I Med Genet 1995;32:557-560 557 A fetus with an X; 1 balanced reciprocal translocation and J Med Genet: first published as 10.1136/jmg.32.7.557 on 1 July 1995. Downloaded from

Mary J Seller, Kalyani Pal, Sharon Horsley, Angela F Davies, A Caroline Berry, R Meredith, Alison C E McCartney

Abstract changes consistent with the early stages ofNor- A 19 week female fetus is described with a rie disease. de novo X;1 reciprocal balanced trans- location, with the breakpoint on the X chromosome at Xpll.4, and eye pathology Case report consistent with the early stages of Norrie This was the sixth pregnancy of a healthy white disease. The fetus seems to be an example couple aged 37 and 41 years. The previous of a female manifesting an X linked re- children, five boys and one girl, are well, in- cessive disease, and it was shown that the cluding a boy, one of twins and now 13 years normal X chromosome was completely old, who had surgery during infancy for trans- inactivated in all cells examined. Norrie position ofthe great vessels. The family history disease has been mapped to Xpll.3, and did not indicate anything significant except for fluorescence in situ hybridisation studies a niece on the paternal side who had multiple showed that the Norrie disease gene had abnormalities and died. No precise diagnosis not obviously been disrupted. had been made but a chromosome abnormality screening by SSCP analysis showed no was suspected. For this reason, and on grounds aberrant fragments of the coding region of maternal age, chorionic villus sampling was ofthe gene. Several eye disease genes map performed in the 11th week of pregnancy. The to the same region of the X chromosome, chromosome complement from both direct and but are excluded on grounds ofpathology. cultured preparations showed a female fetus One possibility is that this fetus has a with a balanced reciprocal translocation be- Norrie-like eye disease caused by the tween the X chromosome and chromosome 1. mutation of another gene located at In view ofthis unexpected finding, and because Xpll.4. Ifthis is so, there are implications the tissue sample had been very small and for prenatal diagnosis. obtained from a single site, an amniocentesis was subsequently performed. The chromo- (JMed Genet 1995;32:557-560) some abnormality was confirmed. Both par- http://jmg.bmj.com/ ents had normal karyotypes, so the fetus ap- peared to have a de novo translocation: 46,X, Genes for a number of eye diseases are located t(X;1)(pl1.4;p36.3). The parents were in- on the X chromosome and several, such as formed that, since the X chromosome was Norrie disease, retinitis pigmentosa type 2, involved, there was some risk of a mental or cone dystrophy, congenital night blindness, and physical abnormality in the child, and because

Aland Island eye syndrome, map to the same the gene for Norrie disease has been mapped on September 26, 2021 by guest. Protected copyright. region of the short arm.' One of these, Norrie to the area of the X chromosome involved in South East Thames disease, has been well characterised. the translocation, there was the possibility that Regional Genetics In Norrie disease, blindness is often present this gene might have been disrupted. The par- Centre, 8th Floor from and a of patients later ents termination of the Guy's Hospital Tower, birth, proportion requested pregnancy. Guy's Hospital, develop mental retardation, psychotic be- The female fetus was of a size and stage of London SE1 9RT, UK haviour, and sensorineural deafness.' Linkage development consistent with its gestational age M J Seller analysis and deletion mapping led to the gene of 19 weeks. There was mild ocular hy- K Pal S Horsley being localised to the region of Xpll.3.?- A pertelorism and small, low set ears. The right A F Davies candidate gene was isolated by positional lung had only two lobes, but the fetus was A C Berry cloning, within which have been otherwise grossly normal except for the eyes. Cytogenetics Services, identified in affected, but not in normal, 48 Wimpole Street, people.67 Despite this, chromosomal studies London WlM 7DG, on a family with affected subjects and an X Pathology of the eyes UK R Meredith chromosome rearrangement suggested that the Both eyes were clearly abnormal (fig 1). On gene was located around Xp 1 4.8 The gene macroscopic examination, the right eye showed Division of product has significant homologies to a number an organised retrolenticular fibrotic mass with Histopathology, St Thomas's Hospital, of extracellular proteins,9 and three di- the vessels being ensheathed in a denser mass London SE1 7EH, UK mensional modelling shows a similarity to of fibrous tissue filling the vitreous and causing A C E McCartney transforming growth factor 1.10 focal detachment of the retina. The left eye Correspondence to: We describe a fetus found at prenatal diag- was similar, but less severely affected. It showed Dr Seller. nosis to have a de novo balanced X; 1 reciprocal a mass of vessels persisting in the vitreous Received 25 February 1994 translocation in whom, following termination forming a conical postlenticular mass, from Revised version accepted for publication of pregnancy at 19 weeks, the eyes were clearly which there had been small focal haemorrhages. 27 February 1995 abnormal and appeared to show pathological The leash of vessels was associated with an 558 Seller, Pal, Horsley, Davies, Berry, Meredith, McCartney

