Molecular Vision 2007; 13:1651-1656 <http://www.molvis.org/molvis/v13/a184/> Received 6 June 2007 | Accepted 10 September 2007 | Published 11 September 2007

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A novel mutation in major intrinsic protein of the lens gene (MIP) underlies autosomal dominant cataract in a Chinese family

Feng Gu,^1,2 Hong Zhai,³ Dan Li,^1,2 Luxin Zhao,³ Chao li,⁴ Shangzhi Huang,^2,5,6 Xu Ma^1,2,7
 
 

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: genetic@263.net.cn

Abstract

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.

Introduction

Congenital cataracts cause 10-30% of all blindness in children, and one-third of these cases are estimated to have a genetic cause [1]. 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 [4], CRYAB [5], CRYBA1/A3 [6], CRYBB1 [7], CRYBB2 [8,9], CRYGC [10], CRYGD [11,12], CRYGS [13], GJA3 [14], GJA8 [15,16], MIP [17], BFSP1, BFSP2 [18,19], PITX3 [20], HSF4 [21], MAF [22], and LIM2 [23]. More genes are yet to be discovered [24].

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.

Methods

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.

Sequence analysis

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 HPLC

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.

Results

Clinical data

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) [27], 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.

Discussion

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 [28]. 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 [17]. 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 [29]. 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) [30], a deletion of four amino acids (the second transmembrane region) [31], 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) [32].

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 [24]. 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 [20], HSF4 [33], GJA3 [34], 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) [26] and other's work in genes which are responsible for cataract (R21L, CRYAA [35]; R49C, CRYAA [36]; R116C, CRYAA [4]; R168W, CRYGC [37]; R14C, CRYGD[12]; R36S, CRYGD [38]; R58H, CRYGD [10]; R288P, MAF [22]; and R120C, HSF4 [22]). 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 [29] 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.

Acknowledgements

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).

References

1. Lund AM, Eiberg H, Rosenberg T, Warburg M. Autosomal dominant congenital cataract; linkage relations; clinical and genetic heterogeneity. Clin Genet 1992; 41:65-9.

2. Scott MH, Hejtmancik JF, Wozencraft LA, Reuter LM, Parks MM, Kaiser-Kupfer MI. Autosomal dominant congenital cataract. Interocular phenotypic variability. Ophthalmology 1994; 101:866-71.

3. Hejtmancik JF. The genetics of cataract: our vision becomes clearer. Am J Hum Genet 1998; 62:520-5.

4. Litt M, Kramer P, LaMorticella DM, Murphey W, Lovrien EW, Weleber RG. Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet 1998; 7:471-4.

5. Berry V, Francis P, Reddy MA, Collyer D, Vithana E, MacKay I, Dawson G, Carey AH, Moore A, Bhattacharya SS, Quinlan RA. Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans. Am J Hum Genet 2001; 69:1141-5.

6. Kannabiran C, Rogan PK, Olmos L, Basti S, Rao GN, Kaiser-Kupfer M, Hejtmancik JF. Autosomal dominant zonular cataract with sutural opacities is associated with a splice mutation in the betaA3/A1-crystallin gene. Mol Vis 1998; 4:21 <http://www.molvis.org/molvis/v4/a21/>.

7. Mackay DS, Boskovska OB, Knopf HL, Lampi KJ, Shiels A. A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q. Am J Hum Genet 2002; 71:1216-21.

8. Litt M, Carrero-Valenzuela R, LaMorticella DM, Schultz DW, Mitchell TN, Kramer P, Maumenee IH. Autosomal dominant cerulean cataract is associated with a chain termination mutation in the human beta-crystallin gene CRYBB2. Hum Mol Genet 1997; 6:665-8.

9. Vanita, Sarhadi V, Reis A, Jung M, Singh D, Sperling K, Singh JR, Burger J. A unique form of autosomal dominant cataract explained by gene conversion between beta-crystallin B2 and its pseudogene. J Med Genet 2001; 38:392-6.

10. Heon E, Priston M, Schorderet DF, Billingsley GD, Girard PO, Lubsen N, Munier FL. The gamma-crystallins and human cataracts: a puzzle made clearer. Am J Hum Genet 1999; 65:1261-7.

