clinical and linkage study of a large family with simple ectopia lentis linked to fbn1

American Journal of Medical Genetics 53:65-71 (1994)

Clinical and Linkage Study of a Large Family With Simple Ectopia Lentis Linked to FBNl

M.J. Edwards, C.J. Challinor, P.W. Colley, J. Roberts, M.W. Partington, G.E. Hollway, H.M. Kozman, and J.C. Mulley Newcastle and Northern New South Wales Genetics Service (M.J.E., J.R., M . W.P.), Department of Ophthalmology (C.J.C.) and Division of Molecular and Cell Biology (P.W.C.), John Hunter Hospital, Newcastle, New South Wales; and Department of Cytogenetics and Molecular Genetics (G.E.H., H.M.K., J.C.M.), Adelaide Children’s Hospital, North Adelaide, South Australia, Australia

Simple ectopia lentis (EL) was studied in a large family, by clinical examination and analysis of linkage to markers in the region of FBNl, the gene for fibrillin which causes Marfan syndrome on chromosome 15. No patient had clinical or echocardiographic evi- dence of Marfan syndrome, although there was a trend towards relatively longer measurements of height; lower segment; arm span; middle finger, hand, and foot length in the affected members of the family, compared with unaffected sibs of the same sex. Analysis of linkage to intragenic FBNl markers was inconclusive because they were relatively uninformative. Construc- tion of a multipoint background map from the CEPH reference families identified microsatellite markers linked closely to FBNl which could demonstrate linkage of EL in this family to the FBNl region. LINKMAP analysis detected a multipoint lod score of 5.68 at D15S119, a marker approximately 6 cM distal to FBN1, and a multipoint lod score of 5.04 at FBNl. The EL gene in this family is likely to be allelic to Marfan syndrome, and molecular characterization of the FBNl mutation should now be possible. 0 1994 Wiley-Liss, Inc.

KEY WORDS: ectopia lentis, autosomal dominant, Marfan syndrome, Fib 15 gene, FBN1, fibrillin, echocardiography, ophthalmology, anthropometry, linkage

Received for publication February 18, 1994; revision received May 20,1994.

Address reprint requests to M.J. Edwards, Newcastle and Northern NSW Genetics Service, Newcastle Western Suburbs Hospital, Post Office Box 84, Waratah, New South Wales 2298, Australia.

0 1994 Wiley-Liss, Inc.

INTRODUCTION Dislocation of the lens of the eye, or ectopia lentis

(EL), may occur in a number of generalised disorders, most notably the Marfan syndrome (MFS) and homo- cystinuria, but also in the Ehlers-Danlos syndrome, the Weil-Marchesani syndrome, a recently described syndrome of oxycephaly and retinal detachment [Reichel et al., 19921, other metabolic disorders such as sulphite oxidase deficiency or molybdenum cofactor deficiency [Beemer et al., 19851, and in association with several local ocular conditions such as ectopic pupils [Waar- denburg et al., 19611, blepharoptosis and myopia [Gillum and Anderson, 19821, aniridia and the Rieger anomaly, as well as trauma and infection [see Jaureguy and Hall, 1979; and Nelson and Maumenee, 1982 for reviews].

Simple EL implies that the lens dislocation is the primary event and, although there may be local secondary consequences (e.g., glaucoma, astigmatism), there are no associated abnormalities elsewhere in the body. Familial cases have been described for many years but as Jaureguy and Hall [19791 point out, there is seldom enough information in the early re- ports to decide whether the EL was simple or part of a generalised disorder. More recently, both auto- soma1 recessive [a1 Salem, 1990; Ruiz et al., 19861 and autosomal dominant [Casper et al., 1985; Jaureguy and Hall, 1979; Sinha and Rahman, 19801 inheritance have been documented. Tsipouras et al. [19921, in a study of 2 families, found a lod score of 3.0 (e = 0.00) using FBNl markers, which strongly sug- gests that the gene for autosomal dominant simple EL is allelic to that of MFS and represents a mutation affecting the fibrillin complex. Allelism is reinforced further by the recent characterization of a FBNl mutation in an EL family [Kainulainen et al., 19941.

We describe clinical and linkage studies of a large Australian family with simple EL. Linkage to the region of FBNl is confirmed, indicating that EL so far represents a homogeneous genetic disorder, and is likely to be allelic to MFS.

66

I

II

111

IV

V

VI

Edwards et al.

I

7 38 3940 41 42 3 4 4

1 2 3 4 5 6 7 8 9 10 1 1 12 1 3 1 4 1 5 16 17 18 19 2 0 2 1 22 23 24 25 IY 26

T Fig. 1. Pedigree. 6: participated in linkage study.

