Original Paper Audiology Neurotology
Received: August 29, 2013 Accepted after revision: January 17, 2014 Published online: April 30, 2014
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
A Novel Missense NDP Mutation [p.(Cys93Arg)] with a Manifesting Carrier in an Austrian Family with Norrie Disease Thomas Parzefall a Trevor Lucas b Markus Ritter c Martin Ludwig a Reinhard Ramsebner a Alexandra Frohne b Christian Schöfer b Markus Hengstschläger d Klemens Frei a
Departments of a Otorhinolaryngology, b Nuclear and Developmental Biology, c Ophthalmology and d Medical Genetics, Medical University of Vienna, Vienna, Austria
Key Words Norrie disease · Syndromic deafness · Hereditary hearing loss · Deafblindness
Abstract Norrie disease is a rare, X-linked genetic syndrome characterized by combined congenital blindness and progressive hearing impairment. Norrie disease is caused by alterations in the NDP gene encoding the growth factor norrin that plays a key role in vascular development and stabilization of the eye, inner ear and brain. We identified a family with 3 affected deafblind males and a single female carrier presenting with a serous retinal detachment but normal hearing. Genetic analysis revealed a novel c.277T>C missense mutation causing the substitution of a hydrophobic cysteine to a hydrophilic arginine [p.(Cys93Arg)] within the highly conserved cysteine knot domain of the norrin protein. These results should expand the scope for amniocentesis and genetic testing for Norrie disease which is gaining in importance due to novel postnatal therapeutic concepts to alleviate the devastating retinal symptoms of Norrie disease. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1420–3030/14/0193–0203$39.50/0 E-Mail [email protected] www.karger.com/aud
Introduction
Combined blindness and hearing impairment is a devastating combination of symptoms depriving affected individuals of normal social interaction, restricting personal mobility and delaying mental development. Deafblindness is a heterogeneous disease that can be caused by infectious agents, trauma, premature birth or genetic syndromes. Norrie disease (MIM No. 310600) is an X-linked hereditary syndrome causing congenital blindness in affected males. The majority of patients suffer from sensorineural hearing loss with an onset in childhood or early adulthood [Halpin et al., 2005; Parving and Warburg, 1977]. Approximately 30–50% of patients have some degree of cognitive retardation [Sims et al., 1989; Smith et al., 2012]. In some cases, a mild clinical phenotype has also been described in female mutation carriers [Halpin et al., 2005; Yamada et al., 2001]. Vision loss in Norrie disease is due to deficient sprouting of the retinal vascular plexus during eye development leading to pseudoglioma, retinal detachment, with cataracts and bulbar atrophy accompanying later stages of the disease [Berger et al., 1996; Richter et al., 1998; Warburg, 1961]. Studies of a mouse model of Norrie disease have Klemens Frei, MD Department of Otorhinolaryngology, Medical University of Vienna AKH-8J, Währinger Gürtel 18–20 AT–1090 Vienna (Austria) E-Mail klemens.frei @ meduniwien.ac.at
shown that hearing loss primarily occurs due to defects in the stria vascularis with consecutive loss of hair cells and spiral ganglion neurons [Rehm et al., 2002]. Norrie disease is caused by mutations in the Norrie disease (pseudoglioma) NDP gene which contains 3 exons spanning 28 kb on the X chromosome [Berger et al., 1992a; Meindl et al., 1992]. This gene encodes the norrin protein, a 133-residue secretory growth factor that promotes vascular development and maintenance in the retina, stria vascularis of the inner ear and brain by activation of the Wnt/β-catenin signaling cascade [Xu et al., 2004]. Norrin is part of a larger family of cysteine-rich growth factors such as transforming growth factor-β and von Willebrand factor that all share a cysteine knot motif [Meitinger et al., 1993]. NDP mutations identified to date in affected human patients include nonsense, missense or frameshift point mutations as well as small intragenic deletions and large deletions abolishing the entire NDP gene and neighboring sequences. The severity and classification of disease correlate approximately with genotype. Whereas mutations near the C terminus of the protein cause Norrie disease, mutations close to the N terminus often cause Xlinked familial exudative vitreoretinopathy (MIM No. 305390), a distinct, milder disease without extraocular manifestations. Large deletions tend to be severest whereas truncating mutations are normally severer than missense mutations [Allen et al., 2006; Smith et al., 2012]. In addition, a number of yet to be identified genetic modifiers presumably also influence the clinical phenotype in Norrie disease since interindividual heterogeneity is a frequent observation in affected families [Allen et al., 2006]. Norrie disease has been reported in different populations worldwide. It is a rare genetic disorder and independently of the population is estimated to have a prevalence below 1:1,000,000. Here, we examined a family with combined hereditary blindness and hearing impairment for alterations in the NDP gene by molecular genetic testing.
