A New Locus for Autosomal Dominant Cataract on Chromosome 12q13

Abstract

purpose. To map the gene for autosomal dominant cataracts (ADC) in an American white family of European descent.

methods. Ophthalmic examinations and linkage analyses using a variety of polymorphisms were performed; two-point lod scores calculated.

results. Affected individuals (14 studied) exhibited variable expressivity of embryonal nuclear opacities based on morphology, location within the lens, and density. This ADC locus to 12q13 was mapped on the basis of statistically significantly positive lod scores and no recombinations (θ_m = θ_f = 0) with markers D12S368, D12S270, D12S96, D12S359, D12S1586, D12S312, D12S1632, D12S90, and D12S83; assuming full penetrance, a maximum lod score of 4.73 was calculated between the disease locus and D12S90.

conclusions. The disease in this family represents the first ADC locus on chromosome 12; major intrinsic protein of lens fiber (MIP) is a candidate gene.

Cataracts in the pediatric population may be caused by intrauterine embryopathies, single gene defects, and chromosomal rearrangements. Immunization programs have reduced the incidence of rubella, commonly associated with congenital cataracts ¹ ; some congenital cataracts, particularly unilateral, are of unknown etiology. Hereditary congenital cataracts account for about one third of pediatric visual loss, ² and nonsyndromal autosomal dominant cataracts (ADC) are the most common. 

Most ADC are congenital, and progression is common. Phenotypic variability has been documented among and within families. ^{3 4 5 6 7} Generally, the cataracts are bilateral and are characterized on the basis of location, size, color, the presence or absence of refractility, and, most notably, shape. ^{8 9} Despite attempts to clinically categorize hereditary cataracts, there is limited correlation of phenotypes with genetic loci. 

ADC is genetically heterogeneous, and 13 loci for ADC have been identified on the basis of linkage analyses and gene mutations; hyperferritinemia, an additional locus, is a systemic disease of autosomal dominant cataracts without other symptoms. Several recent reviews of human cataracts and mouse models are available. ^{10 11} We expanded our clinical study of an American white family of European descent ¹² with some members affected by ADC of embryonal nuclear and pulverulent cortical forms; expressivity was variable. Using linkage analysis, we mapped the disease to 12q13. 

Methods

The family (ADC2) of European extraction (Fig. 1) was ascertained at the Ophthalmology Clinic of the Jules Stein Eye Institute, Department of Ophthalmology, UCLA School of Medicine, through the courtesy of Sherwin J. Isenberg, MD; clinical and negative linkage analyses have been reported. ¹² Informed consent in accordance with the Declaration of Helsinki and with the UCLA Institutional Review Board approval was obtained in all cases. Twenty-seven individuals participated in the study: 14 affected individuals and 13 unaffected individuals of whom 5 were spouses ¹² ; no other diseases aside from age-related disorders were identified. Affected status was determined by pupillary dilation and evaluation of lenses by slit-lamp biomicroscopy or retroillumination in the field, or by a history of cataract extraction before senility (before 60 years of age; JBB); in this family, all aphakic patients were younger than 20 years of age at the time of venipuncture. In all patients except 1 and 6 (categorized originally as unknown based on an examination in the field), the phenotype was determined before genotyping. 

Adults in the family reported that the cataracts were present from early in life (Fig. 2) and are presumed to be congenital based on the examinations of the proband and his sister. ¹² The proband (27) had an embryonal nuclear opacity in each eye with vacuolization; there was mild asymmetry. His affected father (24) had been unaware of his cataracts and had 20/20 vision in each eye. Nystagmus was evident only in those individuals who had undergone cataract surgery early in life and by history had a delay of correction of refractive error. 

