|Molecular Vision 2007;
Received 31 July 2007 | Accepted 22 October 2007 | Published 25 October 2007
A recurrent FBN1 mutation in an autosomal dominant ectopia lentis family of Indian origin
Jai Rup Singh,1
Peter N. Robinson,4
1Centre for Genetic Disorders, Guru Nanak Dev University and 2Dr. Daljit Singh Eye Hospital, Amritsar, India; 3Institute of Human Genetics and 4Institute of Medical Genetics, Charitè, University Medicine of Berlin, Berlin, Germany
Correspondence to: Dr. Vanita Vanita, Centre for Genetic Disorders, Guru Nanak Dev University, Amritsar-143005, Punjab, India; Phone: + 0183-2258802-09, Ext. 3277, 3279; FAX: + 0183-2258863, +0183-2258820; email: firstname.lastname@example.org
Purpose: To identify the genetic defect in an autosomal dominant ectopia lentis (EL) family having 27 affected members in four generations.
Methods: Detailed family history and clinical data were recorded for 48 family members including 24 persons with isolated ectopia lentis. Candidate gene regions at 5q and 15q known to be linked with ectopia lentis were analyzed using fluorescent labeled microsatellite markers. Mutation screening in the candidate gene, fibrillin-1 (FBN1), at 15q was performed by bidirectional sequencing of the amplified products.
Results: A maximum LOD score of 5.74 at θ=0.0 was obtained with marker D15S1024 in close proximity to FBN1 at 15q21. Mutation screening in FBN1 identified a C>T transition at nucleotide position c.718. This nucleotide change resulted in the substitution of highly conserved arginine by cysteine at codon 240 (R240C). This nucleotide substitution was not seen in any unaffected member of the family.
Conclusions: We report a recurrent R240C mutation in FBN1 in an autosomal dominant ectopia lentis family. This mutation has previously been reported in a family with isolated ectopia lentis, in another family with ectopia lentis and involvement of the skeleton and integument, and in one person with classic Marfan syndrome. This is the largest family with isolated ectopia lentis reported to date. The results of the present study provide convincing evidence for a correlation of R240C and isolated ectopia lentis. In addition, this is the first report of molecular characterization in an ectopia lentis family of Indian origin.
Ectopia lentis refers to a displacement or malposition of the lens from its normal location, often in association with stretched or discontinuous zonular fibers. In the majority of cases, ectopia lentis occurs as one symptom of a systemic disease . Most cases of ectopia lentis are associated with Marfan syndrome, an autosomal dominant inherited disorder of connective tissue with prominent manifestations in the cardiovascular system, the skeleton, the eye, and other organs  as well as with autosomal recessive ectopia pupillae et lentis , homocystinuria , sulfite oxidase deficiency , and Weill-Marchesani syndrome [6,7].
Marfan syndrome is caused by mutations in the gene for fibrillin-1 (FBN1), but a range of other phenotypes more or less isolated and involving the aorta, the skeleton, or the eyes have been reported with FBN1 mutations . Over 800 distinct mutations have been entered into the Marfan mutation database . Strong genotype-phenotype correlations have not been found for the majority of mutations . However, mutations associated with neonatal Marfan syndrome and other severe forms of this disorder have been shown to cluster in exons 24-32 [11-13], and several mutations in the 5' and 3' regions of FBN1 have been shown to be associated with ectopia lentis as the sole or predominant manifestation [11,14-17].
We came across a four-generation family affected with bilateral ectopia lentis at the Dr. Daljit Singh Eye Hospital, Amritsar, India. Linkage was obtained with markers at 15q. Sequencing of FBN1 localized at the mapped interval indicated a C>T change at nucleotide position c.718, resulting in the substitution of highly conserved arginine by cysteine at codon 240 (R240C). The mutation cosegregated in all affected individuals and was not observed in any of the unaffected family members. This is the first report of molecular characterization of ectopia lentis in a family of Indian origin.
The index case, a six-year-old child, was diagnosed to have bilateral ectopia lentis. The family history revealed 27 affected members in four generations of which three were deceased (Figure 1). The detailed ophthalmological examination, which included slit lamp examination, performed on 48 members of the family, revealed 24 members as bilaterally affected (some had a history of lens extraction in childhood) and 24 individuals (including seven spouses) were unaffected. The affected unoperated members had myopia in association with ectopia lentis.
Genotyping and linkage analysis
Informed consent was obtained from each individual studied. This study was approved by the Ethics Review Board of the Guru Nanak Dev University, consistent with the provisions of the Declaration of Helsinki. Blood was drawn and DNA isolated by standard methods. Initially, linkage analysis for candidate gene region at 15q and 5q with dinucleotide repeat microsatellite markers (Genethon linkage map)  was done on the DNA samples of 46 ophthalmologically examined individuals (22 affected and 24 unaffected). Microsatellites were amplified in singleplex reactions by "touch-down" polymerase chain reaction (PCR; MJ-Research, Watertown, MA) using fluorescently labeled primers following standard methods. PCR products were pooled and denatured at 95 °C for 1 min and electrophoresed on 4% denaturing polyacrylamide gels using an automated DNA sequencer (ABI-Prism 377; Applied Biosystems, Foster City, CA). Data were collected and analyzed by GENESCAN, version 3.1.2, and genotyping was done using GENOTYPER 2.5.1 software. Autosomal dominant inheritance with complete penetrance of the trait and a disease gene frequency of 0.0001 was assumed. Recombination frequencies were considered to be equal between males and females. Two-point linkage analysis was performed with MLINK from the LINKAGE program package .
