Identification and Functional Analysis of a Novel MIP Gene Mutation Associated with Congenital Cataract in a Chinese Family.

Congenital cataracts are major cause of visual impairment and blindness in children and previous studies have shown about 1/3 of non-syndromic congenital cataracts are inherited. Major intrinsic protein of the lens (MIP), also known as AQP0, plays a critical role in transparency and development of the lens. To date, more than 10 mutations in MIP have been linked to hereditary cataracts in humans. In this study, we investigated the genetic and functional defects underlying a four-generation Chinese family affected with congenital progressive cortical punctate cataract. Mutation screening of the candidate genes revealed a missense mutation at position 448 (c.448G>C) of MIP, which resulted in the substitution of a conserved aspartic acid with histidine at codon 150 (p.D150H). By linkage and haplotype analysis, we obtained positive multipoint logarithm of odds (LOD) scores at microsatellite markers D12S1632 (Zmax = 1.804 at α = 1.000) and D12S1691 (Zmax = 1.806 at α = 1.000), which flanked the candidate locus. The prediction results of PolyPhen-2 and SIFT indicated that the p.D150H mutation was likely to damage to the structure and function of AQP0. The wild type and p.D150H mutant AQP0 were expressed in HeLa cells separately and the immunofluorescence results showed that the WT-AQP0 distributed at the plasma membrane and in cytoplasm, while AQP0-D150H failed to reach the plasma membrane and was mainly retained in the Golgi apparatus. Moreover, protein levels of AQP0-D150H were significantly lower than those of wide type AQP0 in membrane-enriched lysates when the HEK-293T cells were transfected with the same amount of wild type and mutant plasmids individually. Taken together, our data suggest the p.D150H mutation is a novel disease-causing mutation in MIP, which leads to congenital progressive cortical punctate cataract by impairing the trafficking mechanism of AQP0

Family pedigree.

<p>A four-generation Chinese family affected with autosomal dominant cataract is shown. Squares and circles indicate males and females, respectively. The black symbols represent the affected members and open symbols represent the unaffected individuals. The diagonal line indicates a deceased family member and the black arrow indicates the proband. The family members attending this study are marked with asterisks.</p

Mutation screening.

<p>Forward sequence analysis of exon 2 of MIP in the normal and affected members of this family. The DNA sequence chromatogram shows a heterozygous G>C nucleotide change (black arrow) in exon 2 of MIP (c.448G>C), which leads to the replacement of aspartic acid (GAC) with histidine (CAC) at codon 150 (p.D150H).</p

Clinical features of the proband (A, B) and her son (C, D).

<p>Slit-lamp photographs (diffuse illumination and silt lamp) show the phenotype of the congenital cataract is punctate cortical opacities. In younger affected one, the punctate opacities were only seen in the peripheral cortex of the lens. The punctate opacities became denser and invaded into central cortex of the lens gradually with increasing age.</p

Haplotype of the cataractous family.

<p>Eight locus around <i>MIP</i> were genotyped. The disease-susceptibility haplotype (indicated by a vertical box) showed cosegregation with affected members in this family from <i>D12S1632</i> to <i>D12S1691</i>.</p

Bioinformatics analysis of the p.D150H mutation and multiple sequence alignment.

<p>(A) The mutation was predicted to be probably damaging with a score of 1.00 by PolyPhen-2, which meant it may have deleterious effect on the structure and function of the protein. (B) the score and media information content from SIFT was 0.00 and 2.63 respectively, predicting the effect of this amino acid substitution on protein function was damaging. (C) The result of a multiple sequence alignment from various species showed that the aspartic acid at position 150 of AQP0 is highly conserved (marked in green, the mutation in red).</p

Subcellular location of WT-AQP0 and AQP0-D150H in two expressing cells.

<p>(A) Representative fluorescence microscopy images show the distributions of immunoreactive AQP0 and a Golgi apparatus resident protein (GM130) in HeLa cells which were transiently transfected with wild type AQP0 or AQP0-D150H. The wild type AQP0 was detected mainly at the plasma membrane (white arrow) and in cytoplasm. By contrast, AQP0-D150H was not observed at the plasma membrane other than cytoplasmic sites which extensively overlapped with that of GM130. Scale bar = 10μm. (B) The quantities of wild type and p.D150H mutant AQP0 in membrane-enriched lysates of HEK-293T cells were assessed by western blotting, after WT-AQP0 or AQP0-D150H transfected, GAPDH was used as control.</p

Molecular analysis and phenotypic study in 14 Chinese families with Bietti crystalline dystrophy.

To investigate the clinical features and cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene mutations in 14 Chinese families with Bietti crystalline dystrophy (BCD).Seventeen patients from 14 unrelated Chinese families with BCD were recruited for complete clinical ophthalmic examination and genetic study. The 11 exons of CYP4V2 were amplified from genomic DNA of all patients and their family members by polymerase chain reaction (PCR) and then sequenced. Exons of TIMP3 were also sequenced in BCD patient associated with choroidal neovascularization (CNV). One hundred and seventy unrelated healthy Chinese subjects were screened for mutations in CYP4V2.All 17 patients with BCD had mutations in CYP4V2; one of these mutations was novel (c.219T>A, p.F73L) and four other mutations had been reported. The p.F73L mutation was a commonly detected mutation in our study (seven out of 34 alleles), either in the homozygous state or in the heterozygous state. Among the patients, considerable phenotypic variability was detected, both within and between families. Screening of TIMP3 did not find any mutation in the BCD patient associated with CNV.The novel CYP4V2 c.219T>A (p.F73L) mutation may be another recurrent mutation in Chinese patients with BCD. Our study expands the mutation spectrum of CYP4V2 and characterizes novel genotype-phenotype associations in Chinese patients with BCD

Pedigree of the three families in this study with novel <i>CYP4V2</i> mutation c.219T>A (p.F73L).

<p>(A) Pedigree of family 7 (F7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094960#pone-0094960-t001" target="_blank">Table 1</a>). (B) Pedigree of family 10 (F10 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094960#pone-0094960-t001" target="_blank">Table 1</a>). (C) Pedigree of family 12 (F12 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094960#pone-0094960-t001" target="_blank">Table 1</a>). Squares, males; circles, females; filled squares or circles, affected family members; slash, deceased family member; arrow, proband; asterisk, a study participant from whom a blood sample was obtained.</p

Fundus manifestations of three probands with the novel <i>CYP4V2</i> mutation (p.F73L) identified in this study.

<p>(A) Right eye of patient P7. (B) Right eye of patient P10. (C) Left eye of patient P12.1.</p