Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts

Abstract

Purpose: To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family.

Methods: All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model.

Results: Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15) increased after birth to a level that was sustained through the postnatal time points.

Conclusion: A novel missense mutation in LIM2 is responsible for autosomal recessive congenital cataracts.

Fig 1. Pedigree drawing of the family PKCC214 with haplotypes of 6 adjacent chromosome 19q microsatellite markers.

Alleles that constitute the risk haplotype are shaded black and alleles not co-segregating with cataracts are shown in white. Square: male; circle: female; filled symbol: affected individual; double line between symbols: consanguineous mating; diagonal line through symbol: deceased.

Fig 2. Slit-lamp photograph of affected individual 9 of PKCC214.

This photograph depicts a nuclear cataract that developed during infancy.

Fig 3. Identification of the pathogenic missense variation responsible for congenital cataracts in PKCC214.

A) Individual 7 (unaffected; 35 yrs. old) heterozygous carrier and B) individual 11 (affected; 4 yrs. old), homozygous for the c.233G>A mutation. The variation results in a non-conservation substitution in MP19: p.G78D. Arrows point to the allele, c.233G, mutated in PKCC214. C) Sequence alignment of amino acids illustrating conservation of Gly78 among MP19 orthologs. Brown: Primates; green: Euarchontoglires; purple: Laurasiatheria; and orange: Afrotheria.

Fig 4. Investigating the physical characteristics of wild-type and mutant MP19.

Mutant MP19 (p.G78D) exhibited lower hydropathicity (compare A with D), lower hydrophobicity (compare B with E), and higher polarity (compare C with F). The x-axis represents the position of the amino acids while the y-axis represents the hydropathicity, hydrophobicity, and polarity values in a default window size of 9. Arrows point to the difference in their respective hydropathicity (1^st arrow), hydrophobicity (2^nd arrow), and polarity (3^rd arrow).

Fig 5. Missense mutation located in the transmembrane domain of MP19 is responsible for congenital cataracts.

A) LIM2 graphical illustration showing genomic architecture and MP19 protein structure, including the four transmembrane domains. Note: the topology has been obtained from Maher et al., 2012; Exp Eye Res.103:115–6. Molecular interactions of B) wild-type and C) mutant MP19 (harboring Gly78Asp) suggesting interactions of the mutant residue (D78) with the phenylalanine (F10) and cysteine (C74) residues.

Fig 6. Expression profile of lens intrinsic membrane protein 2 (Lim2) in the developing mouse lens.

The expression of Lim2 at different developmental time points was normalized to Gapdh. The x-axis and y-axis represent developmental time points and normalized expression of Lim2 mRNA, respectively.