MATERIAL AND METHODS The family was of Irish descent and lived in New

South Wales. The pedigree is shown in Figure 1. A his- tory and physical assessment, formed with knowledge of whether the person was affected or not, was obtained from all participants. Measurements were made of height; lower segment; occipito-frontal circumference (OFC); ear length; inter-inner canthal; pupillary and outer canthal distances; arm span; and the lengths of the hand, third finger, and foot according to the methods of Hall et al. [1989]. Individuals IV-2, 14-17; V-1, 4-6, 29, 43; and VI-2, 3 all had cardiological assess- ments, including echocardiography. The presence of EL was confirmed or excluded by at least one ophthalmol- ogist and one of us (C.J.C.) examined the eyes of the participating affected parents in generation V and their children.

Blood specimens were collected for DNA extraction using EDTA as the anticoagulant. Southern blots were performed with standard methods [Southern, 19751 using the following probes which were labelled by random priming [Feinberg and Vogelstein, 19841: MF13 [Lee et al., 1991; Tsipouras et al., 19921, pCLM8 [Clark et al., 19911, pCLM9 [Dietz et al., 19911, and pMS1-14 (D15S1) [Barker et al., 19841. The markers defined by MF13, pCLM8, and pCLM 9 are within FBNl. Nylon membranes were washed at medium stringency (final wash 1 x SSPE, 0.1% SDS at 65°C for 5-10 minutes). A (TAAAA)n PCR polymorphism within FBNl [Lee et al., 1991; Tsipouras et al., 19921 was identified by staining of a 12% polyacrylamide gel with ethidium bromide.

The linked microsatellite markers D15S118, D15S119, 0155'123, D15S126, and D15S172 were genotyped in the EL family as described elsewhere [Mulley et al., 19911, using primers listed in the Genome Database. Two-point linkage analysis for the EL family was carried out using MLINK [Lathrop and

Lalouel, 19841. The multipoint background map of genetic markers was constructed using CRIMAP [Lander and Green, 19873 with genotypes from CEPH families. The EL gene was positioned onto this background map using LINKMAP [Lathrop et al., 19841. Risk analysis to determine carrier status for EL with flanking markers was carried out with MLINK using principles described previously [Mulley et al., 19913. Genetic distances for risk analysis were taken from the background map.

RESULTS There were 28 affected individuals in 5 generations.

Nineteen were males and 9 were females, a sex ratio which is within chance expectation (x2 = 2.89: P = 0.09). There were no affected persons without an affected parent. In the offspring of affected individuals the numbers of affected and unaffected males, affected and unaffected females (17:14:8:13) were not signifi- cantly different from the equal numbers expected (x2 = 2.52: P = 0.45). There were 8 instances of male to male transmission. All this indicates autosomal dominant inheritance.

ECTOPIA LENTIS Patient N-2 (Fig. 1) had been told at age 33 years

that the lenses were slightly dislocated. At 51 years there was some blurring of left vision; visual acuities were 616 OD and 6/36 0s. There was almost complete loss of zonules without vertical dislocation of the lenses which appeared to be adherent to the iris. Left cataract extraction and intraocular lens insertion were performed with improvement in vision to 619. Her son (V-4) had iridodonesis at age 2 years; subsequently he had bilateral lens extractions and developed glaucoma. V-6 was first noted to have iridodonesis when blurred vision began at age 13 years; the left lens was extracted and the right one has subluxed superiorly with some

Clinical and Linkage Study of Ectopia Lentis 67

70 - :: 60 ~

a *

$ 4 0 - n

30 -

m

L

C

m

cataract change. V-1 had iridodonesis at birth but eye- sight was not affected until age 15 years. His son (VI-2) had bilateral superiorly subluxed lenses first noted at one year; at 3 years he required bilateral myopic astig- matic correction; the right lens was extracted and he achieved 619 corrected acuity with a contact lens. V-1’s other son VI-3 had a visual acuity of 619 and asymptomatic subcapsular lens opacities; he also had craniosynostosis and postaxial polydactyly of both feet and one hand. V-5 has hypermetropia, nystagmus, and a convergent strabismus, and 2 daughters (VI-4, 5) with the same problems; none of these 3 individuals have lens dislocation. The diagnosis was made in IV-15 at age 50 years; she had a high degree of myopia and bilateral cataracts, small corneas and narrow anterior chamber angles. At the time of left cataract extraction the lens was not subluxed but the zonules were defi- cient and were adherent to the lenses. When the right cataract was later extracted, the lens was noted to be subluxed only at operation. She was treated with corti- costeroid for rheumatoid arthritis, so the cause of the cataracts was uncertain. The age at which the diagnosis was made in the family is shown in Figure 2.