each showed the abnormal karyotype with the breakpoint on the X chromosome clearly at p11.4. X inactivation studies were performed on

fibroblast cells using 5-bromodeoxyuridine J Med Genet: first published as 10.1136/jmg.32.7.557 on 1 July 1995. Downloaded from (BrdU) incorporation, followed by staining with Hoechst 33258 in the dark, then ultra- violet light exposure, and final staining with Leishman's. Since this method uses a B pulse of BrdU, late replicating regions appear pale. In all 43 metaphases studied, the normal X chromosome was the late replicating chro- mosome. This normal X was consistently pale throughout its length and so was considered to be completely inactivated. Neither of the translocated products showed any evidence of late replication. In particular, the derived X was always dark staining and so appeared to be the active X chromosome in every cell examined.

DNA probes and in situ hybridisation Chromosomal fluorescence in situ hy- bridisation (FISH) analysis was performed to determine whether the translocation disrupted the Norrie disease gene. Purified DNA from three cosmid clones, M8, G8, and Al0 which cover the entire Norrie disease gene region (kindly supplied by Dr Wolfgang Berger, Human Genetics Department, University Hospital, Nijmegen, The Netherlands), were labelled with biotin by nick translation (Bio Nick Labelling System, BRL Life Tech- nologies, USA) and used individually as FISH probes on metaphase chromosomes." Hy- bridisation was carried out for 16 hours at

37°C, and signals were developed with fluor- http://jmg.bmj.com/ escein labelled avidin and biotinylated anti- avidin. The slides were mounted in antifading medium containing DAPI as counterstain, and Figure 1 Macroscopic appearance of a transverse section of (above) right eye and (below) left eye, showing an organised retrolenticularfibrotic mass more marked in the viewed using a cooled CCD camera and image right eye than the left. analysis system (Digital Scientific Ltd, UK). G banding was enhanced using the Smartcapture software. organising delicate fibrous membrane. Mi- Each of the three cosmids, Al0, G8, and on September 26, 2021 by guest. Protected copyright. the presence of a croscopically, persistent prim- M8, hybridised to the normal X chromosome ary hyperplastic vitreous in both eyes was at Xp 11.3. The three cosmids also hybridised confirmed, arising as a result of the prolonged to the translocated X proximal to the break- presence of the hyaloid artery within the eyes point. No hybridisation signals were seen on and the subsequent development of a fibro- the derived chromosome 1 (fig 2). This would vascular membrane. In the retinas there were suggest that the Norrie gene is intact in this some artefactual Lange's folds, but no evidence patient. of true . As appropriate for 19 weeks gestation, photoreceptor formation was not complete and active cell division and neuroblastic activity was present. There was Mutation screening thus no evidence of a primary neuroectodermal DNA was prepared from cultured fibroblasts defect at this stage. It was considered that the by standard methods and mutation screening postlenticular masses observed were capable of by single strand conformation polymorphism evolving into an organised vitreous which (SSCP) analysis was kindly performed by Do- would have resulted in retinal traction and rien Van de Pol (Human Genetics Department, detachment. The findings thus appeared to University Hospital, Nijmegen, The Neth- be consistent with the early stages of Norrie erlands). No aberrant fragments for the coding disease. part of the Norrie disease gene were detected, suggesting that there is no mutation in this region in this fetus, although it does not com- Cytogenetic studies pletely exclude the possibility of a mutation Blood, umbilical cord, spleen, ovary, and skin there, or of a mutation elsewhere in the gene from the fetus were cultured separately, and outside the open reading frame. A fetus with an X;1 balanced reciprocal translocation and eye disease 559 excluded that the breakpoint was within the most distal part of exon 3, because that region lies approximately 20 kb from the distal end of cosmid M8, and so a hybridisation signal would

