A Novel MIP Gene Mutation Analysis in a Chinese Family Affected with Congenital Progressive Punctate Cataract

Congenital cataracts are one of the leading causes of visual impairment and blindness in children, and genetic factors play an important role in their development. This study aimed to identify the genetic defects associated with autosomal dominant congenital progressive punctate cataracts in a Chinese family and to explore the potential pathogenesis. Detailed family history and clinical data were recorded, and all the family members’ blood samples were collected for DNA extraction. Linkage analysis was performed by microsatellite markers that are associated with punctate cataracts, and logarithm (base 10) of odds (LOD) scores were calculated using the LINKAGE program. Positive two-point LOD scores were obtained at markers D12S1622 (Zmax = 2.71 at θ = 0.0), D12S1724 (Zmax = 2.71 at θ = 0.0), and D12S90 (Zmax = 2.71 at θ = 0.0), which flank the major intrinsic protein of lens fiber (MIP) gene on chromosomal region 12q13. Direct sequencing of the encoding region of the MIP gene revealed a novel mutation (G>D) in exon 4 at nucleotide 644, which caused a substitution of glycine to aspartic acid at codon 215 (p.G215D) for the MIP protein. The mutation cosegregated with all patients with congenital progressive punctate cataracts, but it was absent in the healthy members. Bioinformatics analysis predicted that the mutation affects the function of the MIP protein. The wild type (WT) and G215D mutant of MIP were transfected with green fluorescent protein (GFP) into Hela cells separately, and it was found that the G215D mutant was aberrantly located in the cytoplasm instead of in the plasma membrane. In summary, our study presented genetic and functional evidence linking the new MIP mutation of G215D to autosomal dominant congenital cataracts, which adds to the list of MIP mutations linked to congenital progressive punctate cataracts.

MIP is expressed in the ocular lens, contributes to over 50% of the total membrane proteins in the lens fiber cell, and plays a role in maintaining lens transparency [24]. Mutations in the MIP gene have been linked to hereditary cataracts in humans and knockout mouse models [25][26][27] [25]).
In the present study, we identified that the p.G215D mutation of MIP was linked to congenital progressive punctate cataracts in a three-generation Chinese family (Figures 1 and 2). All family members with the G215D mutant of MIP have punctate cataracts, and the opacity degree of the lens increases with increasing age. Therefore, the aim of this study was to clone and express the G215D mutant of MIP and compare its properties with those of the normal or wild-type (WT) MIP.

Ethics Statement
After explaining the nature and possible consequences of the study, written informed consents were obtained from subjects including the guardians on behalf of the minors enrolled. All experiments were approved by the Institutional Review Board of Harbin Medical University (Harbin, China) and were conducted according to the principles in the Declaration of Helsinki.

Patient ascertainment and genomic DNA preparation
A three-generation Chinese Han family with congenital progressive punctate cataracts from Shenzhen province was identified as shown in Figure 1. All members of the pedigree were given complete ophthalmologic examinations and did not have any other eye disease. One hundred healthy persons were recruited as the normal controls. After informed consent, 5 ml of venous blood from each subject was collected in a BD Vacutainer (BD, San Jose, CA) containing EDTA. Genomic DNA was extracted by QIAamp DNA Blood Mini Kits (QIAGEN Science, Germantown, MD).

Genotyping and linkage analysis
Fluorescently labeled microsatellite markers, which have been reported to be associated with punctate cataracts, were used for the linkage analysis. Two-point linkage analysis was performed with MLINK from the LINKAGE program package. The disease locus was specified to be an autosomal dominant trait, with a disease allele frequency of 0.0001. The allele frequencies of the markers are considered to be equally distributed.

Mutation screening
The coding exons and the flanking regions of the candidate gene were amplified by polymerase chain reaction (PCR) with the primers listed in Table 1. The sequencing was carried out with an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA), and the results were analyzed using Chromas (version 1.0) software.

Bioinformatics analysis
The WT and mutant MIP protein sequences were analyzed with the PolyPhen tool to predict whether the amino acid substitution affects the structure and function of the protein. The amino acid sequences of MIP from several different species were obtained from NCBI GenBank, and conservation analysis was performed by CLC Main Workbench Software (Aarhus, Denmark). The hydrophobic properties of the mutant and WT amino acid sequences were analyzed by Misc Protein Analysis software, respectively.

Plasmid construction
The expression vector for G215D mutant of MIP was constructed by using site-directed mutagenesis with the following primers: sense primer 59-GGAGGGGGTCTGGA-CAGCCTCCTGTAC-39; antisense primer 59-GTACAG-GAGGCTGTCCAGACCCCCTCC-39. Mutation was confirmed through DNA sequencing. pGFP-WT and pGFP-G215D resultant plasmids were constructed to create GFP-MIP fusion proteins, the open reading frames of WT and G215D mutant of MIP were amplified by PCR with the following primers: sense primer 59-AGT ACC TCG AGA TGT GGG AAC TGC GAT CAG C-39; antisense primer 59-CTA CGA AGC TTC AGG GCC TGG GTG TTC AGT T-39. Products were then cloned into Xho I and Hind III digested pGFP-N1 vector.

