Mutations in FYCO1 cause autosomal-recessive congenital cataracts

Abstract

Congenital cataracts (CCs), responsible for about one-third of blindness in infants, are a major cause of vision loss in children worldwide. Autosomal-recessive congenital cataracts (arCC) form a clinically diverse and genetically heterogeneous group of disorders of the crystalline lens. To identify the genetic cause of arCC in consanguineous Pakistani families, we performed genome-wide linkage analysis and fine mapping and identified linkage to 3p21-p22 with a summed LOD score of 33.42. Mutations in the gene encoding FYVE and coiled-coil domain containing 1 (FYCO1), a PI(3)P-binding protein family member that is associated with the exterior of autophagosomes and mediates microtubule plus-end-directed vesicle transport, were identified in 12 Pakistani families and one Arab Israeli family in which arCC had previously been mapped to the overlapping CATC2 region. Nine different mutations were identified, including c.3755 delC (p.Ala1252AspfsX71), c.3858_3862dupGGAAT (p.Leu1288TrpfsX37), c.1045 C>T (p.Gln349X), c.2206C>T (p.Gln736X), c.2761C>T (p.Arg921X), c.2830C>T (p.Arg944X), c.3150+1 G>T, c.4127T>C (p.Leu1376Pro), and c.1546C>T (p.Gln516X). Fyco1 is expressed in the mouse embryonic and adult lens and peaks at P12d. Expressed mutant proteins p.Leu1288TrpfsX37 and p.Gln736X are truncated on immunoblots. Wild-type and p.L1376P FYCO1, the only missense mutant identified, migrate at the expected molecular mass. Both wild-type and p. Leu1376Pro FYCO1 proteins expressed in human lens epithelial cells partially colocalize to microtubules and are found adjacent to Golgi, but they primarily colocalize to autophagosomes. Thus, FYCO1 is involved in lens development and transparency in humans, and mutations in this gene are one of the most common causes of arCC in the Pakistani population.

Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Slit-Lamp Photograph of a Cataract Patient with FYCO1 Mutations A slit-lamp photograph of affected individual 19 of family 060069 shows a nuclear cataract that developed in early infancy.

Figure 2

Pedigrees and Linkage Intervals for arCC Families (A) Twelve arCC pedigrees collected from Pakistan. Filled symbols denote affected individuals. Eight pedigrees (060003, 060012, 060041, 060058, 060064, 060069, 060091, and 060094) were used for genome-wide linkage scans and fine mapping of arCC intervals and candidate-gene mutation screenings. Four pedigrees (060014, 060031, 060044, and 060054) were used for fine mapping of arCC intervals and candidate-gene mutation screenings. Individuals who were genotyped are marked with an asterisk. (B) Refined arCC interval on the basis of haplotype analysis of patients with recombination events. Markers with the homozygous genotype are boxed so that the region without recombination is defined. Alleles for markers D3S3685 and D3S1289 in patients with recombination events are in bold so that the telomeric and centromeric breakpoints, respectively, are shown. The disease interval was placed between markers D3S3685 and D3S1289.

Figure 3

Sequence Electropherograms of the Eight Homozygous Mutations Found in FYCO1 and Associated with arCC Wild-type sequence from normal control individuals is shown in the top panel for comparison. The c.1045C>T (p.Gln349X), c.1546C>T (p.Gln516X)), c.2206C>T (p.Gln736X), c.2761C>T (p. Arg921X), and c.2830C>T (p. Arg944X) mutations result in the generation of a stop codon; the c.3755 delC (Ala1252AspfsX71) and c.3858_3862dupGGAAT (p.Leu1288TrpfsX37) mutations lead to a frameshift and a premature termination of translation; the c. 3150+1 G>T mutation leads to elimination of the intron 9 donor site; and the c.4127T>C (p.Leu1376Pro) mutation results in the change of a leucine residue to a proline residue in exon 16.

Figure 4

FYCO1 Domain Structure and Mutations (A) Schematic diagram of FYCO1, showing the locations and effects of the recessive mutations. Domain structure of FYCO1. FYVE represents the FYVE zinc-finger domain; LIR represents the LC3-interacting region; and GOLD represents the Golgi dynamics domain. Red arrows show the positions of the truncation mutations associated with arCC, and the blue arrow shows the missense mutation p.Leu1376Pro. (B) Amino acid sequence alignment around the FYCO1 Leu1376 amino acid (asterisk) in eight species, ranging from humans to zebrafish.

Figure 5

FYCO1 Expression in the Lens and Human Lens Cell Culture (A) RT-PCR amplification of Fyco1 mRNA from P3W mouse eye lens. RT (+) and RT (–) denote controls with or without reverse transcription, respectively. Lane 1, full-length Fyco1 transcript; lane 2, negative control for Fyco1 transcript; lane 3, Gapdh transcript; lane 4, negative control for Gapdh transcript. MWM stands for molecular-weight marker. (B) Relative expression of Fyco1 in mouse eye lens tissues at various ages. Expression of Fyco1 was measured in lens tissues by qRT-PCR at different time points during aging. Data represent the mean (±SD) on an arbitrary scale (y axis) representing expression relative to the housekeeping gene Gapdh. (C) Characterization of mutant and wild-type GFP-FYCO1 by immunoblot analysis. Lane 1, transfected wild-type FYCO1-GFP lysate; lane 2, lysate transfected with mutant FYCO1-GFP (p.Leu1376Pro); lane 3, lysate transfected with mutant FYCO1-GFP (p.Leu1288TrpfsX37); lane 4, lysate transfected with mutant FYCO1-GFP (p.Gln736X); lane 5, untransfected lysate. “M” indicates molecular-weight positions of the SeeBlue2 Plus molecular-weight marker. Wild-type proteins migrate at the predicted molecular weight of 197 kDa. Missense mutant proteins migrate at the predicted molecular weight of 197 kDa. Duplication mutant proteins migrate at the predicted molecular weight of 165 kDa. Nonsense mutant proteins migrate at the predicted molecular weight of 105 kDa.

Figure 6

Immunofluorescence Images Showing Localization of Mutant and Wild-Type GFP-FYCO1 Proteins in Human Lens Epithelial Cells Human lens epithelial cells were transfected with pCMV6-AC-FYCO1-GFP in which wild-type (B, D, F, and H) or p.Leu1376Pro mutant FYCO1 (A, C, E, and G) is fused in frame with GFP. Cells were immunostained with organelle-specific antibodies or dyes, including anti-tubulin (red, microtubules) (A and B), anti-golgin 97 (red, Golgi) (C and D), anti-LC3 (red, autophagosomes) (E and F), and LysoTracker Red DND-99 dye (red, endosomes and lysosomes) (G and H). All cells were stained with the nuclear marker DAPI (4′, 6-diamidino-2-phenylindole, blue, nucleus). R + G represents overlay of red and green channels. R + G + B depicts red, green, and blue (DAPI) channels. Scale bars represent 20 μm. Both wild-type (WT) and mutant (Mut) proteins partially colocalize adjacent to Golgi, autophagosomes, endosomes, and lysosomes of the cytoplasm of human lens epithelial cells. Wild-type Xpress-tagged FYCO1 shows similar localization (I).