CARDIAC STATUS No member of the family had symptoms or signs of

aortic root dilatation or aortic valvular disease. One patient died at age 50 years of coronary atherosclerosis, confirmed by a coroner’s examination, and 2 others had aortic aneurysms. 111-3 died of a ruptured abdominal aortic aneurysm at age 73 years. 111-7 survived surgery for an abdominal aortic aneurysm at age 67 years, and a mesenteric artery aneurysm has since been diagnosed. The aortic root diameter of patient V-l was 3.6 cm, at the upper limit of normal a t age 30 years, and he had mitral valve prolapse without regurgitation on echocardiography. His brother (V-4) also had mitral valve prolapse and mild mitral valve regurgitation when assessed at age 30 years. The actuarial mortality curve [Cutler and Ederer, 19581 for the family is shown in Figure 3, with data for the general population of the state of New South Wales.

\

\\\ 50--

U

80 : 2o 1 lo 0 ? 0 1 0 20 30 40 50 60 70 80

Aoe in Years

Fig. 3. Actuarial mortality curves for the affected members of the family and for the general population of the state of New South Wales. -0-, ectopia lentis; -0-, all NSW.

ANTHROPOMETRY All the measurements of physical growth were

within normal limits. The affected individuals were somewhat short in height and none had a Marfanoid habitus (Fig. 4). An unexpected finding was that the adults with EL were, on average, larger in all the bod- ily measurements than those in the family without EL (Fig. 5). These differences were statistically significant for the women (with P values on t-tests < 0.05 or 0.01) but not for the men, probably because there were too few unaffected males for comparison.

LINKAGE STUDIES As the intragenic markers for FBNl were poorly in-

formative for this family, a background map of markers based on the CEPH families was constructed. This de- termined the PCR-based markers that closely flank the Marfan syndrome locus, FBNl, which could be applied to the linkage study in the EL family. The background map is shown in Table I.

1

0.9

0.8 - ? 0.7 -

?3 0.6 -

P m

P

0 0 10 20 3 0 4 0 5 0

Age in years Fig, 4. Family photograph, showing from left to right 111-5 (unaffected), 111-4 (affected), 111-9 (unaffected), 111-8 (unaffected), 111-7 (af-

~

Fig. 2. Age of diagnosis. fected), and 111-3 (affected).

68 Edwards et al.

180 I rn

160 140 120 100 80 60 40 20 0

Mid finger length, cm 10,

Foot length, cm

25

20

15

10

5

0

.-- I

Hand length, an

25 I 20

15

10

5

0

Lower segment length, cm lool

Fig. 5. Anthropometrical measurements of adults with ectopia lentis, compared with adult unaffected relatives of the same sex. k3, Unaffected sister; I, affected sister; U, unaffected brother; 0, affected brother.


explanation might be that the pairs of markers D15Sl- 0158123, and D15S126-Dl5S119 are inverted because of undetected genotyping errors. The purpose of this in- vestigation was to distinguish between the possibility of allelism between EL and MFS versus the possibility that EL mapped elsewhere in the genome. We conclude that in this family the EL mutation is likely to be

TABLE I. Background Genetic Map Showing Markers Flanking FBNl, Chromosome 15q21.1

sex average ( c ~ )

11.6

Odds against inversion

1019:1

Marker

D15S118

D15S172

FBNl within the FBNl gene. 4.1 104:1

D15S1 1.7

2.4 D15S103

D15S123

D15S126

1.2

0.0

1O:l

103:1

1O:l

1:l

1.0 10:l D15S119

6.7 1 0 ' O : l D15S117

Individual VI-17, aged 20 months is either an asymptomatic carrier or a recombinant for FBNl markers (defined by the probes MF13 and pCLM9). Using the informative flanking markers D15S118 and D15S126, and using unknown affection status for VI-17, the chance that she carries the same fibrillin gene as the other affected relatives is 98.9%, by risk analysis. VI-3, aged 9 years, is also either an asymptomatic carrier or a recombinant with 01551. The chance that VI-3 carries the gene, using unknown affection status for him and informative flanking markers D15S172 and D15S126, is 99.7%. With VI-3 and VI-17 re-coded as carriers, the two-point lod scores for linkage to the FBNl region are listed in Table 11. These data show that the gene for EL in this family is either a t the FBNl locus (multipoint lod score 5.04) or slightly distal to FBNl (multipoint lod score 5.68) (Fig. 6) . Although the likelihood in this analysis that EL maps to D15S119 is greater than the likelihood that it maps to FBNl, this observation could be explained by low informativeness of FBNl markers or the possibility that the recombina- tion event between EL and D15Sl and between EL and D15S123 was not detectable using the markers D15S126 and D15S119. This might occur if the individual in which the crossover occurred was homozygous for markers D15S126 and D15S119. Another plausible