11. Ren Z, Li A, Shastry BS, Padma T, Ayyagari R, Scott MH, Parks MM, Kaiser-Kupfer MI, Hejtmancik JF. A 5-base insertion in the gammaC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract. Hum Genet 2000; 106:531-7.

12. Stephan DA, Gillanders E, Vanderveen D, Freas-Lutz D, Wistow G, Baxevanis AD, Robbins CM, VanAuken A, Quesenberry MI, Bailey-Wilson J, Juo SH, Trent JM, Smith L, Brownstein MJ. Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene. Proc Natl Acad Sci U S A 1999; 96:1008-12.

13. Sun H, Ma Z, Li Y, Liu B, Li Z, Ding X, Gao Y, Ma W, Tang X, Li X, Shen Y. Gamma-S crystallin gene (CRYGS) mutation causes dominant progressive cortical cataract in humans. J Med Genet 2005; 42:706-10.

14. Mackay D, Ionides A, Kibar Z, Rouleau G, Berry V, Moore A, Shiels A, Bhattacharya S. Connexin46 mutations in autosomal dominant congenital cataract. Am J Hum Genet 1999; 64:1357-64.

15. Shiels A, Mackay D, Ionides A, Berry V, Moore A, Bhattacharya S. A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonular pulverulent" cataract, on chromosome 1q. Am J Hum Genet 1998; 62:526-32.

16. Polyakov AV, Shagina IA, Khlebnikova OV, Evgrafov OV. Mutation in the connexin 50 gene (GJA8) in a Russian family with zonular pulverulent cataract. Clin Genet 2001; 60:476-8.

17. Berry V, Francis P, Kaushal S, Moore A, Bhattacharya S. Missense mutations in MIP underlie autosomal dominant 'polymorphic' and lamellar cataracts linked to 12q. Nat Genet 2000; 25:15-7.

18. Ramachandran RD, Perumalsamy V, Hejtmancik JF. Autosomal recessive juvenile onset cataract associated with mutation in BFSP1. Hum Genet 2007; 121:475-82.

19. Conley YP, Erturk D, Keverline A, Mah TS, Keravala A, Barnes LR, Bruchis A, Hess JF, FitzGerald PG, Weeks DE, Ferrell RE, Gorin MB. A juvenile-onset, progressive cataract locus on chromosome 3q21-q22 is associated with a missense mutation in the beaded filament structural protein-2. Am J Hum Genet 2000; 66:1426-31.

20. Semina EV, Ferrell RE, Mintz-Hittner HA, Bitoun P, Alward WL, Reiter RS, Funkhauser C, Daack-Hirsch S, Murray JC. A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD. Nat Genet 1998; 19:167-70.

21. Bu L, Jin Y, Shi Y, Chu R, Ban A, Eiberg H, Andres L, Jiang H, Zheng G, Qian M, Cui B, Xia Y, Liu J, Hu L, Zhao G, Hayden MR, Kong X. Mutant DNA-binding domain of HSF4 is associated with autosomal dominant lamellar and Marner cataract. Nat Genet 2002; 31:276-8.

22. Jamieson RV, Perveen R, Kerr B, Carette M, Yardley J, Heon E, Wirth MG, van Heyningen V, Donnai D, Munier F, Black GC. Domain disruption and mutation of the bZIP transcription factor, MAF, associated with cataract, ocular anterior segment dysgenesis and coloboma. Hum Mol Genet 2002; 11:33-42.

23. Pras E, Levy-Nissenbaum E, Bakhan T, Lahat H, Assia E, Geffen-Carmi N, Frydman M, Goldman B, Pras E. A missense mutation in the LIM2 gene is associated with autosomal recessive presenile cataract in an inbred Iraqi Jewish family. Am J Hum Genet 2002; 70:1363-7.

24. Reddy MA, Francis PJ, Berry V, Bhattacharya SS, Moore AT. Molecular genetic basis of inherited cataract and associated phenotypes. Surv Ophthalmol 2004; 49:300-15.

