Genetic study in a tunisian family revealed IVS1+1G>A mutation in the CHM gene

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1 Ann Biol Clin 2015; 73 (4): Genetic study in a tunisian family revealed IVS1+1G>A mutation in the CHM gene La choroïdérémie : révélation de la mutation IVS1+1G >A du gène CHM dans une famille tunisienne Ilhem Ben Charfeddine 1 Taheni Ben Lazreg 1 Narjes Ben Rayana 2 Abdelbasset Amara 1 Ons Mamaï 1 Leila Knani 2 Amira Mili 1 Ahlem M sakni 1 Ali Saad 1 Fafani Ben Hadj Hamida 2 Moez Gribaa 1 1 Laboratoire de cytogénétique, génétique moléculaire et biologie de la reproduction humaines, CHU Farhat Hached, Sousse, Tunisie <ilhem_bc@yahoo.fr> 2 Service d ophtalmologie, CHU Farhat Hached, Sousse, Tunisie Article received May 29, 2014, accepted September 10, 2014 Abstract. Choroideremia is a rare X-linked recessive, hereditary retinal pigment epithelial dystrophy, characterized by night blindness and progressive constriction of the visual fields leading to blindness in young adulthood. In this study, we reported three cases of choroideremia belonging to a Tunisian family. Patients complained of vision loss and night blindness. Fundus examination revealed diffused chorioretenal atrophy. In all cases, there was a visual field constriction and an undetectable electroretinography. Direct sequencing of the CHM gene detected a guanine to adenine transition (G>A) into the donor splice site of intron 1 leads to aberrantly spliced mrna producing a premature stop codon and therefore functional loss of the CHM gene product, REP-1. The diagnosis should be considered in patients with a suitable family history and fundus findings. Key words: choroideremia, CHM gene, Rab escort protein-1 Résumé. La choroïdérémie est une maladie héréditaire rare à transmission récessive liée au chromosome X. De façon caractéristique, les hommes atteints développent une cécité nocturne à l adolescence, suivie d une réduction progressive du champ visuel, aboutissant le plus souvent à la cécité complète vers l âge de 50 ans. Cette pathologie résulte de la mutation du gène CHM, localisé sur le bras long du chromosome X, en Xq21.2. Dans ce travail, nous rapportons l étude clinique et génétique de trois patients atteints de choroïdérémie et faisant partie d une même famille tunisienne. Le séquençage du gène CHM nous a permis de trouver, chez ces trois patients, la même mutation IVS1+1G>A à l état hémizygote. Un conseil génétique s impose pour cette famille dont la prise en charge actuelle repose essentiellement sur l aide visuelle avec un suivi ophtalmologique périodique. Mots clés : choroïdérémie, gène CHM, Rab escort protein-1 doi: /abc Choroideremia (CHM) is an X-linked eye disease characterized by the progressive degeneration of the retinal pigment epithelium (RPE), neurosensory retina, and choroid. Affected hemizygous males develop slowly progressive retinal degeneration leading to night blindness in their teens, loss of peripheral vision, and complete blindness by middle age. Heterozygous females, in contrast, have no or milder symptoms, probably dependent on the degree of X chromosome inactivation. Female carriers can, however, be reliably Reprints: I. Ben Charfeddine identified by ophthalmoscopy [1, 2]. Choroideremia (CHM) is caused by mutations in the widely expressed CHM gene located on chromosome Xq21. The CHM gene comprises 15 coding exons that span a genomic sequence of approximately 186 kb [3]. The product of this gene is a protein of 653 amino acids, designated Rab escort protein isoform 1 (REP)-1, which plays an essential role in the post-translational lipid modification and subsequent membrane targeting of Rab proteins [4]. Rab proteins are low molecular weight GTPases of the Ras superfamily involved in the regulation of exocytic and endocytic cellular pathways. In particular, a reduction To cite this article: Ben Charfeddine I, Ben Lazreg T, Ben Rayana N, Amara A, Mamaï O, Knani L, Mili A, M sakni A, Saad A, Ben Hadj Hamida F, Gribaa M. Genetic study in a tunisian family revealed IVS1+1G>A mutation in the CHM gene. Ann Biol Clin 2015; 73(4): doi: /abc

