A nonsense mutation in the retinal specific guanylate cyclase ... - NCBI

Letters

862 Sincere appreciation is extended to Susanne Emmerich (National Incontinentia Pigmenti Foundation) and the participating families for their willing and continuing cooperation in these investigations. We are grateful to Tracy Jakins, Helen Stewart, and Dian Donnai. We thank John Dean for providing DNA samples. Parts of this work were supported by grants from the Deutsche Forschungsgemeinschaft (DFG), the Genome Analysis Program, Telethon Italy, grant E526 to MD, the UK MRC, and the Foundation Fighting Blindness, Hunt Valley, Maryland, USA. Dr Lewis is a Senior Scientific Investigator of Research to Prevent Blindness, New York, NY, USA. NINA S HEISS ANNEMARIE POUSTKA

Department of Molecular Genome Analysis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany STUART W KNIGHT

Department of Haematology, Imperial College School of Medicine, London, UK SWAROOP ARADHYA DAVID L NELSON

Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, USA RICHARD A LEWIS

Departments of Ophthalmology, Medicine, Pediatrics, and Molecular and Human Genetics, Texas, Houston, USA TERESA ESPOSITO ALFREDO CICCODICOLA MICHELE D’URSO

International Institute of Genetics and Biophysics (IIGB), Via G Marconi 10, 80125 Naples, Italy ASMAE SMAHI SOLANGE HEUERTZ ARNOLD MUNNICH

Hopital des Enfants-Malades, Unité des Recherches sur les Handicaps Génétiques de l’Enfant, Paris, France PIERRE VABRES

Service de Dermatologie, Centre Hospitalier Universitaire, Paris, France HAYLEY WOFFENDIN SUSAN KENWRICK

University of Cambridge, Department of Medicine, Cambridge Institute for Medical Research, Cambridge, UK 1 Heiss NS, Knight SW, Vulliamy TJ, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet 1998;19:32-8. 2 Tantravahi U, Murty VV, Jhanwar SC, et al. Physical mapping of the factor VIII gene proximal to two polymorphic DNA probes in human chromosome band Xq28: implications for factor VIII gene segregation analysis. Cytogenet Cell Genet 1986;42:75-9. 3 Sefiani A, M’rad R, Simard L, et al. Linkage relationship between incontinentia pigmenti (IP2) and nine terminal X long arm markers. Hum Genet 1991;86:297-9. 4 Smahi A, Hyden-Granskog C, Peterlin B, et al. The gene for the familial form of incontinentia pigmenti (IP2) maps to the distal part of Xq28. Hum Mol Genet 1994;3:273-8.

