|Molecular Vision 2007;
Received 6 June 2007 | Accepted 10 September 2007 | Published 11 September 2007
A novel mutation in major intrinsic protein of the lens gene (MIP) underlies autosomal dominant cataract in a Chinese family
Hong Zhai,3 Dan Li,1,2 Luxin
Zhao,3 Chao li,4
1Department of Genetics, National Research Institute for Family Planning; 2Peking Union Medical College, Beijing, China; 3Ophthalmology Department, Central Hospital of Zibo, Shandong, China; 4Third Affiliated Hospital, Sun Yat-Sen University; 5Department of Medical Genetics, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; 6WHO collaborating Center for Community Control of Hereditary Diseases, Beijing, China; 7WHO Collaborative Center for Research in Human Reproduction, Beijing, China
Correspondence to: Xu Ma, Department of Genetics, National Research Institute for Family Planning, 12 Da-hui-si, Hai Dian, Beijing, 100081, China; Phone: +86-10-62176870; FAX: +86-10-62179059; email: firstname.lastname@example.org
Purpose: To identify the causitive mutation in a five-generation family with autosomal dominant congenital total cataract.
Methods: Clinical and ophthalmological examinations were performed on the affected and unaffected family members. All the members were genotyped with microsatellite markers at loci that were considered to be associated with cataracts. Linkage analysis was performed after genotyping. A mutation was detected by direct sequencing using gene specific primers.
Results: Affected individuals in this family showed total cataract. The disease gene was mapping between to a 15.5 Mb interval bounded by D12S368 and D12S1676. A positive two-point LOD score (3.21 at recombination fraction 0) was obtained for the marker D12S90, flanked by D12S368 and D12S1052, on chromosome 12q13.1-21.1. This chromosome encompasses the Major Intrinsic Protein (MIP, MIP26) of the lens, also called aquaporin 0 (AQP0). Sequencing the coding regions of MIP revealed a C>T transition at nucleotide 97 in exon 1 that caused a substitution of arginine (R) to cysteine (C) at codon 33 (p.R33C). This mutation cosegregated with all affected individuals and was not observed in unaffected or in 100 normal unrelated individuals.
Conclusions: This study has identified the first dominant cataract mutation in MIP that is located outside the phylogenetically conserved transmembrane domain.
Congenital cataracts cause 10-30% of all blindness in children, and one-third of these cases are estimated to have a genetic cause . Clinically, cataracts can be classified as anterior polar, posterior polar, nuclear, lamellar (zonular), pulverulent, aculeiform, cerulean, cortical, polymorphic, sutural, and total cataracts [2,3]. Recently, at least 20 independent genetic loci of cataract have been mapped on 14 human chromosomes. Eighteen distinct responsible genes for nonsyndromic hereditary cataracts have been identified including CRYAA , CRYAB , CRYBA1/A3 , CRYBB1 , CRYBB2 [8,9], CRYGC , CRYGD [11,12], CRYGS , GJA3 , GJA8 [15,16], MIP , BFSP1, BFSP2 [18,19], PITX3 , HSF4 , MAF , and LIM2 . More genes are yet to be discovered .
To identify the genetic defect in autosomal dominant congenital cataract in a large Chinese family, allele sharing and linkage analysis methods were used in this study. We linked the disease gene to 12q13.1-14.1, where the MIP loci (MIP) is located and revealed a missense mutation in MIP by sequencing. This mutation cosegregated with all affected individuals and was not observed in unaffected or 100 normal unrelated individuals. To our knowledge, this is the first reported case of cataract caused by a mutation in MIP at a site in the protein that is located outside the transmembrane domain.
Clinical evaluations and DNA specimens
This study conformed to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of our institute. Written informed consent was obtained from all participating individuals or their guardians. The family comprised 22 affected individuals from a five-generation pedigree (Figure 1), originating from the province of Shangdong, China. The study consisted of 18 members including 10 affected individuals, four unaffected individuals, and four spouses (Figure 1). Clinical and ophthalmological examinations were performed by ophthalmologists.
Genotyping and linkage analysis
Genotyping and exclusion analysis was performed as described previously [25,26]. The oligonucleotide primer sequences were obtained from NCBI and GDB. A two-point LOD score (Z) was calculated using the MLINK subprogram of the Linkage package (version 5.1). The mode of inheritance was considered to be autosomal dominant with full penetrance. Disease gene frequency was set to 1/10,000 and the allele frequencies of the markers are considered to be equally distributed.
