CYP1B1, MYOC, and LTBP2 mutations in primary congenital glaucoma patients in the United States

Abstract

Purpose: To screen primary congenital glaucoma patients in the United States for sequence variants within the CYP1B1, LTBP2, and MYOC genes using Sanger and whole exome sequencing.

Design: Retrospective case-control study.

Methods: Fifty-seven primary congenital glaucoma patients (47 families), 71 unaffected family members of the primary congenital glaucoma probands, and 101 healthy unrelated individuals were recruited from a single institution. Sanger sequencing of the primary congenital glaucoma gene, CYP1B1, was performed on 47 proband deoxyribonucleic acid samples. Simultaneously, whole exome sequencing was conducted on 3 families, each including more than 1 affected individual. Concurrently, 33 of 47 primary congenital glaucoma probands with extended family deoxyribonucleic acid samples were screened for LTBP2 and MYOC gene mutations. Exome-sequenced variations were validated by additional Sanger sequencing to confirm segregation of filtered disease-causing single nucleotide variations.

Results: Seven primary congenital glaucoma families (14.9%) manifested disease phenotypes attributable to CYP1B1 mutations. One primary congenital glaucoma family possessed homozygous mutant alleles, whereas 6 families carried compound heterozygous mutations. Five novel combinations of compound heterozygous mutations were identified, of which 2 combinations were found with whole exome sequencing. No disease-causing mutations within the LTBP2 and MYOC genes were discovered.

Conclusions: This study analyzed CYP1B1, LTBP2, and MYOC mutations in a cohort of primary congenital glaucoma patients from the United States, applying whole exome sequencing as a complementary tool to Sanger sequencing. Whole exome sequencing, coupled with Sanger sequencing, may identify novel genes in primary congenital glaucoma patients who have no mutations in known primary congenital glaucoma genes.

FIGURE 1

Pedigrees with CYP1B1 sequence variants in primary congenital glaucoma families 32012, 32016, 32021, and 32071 in the United States. Both alleles of the genotypes are listed below each individual. Deoxyribonucleic acid (DNA) sample from individual 1 was sent for whole exome sequencing. Circles indicate females and squares indicate males. Shaded shapes refer to affected individuals. Arrowheads indicate the proband. WT = wild type; + = DNA was collected and analyzed from that individual.

FIGURE 2

Pedigree showing the CYP1B1 sequence variants in primary congenital glaucoma families 32072, 32057, and 32043 in the United States. Both alleles of the genotypes are listed below each individual. Deoxyribonucleic acid (DNA) samples from individuals 2 through 7 were sent for whole exome sequencing. Circles indicate females and squares indicate males. Shaded shapes refer to affected individuals. Arrowheads indicate the proband. WT =wild type; +=DNA was collected and analyzed from that individual.

FIGURE 3

Locations of identified mutations in the CYP1B1 gene from a study on primary congenital glaucoma individuals in the United States. The locations of the 5 amino acid substitutions, a 10-bp insertion, and a 13-bp deletion identified in this study on primary congenital glaucoma individuals in the United States are annotated in the diagram as p.W57*, p.A106D, c.1064_1076delGAGTGCAGGCAGA, p.R355X, p.E387K, p.R390C, and c.1209_1210insTCATGCCACC. Conserved regions are shown in black with the protein structure annotation listed below.