away at the time of the study (family members 2 and 3). One affected male aged 23 (family member 6), the mother who has a lateonset retinal phenotype (family member 5), the unaffected father (family member 4) and paternal grandmother (family member 1) were available for the study. Complete medical histories were examined including otolaryngological and ophthalmological status in all family members. Informed consent was obtained from all participants in the study, and the study was approved by the Vienna Medical University Ethics Committee (approval No. 198/2004). Mutational Screening of the NDP Gene Genomic DNA of the participants was isolated from peripheral venous blood with the EZNA Blood DNA Kit II (Peqlab, Erlangen, Germany). All 3 exons and splice sites of the NDP gene were analyzed in the affected individuals by direct Sanger sequencing using primers as previously described [Berger et al., 1992b]. To determine the prevalence and the carrier rate of the identified alteration in our population, we genetically screened healthy Austrian control individuals (n = 114). For variant validation, 5′-TGTCGTTCAGCACTGTCCTC-3′ (forward) and 5′-AATTGCATTCCTCGCAGTG-3′ (reverse) primers were used to amplify a 162-bp sequence of the NDP exon 3 containing the identified alteration. Polymerase chain reaction (PCR) was performed in 50-μl aliquots containing 100 ng of chromosomal DNA, 20 pmol of each primer, 200 μM dNTPs, 1.5 mM MgCl2, and 1 unit Taq polymerase (Fermentas, Burlington, Ont., Canada). DNA was amplified by 35 cycles of denaturation at 95 ° C for 30 s, annealing for 30 s at 59 ° C and elongation for 30 s at 72 ° C. The PCR products were purified with a DNA clean and concentrator-5 kit (Zymo Research, Orange, Calif., USA).
Bioinformatics PCR primers were designed using the Primer-3 web interface [Rozen and Skaletsky, 2000]. DNA sequences were read by dye termination sequencing on a CEQ2000XL sequencer (Beckman Coulter, Fullerton, Calif., USA). Sequences were then aligned and compared to the Genome Reference Consortium Human Build 37 sequence (GRCh37/hg19). We used Polyphen-2 to predict the damaging effect of identified amino acid changes in the norrin protein [Adzhubei et al., 2010]. Sequence conservation scores across several species were determined with the Consurf web interface [Ashkenazy et al., 2010]. To predict the effects of identified alterations on mature protein conformation, a schematic model of the predicted mutated protein was generated using the automated mode of the Swiss Model protein model tool [Arnold et al., 2006]. The identified variant was checked using Mutalyzer (http://www. LOVD.nl/mutalyzer/) and named according to the guidelines for the nomenclature of sequence variations of the Human Genome Variation Society (http://www.hgvs.org/mutnomen/).
Material and Methods Patients Members of an affected Austrian family and healthy control individuals (n = 114) were recruited into the study at the Department of Otorhinolaryngology, Medical University Hospital of Vienna. The family has a history of 3 affected male individuals (family members 2, 3 and 6) and 1 female with isolated mild late-onset retinal disease (family member 5). Two affected males had passed
204
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
Results
Clinical Findings in the Study Patients A patient (family member 6) born before term at 37 weeks of gestation presented with congenital blindness. Shortly after birth, this patient was diagnosed with termiParzefall/Lucas/Ritter/Ludwig/Ramsebner/ Frohne/Schöfer/Hengstschläger/Frei
nal stage retinopathy of prematurity. No other symptoms or abnormalities were diagnosed at the time, and genetic testing was not conducted. The patient was educated in a special school for visually impaired children and has normal intelligence. At 8 years of age, left-sided bulbar atrophy and right-sided complete retinal detachment were found at a control ophthalmological examination. The family then stopped regular ophthalmological follow-up examinations. At the age of 19, the patient noticed progressive hearing loss and sought medical advice from an otolaryngology specialist. He received a diagnosis of acute sensorineural hearing loss and underwent standard corticosteroid and rheological therapy without success. On progression of the hearing loss, the patient was referred to the University Clinic. He presented with mild to moderate bilateral sensorineural hearing loss, with lower thresholds at high frequencies (fig. 1a). After careful family anamnesis had been taken, the patient was suspected to suffer from Norrie disease, and genetic testing was initiated. The patient’s mother (family member 5) reported progressive visual deterioration and was first diagnosed with serous retinal detachment at the age of 43. She had no hearing impairment at the time of examination at our clinic (fig. 1b). The patient’s father (family member 4) is a healthy 52-year-old man without any vision or hearing problems confirmed by hearing tests and ophthalmological examination (fig. 1c). The family history (fig. 2a) reveals 2 previous cases of congenital blindness in male family members (family members 2 and 3). One of the 2 males (family member 3) was also reported by the family to have been affected by mental retardation. Both affected male family members had already passed away at the time of the study, and detailed medical records were not available.