Mutations in MIP were screened by direct sequencing. We designed a set of three primer pairs to amplify the four exons and flanking intron sequences of MIP (GenBank NM_012064.2). Primer sequences were: F1: 5'-GTG AAG GGG TTA AGA GGC-3', R1: 5'-GGA GTC AGG GCA ATA GAG-3'; F2-3: 5'-CGG GGA AGT CTT GAG GAG-3', R2-3: 5'-CAC GCA GAA GGA AAG CAG-3'; F4: 5'-CCA CTA AGG TGG CTG GAA-3', R4: 5'-CTC ATG CCC CAA AAC TCA-3'. PCR products were sequenced on an ABI PRISM 3730 DNA Sequencer (Applied Biosystems, Foster City, CA).
Denaturing high-performance liquid chromatography (DHPLC) was used to screen the mutation identified in exon 1 of the MIP gene using a commercial system on the patients, family members, and the 100 normal control subjects (Wave DHPLC; Transgenomic, San Jose, CA). Gene specific PCR primers, which is used in direct sequencing, were used to amplify the fragment. DHPLC was performed as follows: initial concentration at 44% of buffer A (0.1 M triethylammonium acetate, TEAA; Transgenomic) and 56% of buffer B (0.1 M TEAA containing 25% acetonitrile; Transgenomic) at 62.3 °C.
The proband was a 37-year-old male (IV:12). His phenotype is bilateral, complete opacification of the fetal nucleus and the cortex, i.e. the phenotype is total cataract (Figure 2). He was also diagnosed with high myopia, the axial length was 29.39 mm in the right eye and 29.09 mm in the left eye. Affected individuals in the family showed nystagmus, which attests to the severity of the visual deprivation from early infancy. There was no family history of other ocular or systemic abnormalities. Affected individuals in the family showed the same opacification. Hospital records indicated that usually the opacity was either present at birth or developed during the first few months of life but did not progress with age.
Linkage and haplotype analysis
Allele-sharing analysis excluded the linkage of the disease in the family with all known loci of cataract except that for MIP (data not shown). Haplotype analysis showed that the affected individuals in the family shared a common haplotype with markers D12S368, D12S90, and D12S83 at 12q13.1-14.1 (Figure 1). Individuals III:5 and V:4 appear to be recombinants and the disease gene was mapped between a 15.5 Mb interval bounded by D12S368 and D12S1676.
Linkage analysis was performed in this family. We obtained a positive two-point LOD score (3.21 at recombination fraction 0) for the marker D12S90 (Table 1), flanked by D12S368 and D12S1052, on chromosome 12q13.1-21.1. This area encompasses the MIP locus.
Mutation detection for MIP
Direct cycle sequencing of the amplified fragments of MIP in four affected individuals identified a single base alteration c.97C>T (Figure 3) in exon 1 of MIP, which resulted in a substitution of arginine (R) to cysteine (C) at codon 33 (p.R33C). Denaturing HPLC analysis confirmed this mutation (Figure 4), which cosegregated with all affected individuals in the family, and this mutation was not observed in any of the unaffected family members or 100 normal controls. The remainder of the coding sequence did not show any sequence change.
Multiple-sequence alignment and mutation analysis
Using the NCBI and UCSC websites, we obtained multiple-sequence alignment of MIP protein in various species with DNAMAN biosoftware (Lynnon Biosoft, Quebec, Canada) including Homo sapiens, Canis familiaris, Mus musculus, Rattus norvegicus, and Fundulus heteroclitus (Figure 5). We found that codon 33, where the mutation (p.R33C) occurred, was located within a phylogenetically conserved region.
Furthermore, we used online bioinformatics software SIFT (sorting intolerant from tolerant) , which can distinguish between functionally neutral and deleterious amino acid changes in mutagenesis studies and on human polymorphisms. With SIFT, we predicted whether the amino acid substitution in MIP could have a phenotypic effect and found that the substitution at position 33 from R to C is deleterious.
The predicted p.R33C substitution represented a nonconservative amino acid change with the positively charged (basic) polar side group of arginine replaced by the uncharged polar sulfydryl side group of cysteine.