-

DISCUSSION In 1985 Professor T.J.C. Boulton of Newcastle Uni-

versity, New South Wales, suggested that this family might have simple EL with autosomal dominant inheritance. Our studies confirm this but until quite recently several members of the family have been given a mis- taken diagnosis of MFS and warned of a likelihood of early death from aortic rupture, and a need for frequent cardiac monitoring. Clearly the prognosis in simple EL is quite different from that of MFS, even though the gene maps at or close to FBNl. One unexpected finding was the slightly longer measurements of height, hand, and foot lengths in adults with EL compared with the unaffected adults in the family. Conclusions must be limited by the small number of individuals for comparison, but the trend does suggest that comparison of measurements in other families with simple EL would be worthwhile. It also raises the possibility that although the main pathological effects of the gene fall on the suspensory ligament of the lens, minor more gener- alized effects may be present.

Genotypes for MF13 and pCLM9 were identical in all family members tested, and the linkage data for these are presented together in Table I1 as though they rep- resent the same locus. IV-2 and her affected descen- dants had a different D15Sl allele to affected members of the remainder of the family, indicating that a recom- bination had occurred between EL and this locus between the two branches of the family. Rare recombi- nants between FBNl and D15S1 have been reported [Sarfarazi et al., 19921. VI-3 had craniosynostosis and polydactyly, and it is likely that he had EL in addition, with the same unusual ocular manifestations as IV-15, who also had cataracts and minor ectopia lentis with loss of zonules which was not diagnosed until her cataract operation as an adult. VI-17 had a normal phenotype and was apparently recombinant for pCLM9/MF13 and D15Sl. She is only 20 months old,

TABLE 11. Lod Scores Between EL and Closely Linked DNA Markers

Z at 0 of-

Marker .oo .01 .05 .1 .2 .3 .4

D15S118 D15S172 FBNl (TAAAA)n FBNl (MF13/pCLM9) FBNl (pCLM8) D15S1 D15S123 D15S126 D15S119

--m -0.98 0.32 0.73 0.82 --m -0.09 0.58 0.80 0.85

0.83 0.81 0.74 0.64 0.45 1.06 1.04 0.95 0.84 0.63

0.58 0.57 0.52 0.46 0.35 --m 0.47 1.0 1.09 0.93 --m 2.97 3.43 3.33 2.67

5.99 5.89 5.47 4.93 3.77 4.47 4.38 4.04 3.60 2.66

0.59 0.27 0.67 0.38 0.28 0.13 0.42 0.21

0.23 0.11 0.62 0.26 1.78 0.85 2.51 1.18 1.65 0.66

70 Edwards et al.

Generic Dismce (cM)

Fig. 6. Placement of EL on the background map. Maximum location scores were 26.12 at D15DI19 (equivalent to a multipoint lod score of 5.68) and 23.2 at FBNl (equivalent to a multipoint lod score of 5.04).

and probably is an asymptomatic carrier, consistent with incomplete penetrance at an early age.

These results confirm the earlier mapping of simple EL to the FBNl region of chromosome 15, the gene that causes MFS [Tsipouras et al., 19921. Simple ectopia lentis probably represents an allelic variation of the gene that causes MFS. This is reinforced clinically by the anthropological measurements which show a trend towards longer extremities in those who have EL, and by recent immunohistochemical studies of the skin of IV-17, which showed reduced staining of elastin profiles in the reticular dermis with antibodies to fibrillin [M. Godfrey, 1994, University of Nebraska, personal communication]. Allelism is also reinforced by the recent molecular characterization of the first FBNl mutation in another EL family [Kainulainen et al., 19941. It would be of interest to characterize the mutation in the present family in order to determine if the mutations responsible for EL are restricted to certain re- gions of the FBNl gene.

ACKNOWLEDGMENTS We thank the family members for their participation

and Dr. David Reith for help with the clinical studies.

Dr. Maxwell B. Simpson provided valuable operative details of patient IV-15. Probes pCLM8 and 9 were a kind gift of Dr. Cheryl Maslen, and MF13 was received from Dr. Francesco Ramirez. The linkage studies were supported by the National Health and Medical Re- search Council of Australia. We thank Professor Jeffrey Murray for on-line access to the CHLC database used for construction of the multipoint background map.

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clinical and linkage study of a large family with simple ectopia lentis linked to fbn1

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