Blood samples (between 7 and 30 ml depending on ease of venipuncture, level of cooperation, and size of the patient) were collected in EDTA, and genomic DNA was extracted. ¹³ Markers were analyzed for linkage with the ADC2 locus several times as methodology evolved; candidate genes/regions were identified on the basis of expression within the lens or linkage with a chromosomal region in another family. Initially, linkage analysis was based on available polymorphic phenotypic blood markers ¹² ; one individual’s Duffy (FY) genotype was found to be incorrectly coded ¹² and corrected in the present study. Lod scores for haptoglobin (HP) linked to the CTM locus at 16q22.1 ^{4 14 15} and FY, linked to the CZP1 locus at 1q21-25 (formerly CAE1 ¹⁶ ), were recalculated in the present study. New candidate genes were evaluated using restriction fragment length polymorphisms (RFLPs; methods available on request) and short tandem repeat (STR) microsatellite marker loci. The loci for αA-crystallin (CRYAA) ¹⁷ on human chromosome 21q22.3, ¹⁸ αB-crystallin(CRYAB) ¹⁹ on 11q22.3-q23.1, ²⁰ βA1 (formerly βA3/A1)-crystallin(CRYBA1) ²¹ on 17q11.2-q12, ^{22 23} βB2-crystallin(CRYBB2) ²⁴ on 22q11.2-q12.1, ^{25 26} γ-crystallin cluster(CRYG) ²⁷ on 2q33-q35, ²⁸ and ζ-crystallin (CRYZ) ²⁹ on 1p22-p31 ³⁰ were analyzed for linkage to the ADC2 gene using RFLPs. We screened for new RFLPs for the CRYAB, CRYBA1, and CRYBB2 genes in 10 normal and unaffected individuals. Markers for a mapped ADC locus on 17q ³¹ were studied with STR marker loci. 

Once linkage to available candidate genes was excluded, we initiated a genome-wide search using a pooling technique and, thereafter, a systematic approach using the ABI Prism Linkage Mapping Set (version 2; Perkin Elmer–Applied Biosystems, Foster City, CA), with end-labeled fluorescent primers as detailed in the user’s manual. For the pooling method, anonymous markers selected on the basis of predominant alleles in a DNA pool of affected family members compared with an unaffected pool, were amplified using the polymerase chain reaction (PCR). 

The marker loci were localized to chromosomal regions based on data from the Marshfield Institute for Molecular Genetics ³² and the Genome Database. ³³ For linkage analyses, pedigree and genotype data were analyzed with LIPED. ³⁴ Lod scores were calculated using published allele frequencies. ^{33 35} A gene frequency of 0.0001 and penetrance at 1.0 and 0.9 were assumed for the cataract locus; two-point lod scores were calculated for a full range of θ_m and θ_f values. 

Results

Two male siblings (1 and 6), initially coded as unknown based on retroillumination in the field, were restudied by slit-lamp biomicroscopy and recategorized as affected. Both had punctate white opacities of the posterior cortical region, the posterior Y suture, and, to a lesser extent, the anterior cortex; individual 6 had small vertical linear opacities in the inferior cortical region. 

We identified new RFLPs for the CRYBA1 (PstI; 14.0 and 13.5 kb with frequencies of 0.6 and 0.4, respectively) and CRYBB2 (DraI and PstI in linkage disequilibrium; DraI of 10.5 and 9.6 kb with frequencies of 0.55 and 0.45, respectively and PstI of 11.8 and 6.0/4.5 kb with frequencies of 0.55 and 0.45, respectively). No RFLPs for CRYAB were identified. 

Two-point lod score(s) were less than −2.00 (θ_m = θ_f = 0.001) for crystallins CRYAA, CRYBA1, and CRYBB2 as well as for markers flanking both FY and HP and were less than −1.8 (θ_m = θ_f = 0.001) for CRYG flanking markers; multipoint data excluded linkage with flanking markers for CRYZ gene. Using the pooling methods, regions of chromosomes 3, 8, 14, and 19 were excluded. Using the ABI Prism system, markers on chromosomes 1, 12, 13, 17, and 18 were studied and all excluded with the exception of D12S83 and D12S368 (Z _max = 2.33 and 3.76, respectively;θ _m = θ_f = 0); additional markers on chromosome 12 (National Jewish Resource Center, Denver, CO or Research Genetics, Huntsville, AL) were tested (Table 1) . Assuming full penetrance, a maximum lod score of 4.73 (θ_m = θ_f = 0) was calculated for marker D12S90; lod scores without recombinations extended telomeric from D12S368 to D12S83, a distance of 25 to 31 cM. Lod scores at 0.95 penetrance demonstrated linkage and were similar to those calculated with full penetrance; the maximum score for marker D12S90 was 4.62 (θ_m =θ _f = 0). 