Summarily, the cosegregation of the C>T transition in only affected members of the pedigree and its absence in 100 control subjects suggested that the nonconservative p.R33C substitution was a causative mutation rather than a benign polymorphism in linkage disequilibrium with the disease.
The Major Intrinsic Protein (MIP) of the lens (MIP26 or AQP0) is a member of the water-transporting aquaporins, which play critical roles in controlling the water content of cells. It is the most abundant junctional membrane protein in the mature lens, which is encoded by a gene on chromosome 12q13, and is expressed only in terminally differentiated fiber cells, the major cell type of the crystalline lens. Based on amino acid sequence, members of the aquaporin family are predicted to share a common topology consisting of six transmembrane domains joined by five connecting loops . The first extracelluar loop of the five connecting loops contains the residue 33R to 40H.
Several mutations in MIP have been linked to genetic cataracts in humans. The mutations was first reported in 2000 . Since then, few reports about the mutation in this gene have been reported until a novel mutation was recently detected in a four-generation family of European descent . So, four different mutations have been identified giving rise to cataract in four human cataract families (p.E134G, T138R, del3223G, and R33C in this study).
The first two mutations result in amino acid changes within a key part of the water channel, a highly conserved amino acid located within transmembrane helix 4. This mutation would be predicted to affect the water transport function. The third mutation results in a shortened protein and it changes transmembrane helix 6. This mutation is predicted to alter the voltage dependence and calmodulin-binding properties.
Several mutations have been identified in mice including A51R (the second transmembrane region) , a deletion of four amino acids (the second transmembrane region) , and a 76 bp deletion extending from the 39th amino acid of exon 2 to the 59th amino acid of the adjacent intron, which causes the deletion of L121 to G141 (the fourth and fifth transmembrane region) .
So, each of the dominant mutations reported in MIP in humans (E134G, T138R, Del3223G) and in mice lie within the transmembrane helix. In the present study, we have identified the first dominant mutation (p.R33C) in MIP linked with cataract, which lies outside the transmembrane domain.
According to the specific morphology of the lens concerned with position and appearance under slit lamp examination, congenital cataracts were classified as anterior polar, posterior polar, nuclear, lamellar (zonular), pulverulent, aculeiform, cerulean, cortical, polymorphic, sutural, and total cataract . In this Chinese family, the phenotype in this pedigree showed total cataract, which is an uncommon form of congenital cataract and autosomal dominant inheritance is rare. Total cataract is a clinically distinctive opacity that is located at the fetal nucleus and the cortex. Because of its location in the optical center of the eye, total cataract can have a marked effect on visual acuity. So far, four cases with a total cataract phenotype have been reported involving genes PITX3 , HSF4 , GJA3 , and MIP in this study. This study provided further evidence to show genetic heterogeneity with this phenotype.
It is interesting that the same substitution of arginine (R) to cysteine (C) or to other residues have been detected in our recent work in CRYGD (R14C)  and other's work in genes which are responsible for cataract (R21L, CRYAA ; R49C, CRYAA ; R116C, CRYAA ; R168W, CRYGC ; R14C, CRYGD; R36S, CRYGD ; R58H, CRYGD ; R288P, MAF ; and R120C, HSF4 ). Research suggests that more attention should be paid to the arginine residue when we are screening mutations causing cataract.
MIP functional assays in oocyte expression systems  support its role as a water channel while additional roles for this protein have been proposed, including as a structural element and as an adhesion molecule [39,40]. We speculate that mutated MIP, harboring a new exposed cysteine residue at position 33, would allow for the formation of intermolecular disulfide bonds, which would destabilize the native structure of MIP. This wrong disulfide formation may disturb its location at the plasma membrane. Further study is needed to provide insights into the molecular consequence of the mutation identified in this study.
The authors thank the family for their participation in this project and Dr. Siquan Zhu and Dr. Xiaolin Hao (Beijing Tongren Hospital, Capital University of Medical Sciences, Beijing, China) for phenotype identification. This work is partly supported by the National "973" Basic Research Funding Scheme of China (grant number 2001CB5103 and 2007CB5119005) and the National Infrastructure Program of Chinese Genetic Resources (2004DKA30490).
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