5 Jouet M, Stewart H, Landy S, et al. Linkage analysis in 16 families with incontinentia pigmenti. Eur J Hum Genet 1997;5:168-70. 6 Landy SJ, Donnai D. Incontinentia pigmenti (Bloch-Sulzberger syndrome). J Med Genet 1993;30:53-9. 7 Dokal I. Dyskeratosis congenita: an inherited bone marrow failure syndrome. Br J Haematol 1996;92:775-9. 8 GriYths WA. Reticulate pigmentary disorders - a review. Clin Exp Dermatol 1984;9:439-50. 9 Person JR. Incontinentia pigmenti: a failure of immune tolerance? J Am Acad Dermatol 1985;13:120-4. 10 Esterly NB. Nail dystrophy in dyskeratosis congenita and chronic graft-vs-host disease. Arch Dermatol 1986;122:506-7. 11 Ling NS, Fenske NA, Julius RL, Espinoza CG, Drake LA. Dyskeratosis congenita in a girl simulating chronic graft-vs-host disease. Arch Dermatol 1985;121:1424-8. 12 Knight S, Vulliamy T, Copplestone A, Gluckman E, Mason P, Dokal I. Dyskeratosis Congenita (DC) Registry: identification of new features of DC. Br J Haematol 1998;103:990-6. 13 Sölder B, Weiss M, Jager A, Belohradsky BH. Dyskeratosis congenita: multisystemic disorder with special consideration of immunologic aspects. Clin Pediatr 1988;37:521-30. 14 Jessen RT, Van Epps DE, Goodwin JS, Bowerman J. Incontinentia pigmenti. Evidence for both neutrophil and lymphocyte dysfunction. Arch Dermatol 1978;114:1182-6. 15 Ment L, Alper J, Sirota RL, Holmes LB. Infant with abnormal pigmentation, malformations, and immune deficiency. Arch Dermatol 1978;114:1043-4. 16 Brunquell PJ. Recurrent encephalomyelitis associated with incontinentia pigmenti. Pediatr Neurol 1987;3:174-7. 17 Roberts JL, Morrow B, Vega-Rich C, Salafia CM, Nitowsky HM. Incontinentia pigmenti in a newborn male infant with DNA confirmation. Am J Med Genet 1998;75:159-63. 18 Devriendt K, Matthijs G, Legius E, et al. Skewed X-chromosome inactivation in female carriers of dyskeratosis congenita. Am J Hum Genet 1997;60: 581-7. 19 Vulliamy TJ, Knight SW, Dokal I, Mason PJ. Skewed X-inactivation in carriers of X-linked dyskeratosis congenita. Blood 1997;90:2213-16. 20 Migeon BR, Axelman J, de Beur SJ, Valle D, Mitchell GA, Rosenbaum KN. Selection against lethal alleles in females heterozygous for incontinentia pigmenti. Am J Hum Genet 1989;44:100-6. 21 Parrish JE, Scheuerle AE, Lewis RA, Levy ML, Nelson DL. Selection against mutant alleles in blood leukocytes is a consistent feature in incontinentia pigmenti type 2. Hum Mol Genet 1996;5:1777-83. 22 Meier UT, Blobel G. NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J Cell Biol 1994;127:1505-14. 23 Cadwell C, Yoon HJ, Zebarjadian Y, Carbon J. The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3. Mol Cell Biol 1997;17:617583. 24 Lafontaine DLJ, Bousquet-Antonelli C, Henry Y, Caizergues-Ferrer M, Tollervey D. The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev 1998;12:527-37. 25 Knight SW, Heiss NS, Vulliamy TJ, et al. X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene. Am J Hum Genet 1999;65:50-8. 26 WoVendin H, Jakins T, Jouet M, et al. X-inactivation and marker studies in three families with incontinentia pigmenti: implications for counselling and gene localisation. Clin Genet 1999;55:55-60. 27 Jouet M, Moncla A, Paterson J, et al. New domains of neural cell-adhesion molecule L1 implicated in X-linked hydrocephalus and MASA syndrome. Am J Hum Genet 1995;56:1304-14.

J Med Genet 1999;36:862–865

A nonsense mutation in the retinal specific guanylate cyclase gene is the cause of Leber congenital amaurosis in a large inbred kindred from Jordan EDITOR—Leber congenital amaurosis (LCA) (MIM 204000) has the earliest onset and is the most severe form of retinal dystrophy.1–3 It is an autosomal recessive condition that is recognised within the first few months of life because of impaired vision and an extinguished electroretinogram.4 Nystagmus, specifically pendular, and eye poking are frequently observed early on,5 while hypermetropia and keratoconus may develop later during the course of the disease.6 7 Genetic heterogeneity was confirmed when the first gene of LCA was mapped to chromosome 17p13.1 (LCA1) by homozygosity mapping in consanguineous Arab families.8 9 Four diVerent mutations in the retinal specific

guanylate cyclase gene (RETGC) were found in four unrelated probands and thus LCA1 was assumed to result from homozygous alterations in this gene.10 We report here a nonsense mutation in the RETGC gene, which in the homozygous state is responsible for LCA in a large inbred tribe from Jordan. We had already identified a large, highly inbred family from the Jordan valley consisting of about 2000 living subjects, in which aVected members have LCA.11 A 31 member subset of this family was investigated (fig 1). All members were examined by an ophthalmologist and a paediatrician. Four patients had ERG performed (Nos 3, 9, 13, 14). Blood samples were collected from 28 family members after obtaining informed consent from them or their legal guardian. DNA was extracted from peripheral blood samples by standard procedures.12 Seventeen diVerent dinucleotide repeat markers reported to be linked to LCA1 on chromosome 17 were used to test for linkage.8 9 Amplification of these markers was performed according to the manufacturer’s conditions (Research Genetics). Products