from cysteine to arginine [p.(Cys93Arg)]. The cysteine at position 93 was identified to be a highly conserved amino acid in the human, rat (Rattus norvegicus), opossum (Monodelphis domestica), mouse (Mus musculus), dog (Canis familiaris), horse (Equus caballus), cattle (Bos taurus), rhesus monkey (Macaca mulatta) and pig (Sus scrofa), with a Consurf score of 8 (ranging from 1 = lowest to 9 = highest; fig. 2c). With a polyphen-2 score over 0.99, the amino acid exchange from cysteine to arginine is predicted to cause a damaging conformational change within the loop structure that connects the β2- and the β3strands of the mature norrin protein (fig. 2d).
Discussion
A Missense Mutation within the Highly Conserved Cystine Knot Domain of Norrin Mutational analysis of the NDP gene in the proband (family member 6) led to the identification of a single nucleotide change at position 277 of the coding sequence of the NDP gene (fig. 2b). The c.277T>C alteration was not found in unaffected family members (family members 1 and 4) or in 114 healthy individuals (181 chromosomes) screened in the Austrian control population. The mother of this patient (family member 5) was found to be a carrier of the c.277T>C transition (fig. 2b). The missense mutation identified in the family is predicted to cause an amino acid change at position 93 of the mature peptide
In this study, we identify a novel missense point mutation at a highly conserved position within the cysteine knot domain of norrin. The c.277T>C mutation causes an amino acid exchange from a hydrophobic cysteine to a hydrophilic arginine at position 93 [p.(Cys93Arg)] and is predicted to cause a conformational change in the loop that connects the two β-strands of the cysteine knot (fig. 2d). Active norrin is predicted to undergo oligomerization by forming a bridge between 2 cysteines within this loop domain at position 95 of adjacent norrin monomers [Meitinger et al., 1993; Perez-Vilar and Hill, 1997]. It may be speculated that the conformational change caused by the cysteine to arginine at position 93 of the protein due to its close vicinity to the cysteine at position 95 influences the dimerization capacity thereby disrupting the activity of norrin. Furthermore, an intrachain disulfide bridge at position 96 is part of the cysteine knot backbone of norrin. In growth factors with comparable cysteine knot structures, the 3 amino acid residues proximal to the conserved cysteine forming this intrachain disulfide bond are all polar neutral residues as cysteine (p.2771) in von Willebrand factor (NP_000543.2), serine (p.405) in transforming growth factor-β2 (NP_001129071.1) or threonine (p.130) in platelet-derived growth factor (NP_002598.4). It is therefore possible that a hydrophilic residue within the cysteine knot at this position may not be tolerated as shown by the Polyphen-2 analysis for the p.(Cys93Arg) alteration. In contrast to the full manifestation of Norrie disease in males of the family, the mother of proband 6 heterozygous for c.277T>C displayed milder retinal symptoms which were absent in the maternal grandmother and great-grandmother. This phenomenon has been observed
Novel Missense NDP Mutation in Norrie Disease
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
205
Family member 5
dB –10 0 10 20 30 40 50 60 70 80 90 100 110 120
dB –10 0 10 20 30 40 50 60 70 80 90 100 110 120
0
00 ,0
Hz
16
00
0
8,
0
00 4,
00
0
2,
00
0
1,
50
5
0 25
b
Left X Right O
12
00
0
,0
00
0
0
0
Hz
16
8,
00 4,
00 2,
00
0
1,
50
5
0 25
12
a
Left X Right O
Color version available online
Family member 6
Family member 4
206
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
00 ,0
0
0
Hz
16
00 8,
00 4,
0 00 2,
0 00 1,
0
0 50
25
5
c
Left X Right O
12
Fig. 1. Pure-tone audiometry results and ocular phenotypes in the family. a The 23-year-old patient (family member 6) suffers from Norrie disease. Pure-tone audiometry shows bilateral mild to moderate sensorineural hearing loss with severe hearing loss at higher frequencies. Anterior segment photographs of the right (R) and the left (L) eye show a dense lens opacification with posterior synechiae on the right and a cloudy cornea on the left eye. b The mother of family member 6 (family member 5) is a 48-year-old carrier female and presents with normal hearing threshold and progressive visual deterioration. Fundus photographs of the right (R) and the left (L) eye reveal pigmentary changes in the lower nasal quadrant after a resolved serous retinal detachment in the left eye. c Family member 4, the father of the proband, is a healthy 52-year-old male with normal hearing threshold and no visible fundus abnormalities.