Discussion

Eight single genes have been implicated as causative for ADC to date each in a single family with the exception of the βA1-crystallin, gap junction protein α-3(connexin46), γD-crystallin, PAX6, and the β-crystallin gene (CRYBB2) all of which have been reported in two. A chain termination mutation in the β-crystallin gene (CRYBB2) on chromosome 22, ^{36 37} a missense mutation in the gap junction protein α-8 gene (connexin50; MP70) ³⁸ on 1q21.1, ³⁹ and a missense mutation in the human αA-crystallin gene (CRYAA) ⁴⁰ on chromosome 21 have been shown to cause ADC. Activation of the γE-crystallin pseudogene (CRYGEP1) ⁴¹ on 2q33-q35 was reported as the basis of the Coppock-like cataract. Recently, Heon and colleagues ⁴² restudied the family and found that the variation in the pseudogene CRYGEP1, presumed to activate the gene, is a polymorphism and identified a missense mutation in a highly conserved region of exon 2 of the γC-crystallin (CRYGC). Kannabiran and colleagues ^{43 44} demonstrated a mutation of a donor splice junction (intron C) of the βA1-crystallin gene on 17q11.2-q12 as the basis for the ADC in an Indian family ⁴⁵ ; we studied a large Brazilian family with ADC of variable morphology and found a new and different mutation at the same βA1-crystallin splice site (Bateman et al., unpublished data, 2000). Two-point mutations, a missense and a frame-shift, in gap junction protein α-3 (connexin46) on chromosome 13q11-12 have been reported in two families with granular opacities of the fetal nucleus and juvenile cortex. ⁴⁶ Two families with missense mutations of the γD crystallin (CRYGD) gene (2q33-q35) and disparate clinical features have been reported. ^{42 47} Although mutations in the homeobox DNA-binding PAX6 gene usually cause aniridia and/or anterior segment dysgenesis, isolated cataracts have been documented. ^{48 49} Hyperferritinemia, an autosomal dominant systemic disease characterized by elevated serum ferritin, congenital cataracts, and abnormal liver biopsy, ⁵⁰ is caused by mutations of the iron responsive element of ferritin L-subunit gene ⁵¹ and may represent an additional locus. 

There is considerable phenotypic variability in the ADC families that have been studied by linkage analyses. Curiously, similar forms of ADC have been mapped to different chromosomal regions, whereas disparate forms have mapped to the same locus. For example, embryonic/fetal and progressive sutural opacities in one family and stationary posterior polar cataracts in another have been mapped to chromosome 1p36. ^{52 53} Recently, affected members of a family with cerulean cataracts were found to have the identical mutation as a family with Coppock-like cataracts. ^{36 37} The cataracts in our family varied considerably among individuals, and correlation with genotype would not be feasible based on morphology. 

The locus in family ADC2 is in the 12q13 chromosomal region based on linkage analysis and represents the first ADC locus on chromosome 12. There were no recombinations with 9 markers, and lod scores were over 3.0 with the exception of D12S83; differences are based on the number of informative matings. The region spans 25 to 31 cM, ^{32 33} depending on which map was used (Table 1) . 

There are several eye-related genes in the 12q12-14.1 region. Retinol dehydrogenase 1 (RDH1) expressed in the retinal pigment epithelium ⁵⁴ was assigned to chromosome 12q13-q14. ⁵⁵ Diacylglycerol kinase, alpha (DAGK1) is expressed in the retina ⁵⁶ and involved in the regeneration of phosphatidylinositol during transduction; it has been assigned to 12q13.3. ⁵⁷ However, neither is known to be expressed in the lens. The most promising candidate gene is that for the major intrinsic protein of the lens fiber (MP26; MIP; OMIM 154050), ⁵⁸ the predominant membrane protein ^{59 60} that has been mapped to 12q14 (Fig. 3) . ⁶¹ MIP accounts for up to 80% of total lens membrane protein ⁶² and is probably lens-specific. ^{63 64 65} The gene is 3.6 kb and contains four exons; Alu repetitive sequences, which may regulate proliferation, differentiation, and transformation, are found in the 5′-flanking region. ⁶⁰ The gene is regulated spatially and temporally, and the 5′-flanking sequence contains an active promoter region for lens expression, ⁶⁶ including Sp1, AP2, and Sp3 binding sites. ^{67 68} It has channel-forming activity ^{69 70 71 72} and may function as an adhesion molecule ⁶⁴ ; MIP probably maintains lens transparency by reducing interfiber space. ^{73 74} The MIP mRNA is expressed in the lens vesicle early in embryogenesis and in the secondary lens fibers. ^{64 65}  