25. Qi Y, Jia H, Huang S, Lin H, Gu J, Su H, Zhang T, Gao Y, Qu L, Li D, Li Y. A deletion mutation in the betaA1/A3 crystallin gene (CRYBA1/A3) is associated with autosomal dominant congenital nuclear cataract in a Chinese family. Hum Genet 2004; 114:192-7.

26. Gu F, Li R, Ma XX, Shi LS, Huang SZ, Ma X. A missense mutation in the gammaD-crystallin gene CRYGD associated with autosomal dominant congenital cataract in a Chinese family. Mol Vis 2006; 12:26-31 <http://www.molvis.org/molvis/v12/a3/>.

27. Ng PC, Henikoff S. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 2003; 31:3812-4.

28. Francis P, Chung JJ, Yasui M, Berry V, Moore A, Wyatt MK, Wistow G, Bhattacharya SS, Agre P. Functional impairment of lens aquaporin in two families with dominantly inherited cataracts. Hum Mol Genet 2000; 9:2329-34.

29. Geyer DD, Spence MA, Johannes M, Flodman P, Clancy KP, Berry R, Sparkes RS, Jonsen MD, Isenberg SJ, Bateman JB. Novel single-base deletional mutation in major intrinsic protein (MIP) in autosomal dominant cataract. Am J Ophthalmol 2006; 141:761-3.

30. Shiels A, Bassnett S. Mutations in the founder of the MIP gene family underlie cataract development in the mouse. Nat Genet 1996; 12:212-5.

31. Sidjanin DJ, Parker-Wilson DM, Neuhauser-Klaus A, Pretsch W, Favor J, Deen PM, Ohtaka-Maruyama C, Lu Y, Bragin A, Skach WR, Chepelinsky AB, Grimes PA, Stambolian DE. A 76-bp deletion in the Mip gene causes autosomal dominant cataract in Hfi mice. Genomics 2001; 74:313-9.

32. Okamura T, Miyoshi I, Takahashi K, Mototani Y, Ishigaki S, Kon Y, Kasai N. Bilateral congenital cataracts result from a gain-of-function mutation in the gene for aquaporin-0 in mice. Genomics 2003; 81:361-8.

33. Smaoui N, Beltaief O, BenHamed S, M'Rad R, Maazoul F, Ouertani A, Chaabouni H, Hejtmancik JF. A homozygous splice mutation in the HSF4 gene is associated with an autosomal recessive congenital cataract. Invest Ophthalmol Vis Sci 2004; 45:2716-21.

34. Devi RR, Reena C, Vijayalakshmi P. Novel mutations in GJA3 associated with autosomal dominant congenital cataract in the Indian population. Mol Vis 2005; 11:846-52 <http://www.molvis.org/molvis/v11/a100/>.

35. Graw J, Klopp N, Illig T, Preising MN, Lorenz B. Congenital cataract and macular hypoplasia in humans associated with a de novo mutation in CRYAA and compound heterozygous mutations in P. Graefes Arch Clin Exp Ophthalmol 2006; 244:912-9.

36. Mackay DS, Andley UP, Shiels A. Cell death triggered by a novel mutation in the alphaA-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q. Eur J Hum Genet 2003; 11:784-93.

37. Santhiya ST, Shyam Manohar M, Rawlley D, Vijayalakshmi P, Namperumalsamy P, Gopinath PM, Loster J, Graw J. Novel mutations in the gamma-crystallin genes cause autosomal dominant congenital cataracts. J Med Genet 2002; 39:352-8.

38. Kmoch S, Brynda J, Asfaw B, Bezouska K, Novak P, Rezacova P, Ondrova L, Filipec M, Sedlacek J, Elleder M. Link between a novel human gammaD-crystallin allele and a unique cataract phenotype explained by protein crystallography. Hum Mol Genet 2000; 9:1779-86.

39. Gruijters WT. A non-connexon protein (MIP) is involved in eye lens gap-junction formation. J Cell Sci 1989; 93:509-13.

40. Lindsey Rose KM, Gourdie RG, Prescott AR, Quinlan RA, Crouch RK, Schey KL. The C terminus of lens aquaporin 0 interacts with the cytoskeletal proteins filensin and CP49. Invest Ophthalmol Vis Sci 2006; 47:1562-70.