Novel CYP1B1 mutations in consanguineous Pakistani families with primary congenital glaucoma

Abstract

Purpose: To identify the disease-causing mutations in three consanguineous Pakistani families with multiple members affected by primary congenital glaucoma.

Methods: Blood samples were collected, and DNA was extracted. Linkage analysis for reported primary congenital glaucoma loci was performed using closely spaced polymorphic microsatellite markers on genomic DNA from affected and unaffected family members. All coding exons, the exon-intron boundaries, and the 5' untranslated region of CYP1B1 were sequenced.

Results: The alleles of chromosome 2p markers segregate with the disease phenotype in all three families with positive LOD scores. The sequencing results identified three novel mutations (L177R, L487P, and D374E) and one previously reported mutation (E229K) in CYP1B1 that segregate with the disease phenotype in their respective families. None of these sequence variations were present in 96 ethnically matched control samples.

Conclusions: These results strongly suggest that missense mutations in CYP1B1 are most likely to be responsible for primary congenital glaucoma in these families.

Figure 1

Family pedigrees. Pedigrees of A) PKGL021, B) PKGL022 and C) PKGL026. Squares denote males while circles denote females. Filled symbols indicate affected individuals. A double line between individuals signifies consanguinity, and a diagonal line through a symbol indicates a deceased family member. The haplotypes of six adjacent chromosome 2p21 microsatellite markers are shown with alleles forming the risk haplotype shaded black, alleles cosegregating with PCG but not showing homozygosity shaded gray, and alleles not cosegregating with PCG shown in white.

Figure 2

Sequence chromatograms. The forward and reverse sequence chromatograms of (A) unaffected individual 9 of PKGL021, (B) individual 5 of PKGL021, heterozygous and (C) individual 7 of PKGL021, homozygous for a C→G transversion in exon 3, c.1122C>G, resulting in a; p.D374E, (D) individual 8 of PKGL022, heterozygous and (E) individual 10 of PKGL022, homozygous for G→A transition in exon2, c.685G>A, resulting in mutation p.E229K, (F) individual 8 of PKGL022, heterozygous and (G) individual 10 of PKGL022, homozygous for a T→C transition in exon 3: c.1460T>C resulting in a; p.L487P (H) unaffected individual 21 of PKGL026, (I) individual 7 of PKGL026, heterozygous, and (J) individual 16 of PKGL026, homozygous for a T→G transversion in exon 2: c.530T>G: resulting in p. L177R.

Figure 3

Sequence alignment of amino acids of CYP1B1 among higher primate species. The alignment of CYP1B1 among higher primate species shows the conservation of Asp374 (green), Leu487 (blue), and Leu177 (red).