2 in activated Rab27a has been implicated in choroideremia because of preferential binding by REP-1. REP-1 is a protein which escorts the Rab proteins during and after their prenylation to intracellular membranes. This protein also plays an important role in the prenylation reaction itself in retinal tissues as it is indispensable for the activity of Rab geranylgeranyl transferase (RabGGTase) [5]. Here, we describe the clinical features in choroideremia Tunisian family, diagnosed in the service of ophthalmology of the CHU Farhat Hached-Sousse. All patients showed vision loss and night blindness. Heterozygous carrier female also manifested no or milder symptom probably dependent on the degree of X chromosome inactivation. We also reported the results of molecular analysis of the CHM gene in three cases of choroideremia in which a splice site mutation was detected. Materials/methods Patients All our patients are belonging to the same family (figure 1) diagnosed in the laboratory of ophthalmology of the University Hospital Farhat Hached-Sousse, and were examined by fundoscopy and functional ophthalmologic methods by different ophthalmologists. The diagnostic criteria of choroideremia included a history of night blindness and the typical appearance of the fundus, showing peripheral pigmentary retinopathy with areas of pigment epithelial and choroidal atrophy. Appropriate informed consent for the molecular study was obtained only from two brothers (case 1, case 2) and a carrier female (case 3) (figure 1). Case 1 A 51 year-old man (IV.9), a hemizygote in a choroideremia consanguineous family, at first noted night blindness at 20 years old and had since experienced progressive visual field loss. His mother (III.5) and fifth daughters are carriers and his three brothers (IV.11, IV.13 and IV.17) suffered from choroideremia. On diagnosis, the corrected visual acuity was 1/50 in both eyes. Ophthalmoscopic examinations and fluorescein angiograms revealed diffused atrophy of the retinal pigment epithelium and choriocapillaris, islands of relatively less involved areas and many large choroidal vessels remaining intact and electroretinography was undetectable Case 2 A 48-year-old man (IV.11) first noted night blindness at 12 years of age and had since experienced progressive visual field loss. On presentation, the corrected visual acuity was 2/20 in both eyes. Case 3 A 15-year-old female (V.11), the daughter of case 2 was symptomless and she was an obligate carrier of choroideremia (figure 1). PCR amplification The molecular analysis of the CHM gene was carried out after taking informed written consent from all participants. Genomic DNA was isolated from leukocytes of peripheral blood of patients and their relatives by a salt extraction procedure [6] and stored at 4 C. The molecular genetic analysis was performed after polymerase chain reaction (PCR) amplification by direct DNA sequencing of the individual CHM exons and adjacent intronic sequences using primers that were published previously [7]. PCR was performed in 1.5 mm MgCl 2, using 1 U Taq DNA polymerase, Recombinant (Invitogen, Brasil) [8]. Reaction were performed in a ABI 2700 thermocycler (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 94 C for 5 min, then 33 cycles of 94 C denaturing for 30 s, 60 C annealing for 30 s, 72 C extension for 30 s, and a final step of 7 min at 72 C. Products were analyzed on a 1.5% agarose gel stained with ethidium bromide. Nucleotide sequence analysis PCR products were purified by using Charge Switch PCR Clean-Up kit (Invitrogen). Purified fragments were sequenced with the ABI Prism Big Dye Terminator v1.1 Cycle Sequencing Kits, on an ABI PRISM 310 sequencer according to standard procedures as recommended by the manufacturer (Applied Biosystems, Foster City, CA). The samples were purified using Wizard MagneSil TM Sequencing Reaction Clean-Up System kit (Promega), then were electrophoresed on an automated ABI PRISM 310 sequencer and analyzed with the ABI Seq-Scape 2.0 software (PE Applied Biosystems, Foster City, CA). Results The index case (IV.9) has consulted for a progressive and bilateral loss of vision with hemeralopia and night blindness. The anterior segment was examined by slit-lamp biomicroscopy for the presence of any opacity in the lens. It was without particularities. The fundus examinations revealed bilateral and diffuse chorio-retinal atrophy with visualization of the sclera. The macula has a grayish aspect. Goldmann visual fields showed Goldmann peripapillary atrophy and enlarged blind spots in both eyes, as well as a concentric shrinkage of isopters. Fluorescein angiogram confirmed the choroi-retinal atrophy with visibility of the 470 Ann Biol Clin, vol. 73, n 4, juillet-août 2015