AW - have been expected to be visible on the derived J Med Genet: first published as 10.1136/jmg.32.7.557 on 1 July 1995. Downloaded from %, i.0- d Ler(X) chromosome 1 in the FISH analysis. Adjacent to the untranscribed region of exon 1 is a 5' regulatory sequence. Since the 5' end of the gene is orientated towards the centromere,9 it does not seem likely in our case that, even if there was no disruption in the gene itself, this regulatory region has been affected by the trans- location. Another possibility, that the trans- location breakpoint exerts a position effect on the Norrie disease gene, cannot be excluded, a *1' for translocation breakpoints more than 100 kb from SOX9'3 and PAX6'4 are known to be r associated with the respective disease pheno- type. In the latter case, the breakpoint was 3' to the gene. Overall, however, there is no 7.- evidence for a translocation or large deletion of the Norrie disease gene itself in this fetus. I There are several possibilities. There could be a point or other small mutation in a region ofthe Norrie disease gene other than the coding sequence which has arisen de novo in addition -.* to the translocation. However, this is con- sidered unlikely. Another possibility is that the derU b1 fetus has a different eye disorder, either a pre- 77 viously unrecognised "Norrie-like" disease or because one of the other eye disease genes known to be in the same region of the X chromosome, or a dominant gene on chro- mosome 1, has been disrupted by the trans- Figure 2 Fluorescence in situ hybridisation with cosmid G8 showing a sign al on the location. Retinitis pigmentosa 2 is located at short arm of the normal X chromosome, and proximal to the breakpoint on t*he derived X Xp 11.4; however, it, and the other disorders (solid arrow heads), but no signal on the derived chromosome 1. which map to that region of Xp are excluded because their pathology in no way resembles that found in the fetus. There are no known http://jmg.bmj.com/ eye disease genes in the region of chromosome Discussion 1 involved, but a binding pro- This fetus with a balanced X; 1 recipirocal trans- tein is possibly located there. The cloning of location and the breakpoint at Xp 111.4 showed the breakpoints in this case could be a strategy distinctive eye pathology which is consistent to identify a gene for a Norrie-like syndrome. with reported examples of Norrie disease. It maybe ofrelevance to note that it has recently

There are two other published cases of Norrie been found that X linked familial exudative on September 26, 2021 by guest. Protected copyright. disease arising as a result of a chiromosomal vitreoretinopathy, previously thought by link- rearrangement. In one,8 a pericentriic inversion age data to involve a gene close to that ofNorrie of the X chromosome led to the assignment of disease,'5 actually results from a mutation in the gene locus to Xp11.4, while the other a highly conserved region of the Norrie disease case, 12 involving an X;10 balanced trans- gene itself,'617 suggesting allelic heterogeneity location, simply located it at Xp 1. Thus at at the Norrie locus. Because of the lack of the outset it seemed that our feti .is too had knowledge of early pathology, X linked familial Norrie disease. In such cases, it iis assumed exudative vitreoretinopathy cannot be excluded that chromosome breakage occursvwithin, and in this fetus. disrupts, the normal gene. The fact remains that despite the molecular However, linkage studies, usuall)y in micro- genetic findings, the eyes of this fetus clearly deletion cases, have all suggested th,at the Nor- show pathological features consistent with the rie disease gene is located at Xpll1 .3"5 rather presumed early stages of Norrie disease. How- than XpI1.4. In situ hybridisation studies in ever, because of its rarity, the early stages ofthe our case using three cosmids covering the entire pathogenesis are not delineated. A previously Norrie gene confirmed its location at Xp 11.3, reported 11 week fetus,'8 diagnosed as affected and also showed that there was rlo obvious by the use ofgenetic markers, had histologically disruption of the Norrie disease g(ene in this normal eyes, leading to the suggestion that the fetus. In addition, SSCP analysis suggested primary abnormality of vascular proliferation that there is probably no mutation in the coding probably occurs in relation to persistent hy- region of the Norrie disease gene. [he gene is perplastic primary vitreous body after 14 weeks. 27 kb long, and contains three exons;. The open A second case'9 from the third trimester suggests reading frame occurs in the last part of exon 2 that by then overt can-have and the first part of exon 3. The p ossibility is occurred, for diagnosis was made in utero at 34 560 Seller, Pal, Horsley, Davies, Berry, Meredith, McCartney