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Figure 1 Extended partial pedigree of the clan. Note the extensive number of inbreeding loops in every generation. The 31 participating family members are marked in Arabic numerals.

were analysed on 6% denaturing polyacrylamide gels (7.7 mol/l urea). The polyacrylamide gels were silver stained using the protocol of Bassam et al.13 Haplotype analysis was performed and the obligatory cross over events were noted. Since the family was highly inbred, identity by descent was enough to establish linkage. The 20 exons of the RETGC gene were amplified using intronic primers flanking exon sequences using the previously reported conditions.10 When necessary, the fragments were digested by one or more restriction endonucleases to yield fragments suitable for SSCP analysis. Amplified fragments were run on a fan cooled MDE gel for SSCP analysis at 6 W for 14 hours and then silver stained. DNA from regions of the RETGC gene that showed unusual mobility of one allele in carriers and of the two alleles in aVected subjects was sequenced using an automated ABI sequencer with dye terminator chemistry. The reported extended family is a 2000 member tribe inhabiting a village in the Jordan valley, mostly depending on agricultural resources. The successive consanguineous marriages led to an extreme example of inbreeding and is reflected in the high prevalence of blindness which turned out to be LCA. The inbreeding coeYcient in this tribe ranged from 0.037 to 0.09374 with an average of 0.0687. The subset of the family included 13 aVected subjects, their ages ranging from 10 to 45 years at the time of examTable 1

ination. All patients had poor vision noted at birth or shortly afterwards, as well as wandering eyes or pendular nystagmus. The visual acuity ranged from no light perception to 6/60. The majority had attenuated retinal blood vessels on fundus examination, some had pale optic discs, and about two thirds had keratoconus. Two patients had congenital cataracts (Nos 28 and 29) and one patient had bilateral microphthalmia and iris atrophy (No 16). One patient had bilateral macular abnormality similar to target macular lesions (No 17). Extinguished ERG was present in the four patients who underwent the test. The details of the clinical picture in the 13 aVected subjects are summarised in table 1. By analysing the haplotypes, it is quite obvious that the LCA in this family is linked to the LCA1 locus previously described. All aVected members were identical by descent for the disease haplotype (fig 2). The SSCP assay of exon 13 showed a homozygous pattern in aVected subjects and a heterozygous pattern in obligate carriers. This pattern was not present in 10 unrelated controls. The DNA sequencing in all aVected members showed a homozygous Gln to stop mutation in exon 13 at nucleotide position 2646 (cDNA) (CAG→TAG) (fig 3). Obligate carriers were heterozygous for this nonsense mutation. Despite being homozygous for the same mutation, aVected family members showed clinical heterogeneity for

The main clinical manifestations of Leber congenital amaurosis in the 13 aVected subjects Visual acuity

ID

Age/sex

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Left

Keratoconus

Retinal vessels

Optic discs

Remarks

3 9 13 14 15 16 17 21 22 23 28 29 30

45 y/M 22 y/M 14 y/M 15 y/F 19 y/M 20 y/F 23 y/F 15 y/M 13 y/M 10 y/F 11 y/F 13 y/M 16 y/F

No LP HM No LP HM 6/60 LP HM HM HM CF No LP LP LP

No LP HM No LP HM 5/60 LP HM HM HM CF LP LP LP

Yes Yes Yes Yes Yes

Attenuated Attenuated Attenuated Attenuated Attenuated Microphthalmia Attenuated Attenuated Attenuated Attenuated Not seen Not seen Attenuated

Pale Pale Normal Normal Normal Not seen Pale Normal Normal Normal Not seen Not seen Normal

Corneal hydrops

Yes No Yes No No Yes No

M: male; F: female; LP: light perception; HM: hand movement; CF: counting fingers.

