedigrees also showed an unequal loss/gain in TEX28 copies. It was not possible to determine the precise location of this extra gain/loss of TEX28 because of the high sequence similarity.
Additionally, the subject DNA of affected males in the Minnesota pedigree showed an intergenic deletion and duplication downstream from the opsin gene array in the Xq28 region. The deletion was confirmed by a nested PCR approach in the affected males in the Minnesota pedigree. The deletion, however, was not further characterized at its borders because it was exclusive to one family and, hence, may not be a common element that explains the high-grade myopia in these pedigrees. Intergenic deletions are associated with disease and have been proposed to play a role in the regulation of transcription of nearby genes by
cis regulatory action or position effect.
28 However, the potential regulatory effect of this deletion on flanking genes BRCA1-BRCA2–containing complex 3 (BRCC3; 42 kb upstream) and von Hippel-Lindau binding protein 1 (VBP1; 22 kb downstream) requires further investigation. The role of intergenic duplications is unknown, though it is recognized that segmental duplications cause genomic instability.
15 These intergenic alterations were not observed in affected (or unaffected) DNA samples of the BED and the United Kingdom phenotypes (array-CGH and PCR data, respectively).
In addition to CNV, we examined whether TEX28 showed any sequence variants. Mutation screening revealed a novel SNP in the 5′ UTR (nucleotide position 8′ upstream of the initiation codon) of the TEX28 gene that segregated with the affection status in the Minnesota pedigree and one United Kingdom pedigree. However, follow-up study of this SNP in a larger dataset using an allelic discrimination assay (TaqMan; Invitrogen) revealed no significant association (P = 0.6) between the frequencies of the SNP in affected versus unaffected males, thus ruling out any role for this SNP. Several candidate genes on the Xq28 locus were also sequence screened in parallel based on their function and expression profiles and cytogenic location, and no sequence variants were associated with the disease status. The genes that were screened include CTAG1, CTAG2, MPP1, CLIC2, H2AFB3, TMLHE, SPRY3, SYBL1, NT_025307.28, NT_025307.29, AFF2, CXORF1, FMR1, IDS, MECP2, MTM1, RENBP, GABRA3, GDI1, MAGEA10, NSDHL, SLITRK2, SLITRK4, TKTL1, and ZNF275.
Few studies on TEX28 have been published,
9 10 11 and the expression studies we performed revealed that TEX28 is expressed in blood, kidney, and testis at the mRNA level. Although the expression in testis is consistent with previous findings, Chen et al.
29 did not report expression in kidney and did not test for expression in blood. Ocular gene expression experiments conducted in the present study revealed the presence of TEX28 in five ocular tissues, including the retina and the sclera. Interestingly, in a recent study based on ocular-expression profiles using serial analysis of gene expression, TEX28 was reported to be expressed in the human retina.
30 Involvement of the retina through a putative retinoscleral signaling cascade has been proposed to play a role in ocular elongation and myopia development.
31 Consequently, the potential regulatory effects of TEX28 CNV in these layers could be involved in the phenotypes studied.
In summary, our study suggests that array-CGH and real-time PCR are reliable techniques to investigate and validate CNVs. The X-linked high-grade myopia phenotypes with color vision deficiencies studied are associated with TEX28 CNVs. The role of CNVs in genetic disease is a novel area of research and may explain some forms of X-linked myopia.