There are many candidate genes for ADC based on animal models, gene expression within the lens, and chromosomal localization in humans and other mammals. For example, mutations in the Crybb2(βBB2)-, Cryge(γE)-, and ζ-crystallin genes have been found to cause ADC in the Philly mouse, ⁷⁵ eye lens–obsolescence mouse (ELO), ⁷⁶ and 13/N guinea pig, ⁷⁷ respectively. In the mouse, the homologous region of human chromosome 12q13 (region of our ADC2 locus) is chromosome 10 ⁷⁸ and mutations in the Mip (MP26) cause cataracts in the cataract Fraser (Cat^Fr), ^{79 80} lens opacity mutations (Lop), ⁸⁰ and hydropic fibers (Hfi) mice. ⁸¹ In the Cat^Fr mouse, the most abundant Mip mRNA transcript in the adult lens is truncated and is the result of a transposon-induced splicing defect that substitutes a long terminal repeat sequence for the carboxyl-terminal exon of the gene ⁷⁹ ; in this model, the water channel function is disrupted. ⁷² In the Lop mouse, an amino acid substitution inhibits targeting of Mip to the cell membrane. ⁸⁰ In the Hfi mouse, an exon 2 deletion in the transcript is associated with a cataract. ⁸¹ MIP is a candidate gene based on its close location to the cataract locus in our ADC2 family and the reported mutations in the mouse. 

In conclusion, we have identified a new locus for ADC on chromosome 12q13. Affected members of this American family exhibit variable morphology with some opacities in the embryonal nucleus and others in the cortex. MIP is a candidate gene that we are analyzing in this family. 

 Supported by National Eye Institute Grant EY 08282 (JBB).

 Submitted for publication July 6, 1999; revised October 22, 1999 and February 4, 2000; accepted February 15, 2000.

 Commercial relationships policy: N.

 Present affiliations: ⁹QIAGEN, Santa Clarita, California; ¹⁰Genzyme Genetics, Santa Fe, New Mexico.

 

Figure 1.

 

Pedigree of ADC family with haplotypes for the most relevant markers. Only members from whom blood was drawn are included. Solid circles and squares represent affected females and males, respectively; open circles and squares denote unaffected females and males, respectively. The proband is identified by an arrow. The box represents the disease haplotype inherited from the founder.

Figure 2.

 

Photographs of affected members of the family demonstrating phenotypic heterogeneity. Proband (27) had vacuoles in the embryonal nucleus (age, 1 month; A); his father (age, 38 years; 24) has a star-shaped opacity that does not alter vision (B). The proband’s aunt (age, 60 years; no specimen obtained) has a dense cataract in the embryonal nucleus (C).

Table 1.

 

Two-Point Lod Scores between Markers on Chromosome 12 and ADC Loci

Marker	Z max	θm	θf	Marshfield, 32 cM	GDB, 33 Mb
D12S85	0.97	0.11	0.14	61.34	64
D12S368	3.76	0	0	66	72.9–74
D12S270	4.45	0	0	67.63	73.5
D12S96	1.65	0	0	68.16	74.19
D12S359	3.89	0	0	68.16	75.8–77.3
D12S1586	4.39	0	0	69.23	77.3
D12S312	3.09	0	0	71.61	77.68
D12S1632	3.16	0	0	71.61	78.87
D12S90	4.73	0	0	71.61	79.1
D12S83	2.33	0	0	75.17	83.2
D12S326	1.13	0	0.34	86.4	93.3–93.9

Figure 3.

 

Ideogram of chromosome 12 with linked markers and MIP. The underlined markers recombined with the ADC locus and identify the interval. ^{32 33}

The authors thank Sherwin Isenberg, MD, for referring the proband and his family for genetic counseling and Cindy Jaworski, PhD, Joram Piatigorsky, PhD, Michael Gorin, MD, PhD, Suraj Bhat, PhD, and J. Samuel Zigler, Jr, PhD, for providing the CRYAA, CRYAB, CRYBA1, CRYBB2, CRYGA, and CRYZ clones, respectively.