Dissecting the genetics of human high myopia: a molecular biologic approach

Abstract

Purpose: Despite the plethora of experimental myopia animal studies that demonstrate biochemical factor changes in various eye tissues, and limited human studies utilizing pharmacologic agents to thwart axial elongation, we have little knowledge of the basic physiology that drives myopic development. Identifying the implicated genes for myopia susceptibility will provide a fundamental molecular understanding of how myopia occurs and may lead to directed physiologic (ie, pharmacologic, gene therapy) interventions. The purpose of this proposal is to describe the results of positional candidate gene screening of selected genes within the autosomal dominant high-grade myopia-2 locus (MYP2) on chromosome 18p11.31.

Methods: A physical map of a contracted MYP2 interval was compiled, and gene expression studies in ocular tissues using complementary DNA library screens, microarray matches, and reverse-transcription techniques aided in prioritizing gene selection for screening. The TGIF, EMLIN-2, MLCB, and CLUL1 genes were screened in DNA samples from unrelated controls and in high-myopia affected and unaffected family members from the original seven MYP2 pedigrees. All candidate genes were screened by direct base pair sequence analysis.

Results: Consistent segregation of a gene sequence alteration (polymorphism) with myopia was not demonstrated in any of the seven families. Novel single nucleotide polymorphisms were found.

Conclusion: The positional candidate genes TGIF, EMLIN-2, MLCB, and CLUL1 are not associated with MYP2-linked high-grade myopia. Base change polymorphisms discovered with base sequence screening of these genes were submitted to an Internet database. Other genes that also map within the interval are currently undergoing mutation screening.

Figure 1

Physical map of the chromosome 18p11.31 critical region. The horizontal scale in megabases (M) is at the top. Polymorphic microsatellite DNA markers are labeled above the scale in red. Below the scale, finished (phase 3) bacterial artificial clones (BAC) clones are labeled in blue, working draft (phase 2 or 1) BAC clones are in black, known genes are in dark blue, insilico predicted genes by the public databases GENSCAN (http://genes.mit.edu/GENSCAN.htm) and OTTO* (http://cds.celera.com/biolib/info) are in light blue and green, respectively. The gene myomesin 1 (MYOM1) has been mapped to two different positions. In the NCBI database, MYOM1 maps distally with overlap just outside of the critical region. The Celera assembly (*) shows that MYOM1 spans the gap between BAC clones AP001024 and AP002471 in a more centromeric position.

Figure 2

Polymerase chain reaction amplicons of MYP2 candidate gene complementary DNA (cDNA) from reverse-transcribed RNA from human ocular tissues and commercially available RNA from various human tissue types (Ambion). 1-sclera, 2-cornea, 3-optic nerve, 4-retina, 5-lung, 6-skeletal muscle, 7-heart, 8-trachea, 9-kidney, 10-brain. Expected amplicon size based on primer selection encompassing exonic sequence is shown. Note that TGIF transcripts show two variant spliced isoforms. The commercial DNA ladder has standard-sized DNA molecular weights spaced at 100 base pairs apart and is used to determine the approximate molecular weights of the test amplicons.

Figure 3

Pedigree and haplotype of family with significant linkage to a 7.71-cM interval on chromosome 17q21-q22 after a genome screen.

Figure 4

Flow chart of standard methods for candidate gene mutation identification and functional verification.

Figure 5

Genomic structure of the EMLIN-2 gene. The exons are in boxes, intronic sequences are shown as horizontal connecting lines. Base pair size is provided as adjoining numbers. The arrows indicate primer locations.

Figure 6

Genomic structure of the 11-exon CLUL1 gene.

Figure 7

Genomic structure of the 4-exon MLCB gene.

Figure 8

Genomic structure of the 10-exon TGIF gene. The associated ~47.6kb region of NT_010859 on chromosome 18p11.31 of TGIF, showing 10 exons with alternative start sites and splicing that generates eight transcript variants. The exons are represented as boxes, initiation codons are represented by a vertical line with arrow, and stop codons are represented by a vertical line with a black square.