dB –10 0 10 20 30 40 50 60 70 80 90 100 110 120
Parzefall/Lucas/Ritter/Ludwig/Ramsebner/ Frohne/Schöfer/Hengstschläger/Frei
Color version available online
Norrie disease Late-onset mild retinal phenotype
1*
2
4*
a
3
5*
6* hemi c.277T
hemi c.277C
het c.277T/C
Cys
Arg
Cys Arg
b
Leader peptide
Cysteine knot domain 133 aa aa 93
Human Rat Opossum Mouse Dog Horse Cattle Rhesus monkey Pig
Fig. 2. a The family contains 3 males with
Norrie disease (family members 2, 3 and 6) and a female carrier with a late-onset mild retinal phenotype (family member 5). Individuals indicated with an asterisk were available for this study. b Mutational analysis of the NDP gene in the family identified a c.277T>C missense mutation transmitted by the carrier mother. c The mature norrin protein contains a leader peptide and a cysteine knot domain. c.277T>C leads to an amino acid exchange at residue 93 of norrin [p.(Cys93Arg)]. The residue 93 is a highly conserved amino acid across several species. d The p.(Cys93Arg) mutation is predicted to cause a conformational change of the loop structure connecting the β2- and β3-strands of the mature norrin protein. WT = Wild type.
Novel Missense NDP Mutation in Norrie Disease
c
Variable
Average WT
Conserved p.(Cys93Arg)
d
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
207
previously in families with Norrie mutations and has been attributed to clinical mosaicism caused by stochastic X chromosomal inactivation [Kondo et al., 2007; Yamada et al., 2001]. The progressive introduction of routine molecular genetic testing for hereditary diseases is becoming a powerful tool in the diagnosis and therapy of Norrie disease. As in many congenital disorders, adaptation to congenital or early-onset blindness was less problematic for these patients than adjustment to the later-onset, slowly progressive hearing loss. For this reason, the proband (family member 6) sought medical advice when hearing loss occurred later in adolescence. A recent study of 59 patients affected with Norrie syndrome reported the manifestation of depression in nearly all cognitively normal Norrie disease patients at the onset of hearing loss in that cohort [Smith et al., 2012]. Our patient (family member 6) initially received the diagnosis of idiopathic sensorineural hearing loss and underwent recurrent standard treatment including repeated high-dose intravenous corticoids and pentoxifyllin without effect. It is essential for the otolaryngologist to be aware of this disease when being consulted by affected individuals and to take into consideration genetic testing. There has been a recent report that blindness could be effectively treated in a preterm neonate with Norrie syndrome within the first few days after birth (at a gestational age of 37 weeks) with laser photocoagulation therapy [Chow et al., 2010]. This patient was tested genetically after amniocentesis due to occurrence of Norrie disease in the family. This report supports a further series of cases of successful vitrectomy before 12 months of age in 14 boys with Norrie disease. In 7 of the 14 probands light perception acuity could be maintained by this treatment method [Walsh et al.,
2010]. These reports underline that genetic diagnosis is crucial for potential treatment options in families with Norrie disease. Norrie disease is a rare genetic disorder and has been reported in several populations worldwide. Here, we report the first case of Norrie disease in the Austrian population. Since the catchment area of our clinic includes approximately 3 million patients and our institutes are the only tertiary medical settings offering genetic testing in this area, we estimate that the prevalence in the Austrian population is similar to the prevalence of below 1/1,000,000 inhabitants found in previously reported populations.
Conclusion
In summary, we report data regarding the prevalence of Norrie disease in the Austrian population and report a novel missense mutation within the highly conserved cysteine knot domain of norrin. This is the first case of Norrie disease in Austria and a rare case of a disease-affected female carrier.
Acknowledgments We wish to thank the participating family members and Oliver Brandau for genetic counseling of the patients. This project was supported by funds of the Oesterreichische Nationalbank (Anniversary Fund, project No. AP14058ONB).
Disclosure Statement The authors declare no conflict of interest.
References Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR: A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248–249. Allen RC, Russell SR, Streb LM, Alsheikheh A, Stone EM: Phenotypic heterogeneity associated with a novel mutation (Gly112Glu) in the Norrie disease protein. Eye (Lond) 2006; 20: 234–241. Arnold K, Bordoli L, Kopp J, Schwede T: The Swiss-model workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006;22:195–201. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N: Consurf 2010: calculating evolutionary
208
conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res 2010;38:W529–W533. Berger W, Meindl A, van de Pol TJ, Cremers FP, Ropers HH, Doerner C, Monaco A, Bergen AA, Lebo R, Warburg M, et al: Isolation of a candidate gene for Norrie disease by positional cloning. Nat Genet 1992a;1:199–203. Berger W, van de Pol D, Bachner D, Oerlemans F, Winkens H, Hameister H, Wieringa B, Hendriks W, Ropers HH: An animal model for Norrie disease (ND): gene targeting of the mouse ND gene. Hum Mol Genet 1996;5:51–59. Berger W, van de Pol D, Warburg M, Gal A, Bleeker-Wagemakers L, de Silva H, Meindl A, Meitinger T, Cremers F, Ropers HH: Muta-
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
tions in the candidate gene for Norrie disease. Hum Mol Genet 1992b;1:461–465. Chow CC, Kiernan DF, Chau FY, Blair MP, Ticho BH, Galasso JM, Shapiro MJ: Laser photocoagulation at birth prevents blindness in Norrie’s disease diagnosed using amniocentesis. Ophthalmology 2010;117:2402–2406. Halpin C, Owen G, Gutierrez-Espeleta GA, Sims K, Rehm HL: Audiologic features of Norrie disease. Ann Otol Rhinol Laryngol 2005;114:533–538. Kondo H, Qin M, Kusaka S, Tahira T, Hasebe H, Hayashi H, Uchio E, Hayashi K: Novel mutations in Norrie disease gene in Japanese patients with Norrie disease and familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci 2007;48:1276–1282.
Parzefall/Lucas/Ritter/Ludwig/Ramsebner/ Frohne/Schöfer/Hengstschläger/Frei
Meindl A, Berger W, Meitinger T, van de Pol D, Achatz H, Dorner C, Haasemann M, Hellebrand H, Gal A, Cremers F, et al: Norrie disease is caused by mutations in an extracellular protein resembling C-terminal globular domain of mucins. Nat Genet 1992;2:139–143. Meitinger T, Meindl A, Bork P, Rost B, Sander C, Haasemann M, Murken J: Molecular modelling of the Norrie disease protein predicts a cystine knot growth factor tertiary structure. Nat Genet 1993;5:376–380. Parving A, Warburg M: Audiological findings in Norrie’s disease. Audiology 1977;16:124–131. Perez-Vilar J, Hill RL: Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix. J Biol Chem 1997;272:33410–33415.
Novel Missense NDP Mutation in Norrie Disease
Rehm HL, Zhang DS, Brown MC, Burgess B, Halpin C, Berger W, Morton CC, Corey DP, Chen ZY: Vascular defects and sensorineural deafness in a mouse model of Norrie disease. J Neurosci 2002;22:4286–4292. Richter M, Gottanka J, May CA, Welge-Lussen U, Berger W, Lutjen-Drecoll E: Retinal vasculature changes in Norrie disease mice. Invest Ophthalmol Vis Sci 1998;39:2450–2457. Rozen S, Skaletsky H: Primer3 on the www for general users and for biologist programmers. Methods Mol Biol 2000;132:365–386. Sims KB, de la Chapelle A, Norio R, Sankila EM, Hsu YP, Rinehart WB, Corey TJ, Ozelius L, Powell JF, Bruns G, et al: Monoamine oxidase deficiency in males with an X chromosome deletion. Neuron 1989;2:1069–1076. Smith SE, Mullen TE, Graham D, Sims KB, Rehm HL: Norrie disease: extraocular clinical manifestations in 56 patients. Am J Med Genet A 2012;158A:1909–1917. Walsh MK, Drenser KA, Capone A Jr, Trese MT: Early vitrectomy effective for Norrie disease. Arch Ophthalmol 2010;128:456–460.
Warburg M: Norrie’s disease: a new hereditary bilateral pseudo-tumor of the retina. Acta Ophthalmol 1961;39:757–772. Xu Q, Wang Y, Dabdoub A, Smallwood PM, Williams J, Woods C, Kelley MW, Jiang L, Tasman W, Zhang K, Nathans J: Vascular development in the retina and inner ear: control by norrin and frizzled-4, a high-affinity ligandreceptor pair. Cell 2004;116:883–895. Yamada K, Limprasert P, Ratanasukon M, Tengtrisorn S, Yingchareonpukdee J, Vasiknanonte P, Kitaoka T, Ghadami M, Niikawa N, Kishino T: Two Thai families with Norrie disease (ND): association of two novel missense mutations with severe ND phenotype, seizures, and a manifesting carrier. Am J Med Genet 2001a;100:52–55.