Expression and subcellular localization
Hela cells were plated in 6-well plates 24 h prior to transfection at approximately 60% confluency. According to the manufacturer's protocols, GFP-MIP expression constructs containing WT and G215D mutant were transfected using Lipofectamine 2000, separately. The empty vector pGFP-N1 was transfected as a control. 48 hours after transfection, the cells were analyzed by fluorescence microscopy.

Western blot analysis
After transfected with MIP-WT or MIP-G215D plasmids separately, HEK 293t cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, 1% NP40, 150 mM NaCl, 1 mM EDTA and 1 mM PMSF) for 30 min at 4 uC. Total proteins were extracted and separated by 12% SDS/PAGE gels, and then transferred to PVDF membranes, separately. The membranes were incubated with antibodies of anti-b-actin (Cell Signaling Technology, Inc, USA) and anti-GFP (Abcam, Inc, Cambridge,
Direct sequencing revealed a heterozygous change, G.A, at position 644 (c.644G.A) of the MIP gene, which leads to the substitution of a glycine in the WT to an aspartic acid at codon 215 (p.G215D). Glycine is highly conserved across various species, as determined from the NCBI database. This mutation cosegregated well with all affected individuals and was not found in  unaffected family members or in the 100 unrelated normal controls (Figure 4). PolyPhen analysis demonstrated that the substitution at position 215 of MIP from glycine to aspartic acid scored 0.811, which meant that this mutation is predicted to be ''possibly damaging''. In comparison with the WT-MIP protein, it is obvious that the mutant has a lower hydrophobicity in this region ( Figure 5).
We tested the subcellular location of WT-MIP and G215D-MIP after transient transfection of Hela cells by fluorescence microscopy. As shown in Figure 6, the empty vector pGFP-N1 was located in both the nucleus and cytoplasm, while the expressed WT-MIP was located in the plasma membrane. In contrast, G215D-MIP was aberrantly located in the cytoplasm, indicating that the G215D mutation prevented its localization in the plasma membrane, accurately.
Western blot analysis revealed that WT-MIP and G215D-MIP had similar protein levels in western blot analysis of the cell lysates (Figure 7), indicating that mutation did not result in the expression or instability of the protein.

Discussion
In the present study, a novel mutation of the MIP gene was identified in exon 4 at nucleotide 644, which caused a substitution of glycine to aspartic acid at codon 215 (p.G215D) in a Chinese family with congenital progressive punctate cataracts. The G215D MIP mutant was found to cosegregate with the disease phenotype in the family and was not present in unaffected family members or in 100 normal controls.
The MIP gene, also known as the MIP26 or AQP0 gene, encodes a 263-amino acid peptide. AQP0 is abundant and  contributes to over 50% of total membrane fiber proteins [37]. As water pores confer rapid movements of water across plasma membranes, AQP0 is considered to be essential for the lens microcirculation system and the maintenance of lens transparency [38,39]. It has been widely reported that mutations of MIP in humans and mice are involved in the induction of cataracts [40][41][42][43]. As aquaporin, MIP has a unique structure with six transmembrane bilayerspanning domains (H1-H6), three extracellular loops (A, C, and E), two intracellular loops (B and D), and the NH2-and COOH-terminal intracellular domains. To the best of our knowledge, this is the third mutation of the MIP gene in transmembrane bilayer-spanning domains (H6) and the first substitution mutation in H6 that can cause autosomal dominant congenital cataracts. The first mutation in H6 of MIP (C.638delG) was reported to be associated with polymorphic cataracts in 2006 [36], and the second mutation of MIP (IVS-1G.A) was found to be associated with nuclear ''snail-like'' cataracts in 2009 [35]. The functional consequences of the 638G deletion have been identified, showing that the mutant protein was located in the  endoplasmic reticulum and induced cellular cytotoxicity [36]. Similarly, Prior studies of mutant MIP proteins have shown a loss of water permeability function and impaired trafficking in expression systems [43,44]. Our findings were consistent with the results of Senthil Kumar et al [43], who identified the G165D MIP mutation and confirmed that this mutant was involved in congenital lamellar cataracts in a South Indian family. In addition, the expression of MIP and G165D-MIP in Xenopus oocytes and Hela cells suggested that substitution of the conserved glycine residue leads to improper trafficking of AQP0 and destruction of water channel function.
In summary, the current study presented genetic and functional evidence linking a new mutation of MIP (G215D) to autosomal dominant congenital cataracts. Our results showed that G215D-MIP had a lower hydrophobicity and was aberrantly located in the cytoplasm of Hela cells, while WT-MIP was mainly detected in the plasma membrane. We speculate that G215D-MIP would be unable to exert channel functions. Since AQP0 has several functions and plays an adhesive molecule role in the lens, it is likely that, besides channel functions, G215D-MIP would be unable to exert many other functions. Further studies, such as research on protein aggregation in solution and animal models, are needed in order to provide insights into the molecular consequence of this mutation.