We are extremely grateful to Dr Wolfgang Berger for the gift of weeks by sonography. The globes had appeared the cosmids and to Dorien Van de Pol for performing the SSCP normal on the scan at 28 weeks but the de- studies. tachments were total at 34 weeks. The findings in our case at 19 weeks were consistent with an 1 McKusick VA. Mendelian inheritance in man. Catalogs of autosomal dominant, autosomal recessive and X-linkedpheno- intermediate stage, with the pathogenetic pro- types. 10th ed. Baltimore: Johns Hopkins University Press, J Med Genet: first published as 10.1136/jmg.32.7.557 on 1 July 1995. Downloaded from cess initiated. There was persistent primary hy- 1992. 2 Warburg M. Retinal malformations. Trans Ophthal Soc UK perplastic vitreous body but no macroscopic 1979;99:272-83. retinal detachment. The X inactivation studies 3 de la Chapelle A, Sankila EM, Lindlof M, Aula P, Norio R. Norrie disease caused by a gene deletion allowing showed that in all cells examined, as far as it is carrier detection and prenatal diagnosis. Clin Genet 1985; possible to ascertain, the normal X was com- 28:317-20. 4 Donnai D, Mountford RG, Read AP. Norrie disease re- pletely inactivated. Ifthis is reflected throughout sulting from a gene deletion: clinical features and DNA the body, then the milder phenotype observed studies. Jf Med Genet 1988;25:73-8. 5 Sims KB, Lebo RV, Benson G, et al. The Norrie disease in the eyes compared with what is seen later in gene maps to a 150kb region on chromosome Xpll.3. gestation and postnatally cannot be explained Hum Mol Genet 1992;1:83-9. 6 Berger W, Meindl A, van de Pol TJR, et al. Isolation of a by incomplete X inactivation, and is a true state- candidate gene for Norrie disease by positional cloning. ment of early pathogenesis. Nature Genet 1992;1:199-203. 7 Berger W, van de Pol D, Warburg M, et al. Mutations in Our case is also interesting because, like the candidate gene for Norrie disease. Hum Mol Genet that of Ohba and Yamashita'2 involving Norrie 1992;1:461-5. 8 Pettenati MJ, Rao PN, Weaver RG, Thomas IT, McMahan disease and an X; 10 translocation, the fetus MR. Inversion (X) (p11.4q22) associated with Norrie was female. These are rare examples of an X disease in a four generation family. Am J Med Genet 1993; 45:577-80. linked recessive gene being manifest in females. 9 Meindl A, Berger W, Meitinger T, et al. Norrie disease is Usually this is because of what is termed "non- caused by mutation in an extracellular protein resembling C-terminal globular domain ofmucins. Nature Genet 1992; random X inactivation".20 This has been con- 2:139-43. firmed in this fetus, where every cell examined 10 Meitinger T, Meindl A, Bork P, et al. Molecular modelling ofthe Norrie disease protein predicts a cystine knot growth has the normal X chromosome inactivated. factor tertiary structure. Nature Genet 1 993;5:376-80. However, it is important to recognise that the 11 Adinolfi M, Davies AF. Non-isotopic in situ hybridisation. Applications to clinical and moleculargenetics. Austin, Texas: actual origin of this observation is probably R G Landes, 1994. not preferential inactivation of one of the X 12 Ohba N, Yamashita T. Primary vitreoretinal dysplasia re- sembling Norrie's disease in a female: association with X chromosomes. Rather, it is believed that at autosome chromosomal translocation. Br J Ophthalmol the outset, inactivation occurred randomly, but 1986;70:64-7 1. 13 Wagner T, Wirth J, Meyer J, et al. Autosomal sex reversal subsequently there was cellular selection in and campomelic dysplasia are caused by mutations in favour of those cells with the most complete and around the SRY-related gene SOX9. Cell 1994;79: 1111-20. genetic constitution.2' This is represented by 14 Jordan T, Hanson I, Zaletayev D, et al. The human PAX6 the cells with the translocated X which bears gene is mutated in two patients with . Nature Genet 1992;1:328-32. the major part of an autosome, without which 15 Fullwood P, Jones J, Bundy S, Dudgeon J, Fielder AR, a person could not survive. Ultimately, in all Kilpatrick MW. X-linked exudative vitreoretinopathy, clin- ical features and genetic linkage analysis. Br J Ophthalmol cells, the normal X chromosome bearing the 1993;77: 168-70. normal allele at the relevant locus is thus in- 16 Chen ZY, Battinelli EM, Fielder A, et al. A mutation in

the Norrie disease gene (NDP) associated with X-linked http://jmg.bmj.com/ active, and the disrupted allele (or other muta- familial exudative vitreoretinopathy. Nature Genet 1993;5: tion ifthis is the case) on the active translocated 180-3. 17 Black G, Redmond RM. Molecular biology of Norrie dis- chromosome is expressed. ease. Eye 1994;8:491-6. Finally, since this case suggests the possibility 18 Parsons MA, Curtis D, Blank CE, Hughes HN, McCartney AC. The ocular pathology of Norrie disease in a fetus of that what is currently regarded as one severe, 11 weeks gestation. Graefes Arch Clin Exp Ophthalmol early onset eye disease may actually be two 1992;230:248-5 1. 19 Redmond RM, Vaughan JI, Jay M, Jay B. In utero diagnosis similar disorders caused by mutations at sep- of Norrie disease by ultrasonography. Ophthal Paediatr

arate but relatively close loci, there are practical Genet 1993;14:1-3. on September 26, 2021 by guest. Protected copyright. 20 Mattei MG, Mattei JF, Ayme S, Giraud F. X-autosome implications. It is important that this possibility translocations: cytogenetic characteristics and their con- is taken into account, particularly by those sequences. Hum Genet 1982;61:295-309. 21 Gartler SM, Sparkes RS. The Lyon-Beutler hypothesis and undertaking the prenatal diagnosis of Norrie isochromosome X patients with the Turner syndrome. disease by linkage analysis. Lancet 1963;ii:411.