All 65 exons and splice sites of FBN1 were amplified using exon-specific primers. Genomic DNA from two affected and one unaffected individual were amplified as described elsewhere . PCR products were purified using a PCR product purification kit (QIAquick; Qiagen, Valencia, CA). Purified PCR products were sequenced bidirectionally with the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit version 3.1 (Applied Biosystems, Foster City, CA). In a 10 μl final reaction volume, 5.0 μl purified PCR product, 4.0 μl BigDye Terminator ready reaction mix, and 3.2 pmoles of primer were used. Cycling conditions for sequencing reactions, purification of sequencing reaction products using isopropanol (Applied Biosystems), and electrophoresis were performed as described elsewhere . Sequencing results were assembled and analyzed using the SeqMan II program of the Lasergene package (DNA STAR Inc, Madison, WI).
We identified a four-generation family with 27 individualss affected with isolated ectopia lentis. Twenty-four people presented for complete ophthalmological workups. In addition, complete detailed clinical examinations including echocardiography could be performed in six people (Table 1). All mutation carriers showed ocular involvement only. Abnormalities of the cardiovascular system, the skeleton, and the integument were excluded as well as striae atrophicae or a history of hernia.
Linkage and haplotype analysis
We obtained positive lod scores with markers at 15q (Table 2). A maximum positive lod score of 5.7 at θ=0.000 was obtained with marker D15S1024. The relevant risk haplotype showed complete cosegregation in all the 22 analyzed affected individuals and in none of the 24 unaffected individuals (Figure 1).
Sequencing of FBN1 in two affected (III-28, IV-56) and one unaffected individual (II-6) showed a heterozygous C>T change in the affected individuals at nucleotide position c.718 from the translation start site (Figure 2). This nucleotide change resulted in the substitution of highly conserved arginine by cysteine at codon 240 (R240C). All 23 affected family members showed this nucleotide change, which was not seen in any of the 24 unaffected individuals. Further, this nucleotide substitution was not observed in 50 control subjects from the same ethnic background (data not shown).
In the present study, we have characterized the largest family to date with isolated ectopia lentis related to a FBN1 mutation. The mutation identified, R240C, leads to an additional cysteine residue by substitution of a highly conserved arginine residue within the 9-cysteine Fib-like module encoded by FBN1 exon 6. The same mutation has been previously reported in four other families (Table 3). In one family, 12 affected persons showed isolated ectopia lentis. In another family, there was involvement of the integument and joint hypermobility in addition to ectopia lentis. Interestingly, Loeys and colleagues describe an individual with the mutation R240C who had classic Marfan syndrome including ectopia lentis as well as skeletal and cardiovascular involvement .
Several mutations in FBN1 have been reported in patients affected by isolated ectopia lentis including E2447K , R62C [17,22], R545C , and R240C [16,23]. Additionally, FBN1 mutations have been reported in several families with predominant ectopia lentis as well as other skeletal findings , glaucoma , and mild cardiovascular involvement (reviewed in ).
All reported mutations leading to isolated ectopia lentis were arginine to cysteine substitutions mostly confined to the 15 exons of FBN1 . A recent study of over thousand probands with Marfan syndrome and FBN1 mutations demonstrated a striking correlation between ectopia lentis and mutations affecting cysteine residues, leading to the speculation that the pathophysiology of ectopia lentis is more related to a disruption of the structural function of fibrillin-1 in the 10-12 nm extracellular microfibrils in the ciliary zonule rather than a disruption of TBFβ signaling . Disruptions in TGFβ signaling have been demonstrated in the pulmonary and cardiovascular systems in mouse models of Marfan syndrome [26,27] and are thought to represent a major component of the molecular pathogenesis of Marfan syndrome [27,28].
The correlation between the R240C mutation and a purely ocular phenotype in the present family and in the family reported by Adès and coworkers , therefore, might suggest that the R240C mutation primarily disrupts a structural function of fibrillin-1 without having a major effect on TGFβ metabolism. The reasons for the interfamilial variability leading to additional symptoms in several other affected persons (Table 3) are unclear but could speculatively be related to modifying genes involved in the extracellular matrix or TGFβ metabolism.
We wish to thank the patients and their relatives for their cooperation. This work was in part supported by grant number DBT/BT/IC/71/89/Pt from the Department of Biotechnology sanctioned to J.R.S. and INI 331 from Bundesministerium fur Bildung und Forschung to K.S.
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