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
209
Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Received: August 29, 2013 Accepted after revision: January 17, 2014 Published online: April 30, 2014
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
A Novel Missense NDP Mutation [p.(Cys93Arg)] with a Manifesting Carrier in an Austrian Family with Norrie Disease Thomas Parzefall a Trevor Lucas b Markus Ritter c Martin Ludwig a Reinhard Ramsebner a Alexandra Frohne b Christian Schöfer b Markus Hengstschläger d Klemens Frei a
Departments of a Otorhinolaryngology, b Nuclear and Developmental Biology, c Ophthalmology and d Medical Genetics, Medical University of Vienna, Vienna, Austria
Key Words Norrie disease · Syndromic deafness · Hereditary hearing loss · Deafblindness
Abstract Norrie disease is a rare, X-linked genetic syndrome characterized by combined congenital blindness and progressive hearing impairment. Norrie disease is caused by alterations in the NDP gene encoding the growth factor norrin that plays a key role in vascular development and stabilization of the eye, inner ear and brain. We identified a family with 3 affected deafblind males and a single female carrier presenting with a serous retinal detachment but normal hearing. Genetic analysis revealed a novel c.277T>C missense mutation causing the substitution of a hydrophobic cysteine to a hydrophilic arginine [p.(Cys93Arg)] within the highly conserved cysteine knot domain of the norrin protein. These results should expand the scope for amniocentesis and genetic testing for Norrie disease which is gaining in importance due to novel postnatal therapeutic concepts to alleviate the devastating retinal symptoms of Norrie disease. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1420–3030/14/0193–0203$39.50/0 E-Mail [email protected] www.karger.com/aud
Introduction
Combined blindness and hearing impairment is a devastating combination of symptoms depriving affected individuals of normal social interaction, restricting personal mobility and delaying mental development. Deafblindness is a heterogeneous disease that can be caused by infectious agents, trauma, premature birth or genetic syndromes. Norrie disease (MIM No. 310600) is an X-linked hereditary syndrome causing congenital blindness in affected males. The majority of patients suffer from sensorineural hearing loss with an onset in childhood or early adulthood [Halpin et al., 2005; Parving and Warburg, 1977]. Approximately 30–50% of patients have some degree of cognitive retardation [Sims et al., 1989; Smith et al., 2012]. In some cases, a mild clinical phenotype has also been described in female mutation carriers [Halpin et al., 2005; Yamada et al., 2001]. Vision loss in Norrie disease is due to deficient sprouting of the retinal vascular plexus during eye development leading to pseudoglioma, retinal detachment, with cataracts and bulbar atrophy accompanying later stages of the disease [Berger et al., 1996; Richter et al., 1998; Warburg, 1961]. Studies of a mouse model of Norrie disease have Klemens Frei, MD Department of Otorhinolaryngology, Medical University of Vienna AKH-8J, Währinger Gürtel 18–20 AT–1090 Vienna (Austria) E-Mail klemens.frei @ meduniwien.ac.at
shown that hearing loss primarily occurs due to defects in the stria vascularis with consecutive loss of hair cells and spiral ganglion neurons [Rehm et al., 2002]. Norrie disease is caused by mutations in the Norrie disease (pseudoglioma) NDP gene which contains 3 exons spanning 28 kb on the X chromosome [Berger et al., 1992a; Meindl et al., 1992]. This gene encodes the norrin protein, a 133-residue secretory growth factor that promotes vascular development and maintenance in the retina, stria vascularis of the inner ear and brain by activation of the Wnt/β-catenin signaling cascade [Xu et al., 2004]. Norrin is part of a larger family of cysteine-rich growth factors such as transforming growth factor-β and von Willebrand factor that all share a cysteine knot motif [Meitinger et al., 1993]. NDP mutations identified to date in affected human patients include nonsense, missense or frameshift point mutations as well as small intragenic deletions and large deletions abolishing the entire NDP gene and neighboring sequences. The severity and classification of disease correlate approximately with genotype. Whereas mutations near the C terminus of the protein cause Norrie disease, mutations close to the N terminus often cause Xlinked familial exudative vitreoretinopathy (MIM No. 305390), a distinct, milder disease without extraocular manifestations. Large deletions tend to be severest whereas truncating mutations are normally severer than missense mutations [Allen et al., 2006; Smith et al., 2012]. In addition, a number of yet to be identified genetic modifiers presumably also influence the clinical phenotype in Norrie disease since interindividual heterogeneity is a frequent observation in affected families [Allen et al., 2006]. Norrie disease has been reported in different populations worldwide. It is a rare genetic disorder and independently of the population is estimated to have a prevalence below 1:1,000,000. Here, we examined a family with combined hereditary blindness and hearing impairment for alterations in the NDP gene by molecular genetic testing.