Corneal hydrops Iris atrophy Macular lesions

Cataracts Cataracts

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D17S796 D17S720 D17S960 D17S1796 D17S1353 D17S1881

Figure 2 Haplotypes of six selected dinucleotide markers within the region of linkage on 17p13.1. The markers are arranged from telomere to centromere. The marker names are shown in the closed box. The aVected subjects are homozygous for the extent of the haplotype.

symptoms and signs other than the impaired visual acuity and nystagmus. This suggests that other factors, possibly environmental, as well as genetic play a role in the variability in clinical expression of this monogenic disorder. Since the mutations detected so far denote either profound instability of the protein or premature translation termination, it strongly suggested that LCA is the result of abolished production in cGMP in photoreceptor cells.10 The mutation in our family produces premature termination in translation, which strengthens this suggestion. The presence of congenital cataracts and congenital microphthalmia in this family suggest that the RETGC gene may play a role in eye development in utero as well. Linkage analysis in this family followed by the detection of the mutation provides us with a potent set of tools for carrier identification. This can be applied to premarital testing and counselling, which provides a socially acceptable solution to this problem in a large family with widely practised intermarriage. Prenatal diagnosis can also be provided to married couples who are known carriers. The authors are very grateful to Jordan University of Science and Technology, Yarmouk University, and the University of Jordan for support. This study was partially funded by the Higher Council for Science and Technology. We wish to

acknowledge support from the Grousbeck Family Foundation and the Roy J Carver Charitable Trust (support for VCS, JB, and EMS). We would also like to thank Dr J Kaplan and her coworkers for sharing information about the RETGC gene with us. HATEM EL-SHANTI

Departments of Paediatrics and Medical Laboratory Sciences, School of Medicine, Jordan University of Science and Technology, Irbid, Jordan and National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan MAHMOUD AL-SALEM

Department of Ophthalmology, School of Medicine, Jordan University of Science and Technology, Irbid, Jordan MAHMOUD EL-NAJJAR

Department of Physical Anthropology, Yarmouk University, Irbid, Jordan KAMEL AJLOUNI

Department of Internal Medicine, School of Medicine, Jordan University, Amman, Jordan and National Center for Diabetes, Endocrinology and Genetics, Amman, Jordan JOHN BECK VAL C SHEFFIELD

Howard Hughes Medical Institute and Department of Pediatrics, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA EDWIN M STONE

Department of Ophthalmology, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA

Figure 3 Sequence from forward (above) and reverse (below) directions. The mutation is homozygous and lies within exon 13. The change is a T (TAG=stop) in place of C (CAG=Gln). The T is flanked by As and is marked with a cap.

Letters

1 Leber T. Ueber Retinitis pigmentosa und angeborene Amaurose. Albrecht von Graefes Arch Ophthalmol 1869;15:1-25. 2 Foxman SG, Hechenlively JR, Batemen BJ, Wirstschafter JD. Classification of congenital and early-onset retinitis pigmentosa. Arch Ophthalmol 1985;103:1502-7. 3 Kaplan J, Bonneau D, Frezal J, Munnich A, Dufier JL. Clinical and genetic heterogeneity in retinitis pigmentosa. Hum Genet 1990;85:635-42. 4 Franceschetti A, Deiterie P. L’importance diagnostique de l’electoretinogramme dans les degenerescence tapeto-retiniennes avec retrecissement du champ visuel et hemeralopie. Conf Neurol 1954;14:184-6. 5 Franceschetti A, Forni S. Degenerescence tapeto-retinienne (type Leber) avec aspect marbre du fond de l’oeil peripherique. Ophthalmologica 1958;135:610-16. 6 Wagner RS, Caputo AR, Nelson LB, Zanoni D. High hyperopia in Leber’s congenital amaurosis. Arch Ophthalmol 1985;103:1507-9.