3 Tunisian choroideremia family with IVS1+1G>A mutation I. II. III. P IV. case 2 V case 3 adoptative child Male Affected P Proband Female Unknown sex Unaffected Deceased Obligat carrier Figure 1. Pedigree of the Tunisian choroideremia family. A G G G A C G G A T A T G T G B T A G G G G A C G G N T A T G T G T A Figure 2. Profile of hemizygote and heterozygous IVS1+1G>A mutation. A: hemizygote patients; B: heterozygous female carrier. choroidal vascularisation. The electroretinogram (ERG) was undetectable. For the other three brothers (IV.11, IV.13 and IV.17), ophthalmological tests were performed. Characteristic fundus, night blindness, peripheral visual field loss, electroretinography and other manifestations led us to a diagnosis of choroideremia. The niece of the index case (V.11) was symptomless and show normal fundus. In this study we performed the molecular genetic analysis of the CHM gene in two brothers (IV.9 and IV.11) suffering from choroideremia and a carrier female (V.11). Direct sequencing of the PCR products from this affected males revealed a single guanine (G) to adenine (A) transition at nucleotide 146+1, splicing junction signal in intron 1: IVS1+1G>A(figure 2A). This mutation was described for the first time and only by Fujiki in 1998 (5, 19). Since, no other articles concerning this mutation was been published. The IVS1+1G>A mutation, and because of its localization in the consensus sequence of the donor splice site, leads to aberrantly spliced mrna producing a premature stop codon and consequently a truncated protein that is rapidly degraded and lost. Note also that the female carrier is heterozygous for the mutation (figure 2B). Discussion Choroideremia is a progressive, diffuse, bilateral chorioretinal dystrophy with an X-linked recessive mode of inheritance. Patients generally present in their first or Ann Biol Clin, vol. 73, n 4, juillet-août

4 second decades, with problems in dark adaptation, which progresses to night blindness, followed closely by the significant constriction of visual fields, and with severe impairment in central visual acuity by the fifth to seven decades. There is progression of these defects in the central 10-degree visual fieled [9]. Clinical diagnosis is based on the presence of night blindness and peripheral visual field defects, lesions in fundus, hypovolted ERG traces, and progressive worsening of these signs. Initial fundus changes begin in the midperipheral or the far peripheral retina in the form of patches of pigment atrophy and clumping. In the intermediate stages of the disease, the atrophy of the retinal pigment epithelium and choriocapillaris become more diffuse, while the intermediate and larger choroidal vessels remain relatively better preserved [2, 10]. As the disease progresses, both the intermediate and large sized choroidal vessels become more atrophic, exposing the underlying sclera. Full field ERG is the key test, particularly when patients are asymptomatic and show normal fundus at early stages of the disease or in autosomal dominant forms with variable penetrance, since it is usually hypovolted before the appearance of clinical signs (night blindness). It is important to ascertain the diagnosis by repeating the examination one or two years after it has been first established. The pathology in affected males is similar to that seen in other choroidal dystrophies, ranging from mild abnormalities in the retinal pigment epithelium and choriocapillaris, to complete absence of the choroid and outer retina [11]. As a result of the random inactivation of one X chromosome in each cell early in fetal development, that is, lyonization, the retinas of female carriers are mosaics, containing patches of both mutant and normal cells. Carrier females usually show fundus changes, including