away at the time of the study (family members 2 and 3). One affected male aged 23 (family member 6), the mother who has a lateonset retinal phenotype (family member 5), the unaffected father (family member 4) and paternal grandmother (family member 1) were available for the study. Complete medical histories were examined including otolaryngological and ophthalmological status in all family members. Informed consent was obtained from all participants in the study, and the study was approved by the Vienna Medical University Ethics Committee (approval No. 198/2004). Mutational Screening of the NDP Gene Genomic DNA of the participants was isolated from peripheral venous blood with the EZNA Blood DNA Kit II (Peqlab, Erlangen, Germany). All 3 exons and splice sites of the NDP gene were analyzed in the affected individuals by direct Sanger sequencing using primers as previously described [Berger et al., 1992b]. To determine the prevalence and the carrier rate of the identified alteration in our population, we genetically screened healthy Austrian control individuals (n = 114). For variant validation, 5′-TGTCGTTCAGCACTGTCCTC-3′ (forward) and 5′-AATTGCATTCCTCGCAGTG-3′ (reverse) primers were used to amplify a 162-bp sequence of the NDP exon 3 containing the identified alteration. Polymerase chain reaction (PCR) was performed in 50-μl aliquots containing 100 ng of chromosomal DNA, 20 pmol of each primer, 200 μM dNTPs, 1.5 mM MgCl2, and 1 unit Taq polymerase (Fermentas, Burlington, Ont., Canada). DNA was amplified by 35 cycles of denaturation at 95 ° C for 30 s, annealing for 30 s at 59 ° C and elongation for 30 s at 72 ° C. The PCR products were purified with a DNA clean and concentrator-5 kit (Zymo Research, Orange, Calif., USA).
Bioinformatics PCR primers were designed using the Primer-3 web interface [Rozen and Skaletsky, 2000]. DNA sequences were read by dye termination sequencing on a CEQ2000XL sequencer (Beckman Coulter, Fullerton, Calif., USA). Sequences were then aligned and compared to the Genome Reference Consortium Human Build 37 sequence (GRCh37/hg19). We used Polyphen-2 to predict the damaging effect of identified amino acid changes in the norrin protein [Adzhubei et al., 2010]. Sequence conservation scores across several species were determined with the Consurf web interface [Ashkenazy et al., 2010]. To predict the effects of identified alterations on mature protein conformation, a schematic model of the predicted mutated protein was generated using the automated mode of the Swiss Model protein model tool [Arnold et al., 2006]. The identified variant was checked using Mutalyzer (http://www. LOVD.nl/mutalyzer/) and named according to the guidelines for the nomenclature of sequence variations of the Human Genome Variation Society (http://www.hgvs.org/mutnomen/).
Material and Methods Patients Members of an affected Austrian family and healthy control individuals (n = 114) were recruited into the study at the Department of Otorhinolaryngology, Medical University Hospital of Vienna. The family has a history of 3 affected male individuals (family members 2, 3 and 6) and 1 female with isolated mild late-onset retinal disease (family member 5). Two affected males had passed
204
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
Results
Clinical Findings in the Study Patients A patient (family member 6) born before term at 37 weeks of gestation presented with congenital blindness. Shortly after birth, this patient was diagnosed with termiParzefall/Lucas/Ritter/Ludwig/Ramsebner/ Frohne/Schöfer/Hengstschläger/Frei
nal stage retinopathy of prematurity. No other symptoms or abnormalities were diagnosed at the time, and genetic testing was not conducted. The patient was educated in a special school for visually impaired children and has normal intelligence. At 8 years of age, left-sided bulbar atrophy and right-sided complete retinal detachment were found at a control ophthalmological examination. The family then stopped regular ophthalmological follow-up examinations. At the age of 19, the patient noticed progressive hearing loss and sought medical advice from an otolaryngology specialist. He received a diagnosis of acute sensorineural hearing loss and underwent standard corticosteroid and rheological therapy without success. On progression of the hearing loss, the patient was referred to the University Clinic. He presented with mild to moderate bilateral sensorineural hearing loss, with lower thresholds at high frequencies (fig. 1a). After careful family anamnesis had been taken, the patient was suspected to suffer from Norrie disease, and genetic testing was initiated. The patient’s mother (family member 5) reported progressive visual deterioration and was first diagnosed with serous retinal detachment at the age of 43. She had no hearing impairment at the time of examination at our clinic (fig. 1b). The patient’s father (family member 4) is a healthy 52-year-old man without any vision or hearing problems confirmed by hearing tests and ophthalmological examination (fig. 1c). The family history (fig. 2a) reveals 2 previous cases of congenital blindness in male family members (family members 2 and 3). One of the 2 males (family member 3) was also reported by the family to have been affected by mental retardation. Both affected male family members had already passed away at the time of the study, and detailed medical records were not available.