865 7 Karel I. Keratoconus in congenital diVuse tapetoretinal degeneration. Ophthalmologica 1968;155:8-15. 8 Camuzat A, Dollfus H, Rozet JM, et al. A gene for Leber’s congenital amaurosis maps to chromosome 17p. Hum Mol Genet 1995;4:1447-52. 9 Camuzat A, Rozet JM, Dollfus H, et al. Evidence of genetic heterogeneity of Leber’s congenital amaurosis (LCA) and mapping of LCA1 to chromosome 17p13. Hum Genet 1996;97:798-801. 10 Perrault I, Rozet JM, Calvas P, et al. Retinal-specific guanylate cyclase gene mutations in Leber’s congenital amaurosis. Nat Genet 1996;14:461-4. 11 Al-Salem M. Leber’s congenital amaurosis in 22 aVected members of one family. J Pediatr Ophthal Strabismus 1997;34:254-7. 12 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. 13 Bassam BJ, Caetano-Anolles G, GresshoV PM. Fast and sensitive silver-staining of DNA in polyacrylamide gels. Anal Biochem 1991;196:80-3.

J Med Genet 1999;36:865–866

Skewed X chromosome inactivation in a female with haemophilia B and in her non-carrier daughter: a genetic influence on X chromosome inactivation?

patient’s DNA with HpaII one double band only, the band inherited from the father, was visible. The patient therefore had an extremely skewed XIP with the maternally inherited X chromosome as the active X chromosome in the majority of the cells. Identical results were obtained with DNA from

I 1

EDITOR—Phenotypic expression of X linked disorders in females may be the result of an X chromosome anomaly or homozygosity for the mutated gene, but is probably most frequently the result of skewed X chromosome inactivation. Skewed X inactivation may be the result of a chance event, but may also be because of genetic factors.1 We report here the results of X inactivation analysis in a family with haemophilia B, which showed extremely skewed X inactivation both in a female haemophilia patient and in her non-carrier daughter, indicating a possible genetic influence on X chromosome inactivation in this family. Familial skewed X inactivation may interfere with carrier detection, since skewed X inactivation with the mutant gene on the inactive X chromosome may lead to a normal phenotype in a carrier. The patient was a 40 year old female who belonged to a family with moderate haemophilia B2 (fig 1). Plasma factor IX (FIX) activity was 0.02-0.03 units/ml, which was identical to the FIX activity in the aVected male relatives. She had suffered from bleeding episodes after tooth extraction as a child and heavy bleeding from the episiotomy after her first delivery. A C→T transition, causing mutation S360L in exon 8 of the factor IX gene, was found in the male relative with haemophilia using single strand conformation analysis (fig 1, II.4). Both the female haemophilia patient and her mother (III.1 and II.3) were heterozygous for the mutation, whereas her two daughters (IV.1 and IV.2) did not have the mutation. The X chromosome inactivation pattern (XIP) was determined by PCR analysis of a polymorphic trinucleotide repeat in the first exon of the androgen receptor (AR) gene which, after digestion with the methylation sensitive enzyme HpaII, gives a PCR product from the inactive X chromosome only.3 XIP was measured as the ratio between the PCR products from the two X chromosomes and scored as random (ratios 50:50-80:20) or skewed (ratios >80:2095:5). When one band only was visible, the pattern was scored as extremely skewed (ratios >95:5). DNA from a girl with a deletion within chromosome Xp22 who had previously been shown to have a skewed XIP was used as a control. Samples containing mixtures of DNA from two males with well separated bands were run as quantitative controls. The most extreme mixture that gave two visible bands was 95:5. The results of X inactivation analysis are shown in fig 2. Each allele at the AR locus gives two equally strong bands on a denaturing polyacrylamide gel owing to the two complementary DNA strands migrating slightly diVerently during electrophoresis. After digestion of the

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DXS 559 DXS 56 DXS 6789 DXS 6799 DXS 6797 DXS 6804 DXS 1001 DXS 1047 GATA 31 E08 DXS 1193

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Active allele in X inactivation analysis

Figure 1 Pedigree of haemophilia family modified from Ørstavik et al.2 The mutation 360L was identified in II.4, II.3, and III.1, but not in IV.1 or IV.2.