patchy depigmentation of the retinal pigment epithelium (RPE) and coarse pigmentary granularity in the periphery, but can retain good visual function throughout life. The severity of retinal disease in carriers is variable and depends on the proportion of cells expressing the mutant X chromosome. Choroideremia is caused by a lack of a functional gene product. With the exception of large deletions, punctual mutations (substitutions, insertions or deletions of bases) constitute the major molecular defects identified in the CHM gene All of the REP-1 mutations identified so far in affected individuals are predicted to lead to premature stop codon during translation and to result either in a protein truncated at the carboxy-terminus or in no protein at all. Mutation detected by analysis of genomic DNA was an IVS1+1G>A mutation that alters a strictly conserved nucleotide of the splice site donor sequence in intron 1 was identified in all our patients. This mutation involves G>A transition at CpG dinucleotides and may thus reflect the hypermutability of this site and was described for the first time by Fujiki in 1998[12]. Female carrier is heterozygous for the mutation. Conclusion At present, there is neither a treatment nor a cure for CHM. Management is based on lows vision-aids but ongoing studies are developing therapeutic strategies that target REP Rab proteins and modulate the endocytic pathway. In addition, the results of a recent study of gene therapy, performed in Oxford elicit much hope [13]. Once the diagnosis is made, patients should be informed and familial surveys recommended. Genetic counseling is always advised. A precise phenotypic diagnosis is always mandatory and is particularly useful in the absence of familial history or in sporadic cases. Acknowledgements. We wish to express our appreciation for the co-operation and generosity of the patient and his family members. We appreciate the expert technical assistance of Siham Sassi and Safa Bouker and Hedi Laatiri. Conflicts of interest: None of the authors have any conflict of interest to disclose. References 1. Sankila EM, Sistonen P, Cremers F, de la Chapelle A. Choroideremia: linkage analysis with physically mapped close DNA-markers. Hum Genet 1991 ; 87 : van den Hurk JA, van de Pol DJ, Wissinger B, van Driel MA, Hoefsloot LH, de Wijs IJ, et al. Novel types of mutation in the choroideremia (CHM) gene: a full-length L1 insertion and an intronic mutation activating a cryptic exon. Hum Genet 2003 ; 113 : Cremers FP, van de Pol DJ, van Kerkhoff LP, Wieringa B, Ropers HH. Cloning of a gene that is rearranged in patients with choroideraemia. Nature 1990 ; 347 : Rak A, Pylypenko O, Niculae A, Pyatkov K, Goody RS, Alexandrov K. Structure of the Rab7:REP-1 complex: insights into the mechanism of Rab prenylation and choroideremia disease. Cell 2004 ; 117 : Seabra MC, Ho YK, Anant JS. Deficient geranylgeranylation of Ram/Rab27 in choroideremia. J Biol Chem 1995 ; 270 : Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988 ; 16 : van Bokhoven H, Schwartz M, Andréasson S, van den Hurk JA, Bogerd L, Jay M, et al. Mutation spectrum in the CHM gene of Danish and Swedish choroideremia patients. Hum Mol Genet 1994;3: Innis MA GDH, Sninsky JJ, WhiteF T.J. JJ. PCR protocols: a guide to methods and applications. San Diego, Ca : Academic Press, Ann Biol Clin, vol. 73, n 4, juillet-août 2015

5 Tunisian choroideremia family with IVS1+1G>A mutation 9. Hirakawa H, Iijima H, Gohdo T, Imai M, Tsukahara S. Progression of defects in the central 10-degree visual field of patients with retinitis pigmentosa and choroideremia. Am J Ophthalmol 1999 ; 127 : McCulloch C. Choroideremia: a clinical and pathologic review. Trans Am Ophthalmol Soc 1969 ; 67 : O SJ, Kim SH, Lee HY. A case of choroideremia with recurrent anterior uveitis. Korean J Ophthalmol 2003 ; 17 : Fujiki K, Hotta Y, Hayakawa M, Saito A, Mashima Y, Mori M, et al. REP-1 gene mutations in Japanese patients with choroideremia. Graefes Arch Clin Exp Ophthalmol 1999 ; 237 : MacLaren RE, Groppe M, Barnard AR, Cottriall CL, Tolmachova T, Seymour L, et al. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial. Lancet 2014 ; 383 : Ann Biol Clin, vol. 73, n 4, juillet-août