from cysteine to arginine [p.(Cys93Arg)]. The cysteine at position 93 was identified to be a highly conserved amino acid in the human, rat (Rattus norvegicus), opossum (Monodelphis domestica), mouse (Mus musculus), dog (Canis familiaris), horse (Equus caballus), cattle (Bos taurus), rhesus monkey (Macaca mulatta) and pig (Sus scrofa), with a Consurf score of 8 (ranging from 1 = lowest to 9 = highest; fig. 2c). With a polyphen-2 score over 0.99, the amino acid exchange from cysteine to arginine is predicted to cause a damaging conformational change within the loop structure that connects the β2- and the β3strands of the mature norrin protein (fig. 2d).
Discussion
A Missense Mutation within the Highly Conserved Cystine Knot Domain of Norrin Mutational analysis of the NDP gene in the proband (family member 6) led to the identification of a single nucleotide change at position 277 of the coding sequence of the NDP gene (fig. 2b). The c.277T>C alteration was not found in unaffected family members (family members 1 and 4) or in 114 healthy individuals (181 chromosomes) screened in the Austrian control population. The mother of this patient (family member 5) was found to be a carrier of the c.277T>C transition (fig. 2b). The missense mutation identified in the family is predicted to cause an amino acid change at position 93 of the mature peptide
In this study, we identify a novel missense point mutation at a highly conserved position within the cysteine knot domain of norrin. The c.277T>C mutation causes an amino acid exchange from a hydrophobic cysteine to a hydrophilic arginine at position 93 [p.(Cys93Arg)] and is predicted to cause a conformational change in the loop that connects the two β-strands of the cysteine knot (fig. 2d). Active norrin is predicted to undergo oligomerization by forming a bridge between 2 cysteines within this loop domain at position 95 of adjacent norrin monomers [Meitinger et al., 1993; Perez-Vilar and Hill, 1997]. It may be speculated that the conformational change caused by the cysteine to arginine at position 93 of the protein due to its close vicinity to the cysteine at position 95 influences the dimerization capacity thereby disrupting the activity of norrin. Furthermore, an intrachain disulfide bridge at position 96 is part of the cysteine knot backbone of norrin. In growth factors with comparable cysteine knot structures, the 3 amino acid residues proximal to the conserved cysteine forming this intrachain disulfide bond are all polar neutral residues as cysteine (p.2771) in von Willebrand factor (NP_000543.2), serine (p.405) in transforming growth factor-β2 (NP_001129071.1) or threonine (p.130) in platelet-derived growth factor (NP_002598.4). It is therefore possible that a hydrophilic residue within the cysteine knot at this position may not be tolerated as shown by the Polyphen-2 analysis for the p.(Cys93Arg) alteration. In contrast to the full manifestation of Norrie disease in males of the family, the mother of proband 6 heterozygous for c.277T>C displayed milder retinal symptoms which were absent in the maternal grandmother and great-grandmother. This phenomenon has been observed
Novel Missense NDP Mutation in Norrie Disease
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
205
Family member 5
dB –10 0 10 20 30 40 50 60 70 80 90 100 110 120
dB –10 0 10 20 30 40 50 60 70 80 90 100 110 120
0
00 ,0
Hz
16
00
0
8,
0
00 4,
00
0
2,
00
0
1,
50
5
0 25
b
Left X Right O
12
00
0
,0
00
0
0
0
Hz
16
8,
00 4,
00 2,
00
0
1,
50
5
0 25
12
a
Left X Right O
Color version available online
Family member 6
Family member 4
206
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
00 ,0
0
0
Hz
16
00 8,
00 4,
0 00 2,
0 00 1,
0
0 50
25
5
c
Left X Right O
12
Fig. 1. Pure-tone audiometry results and ocular phenotypes in the family. a The 23-year-old patient (family member 6) suffers from Norrie disease. Pure-tone audiometry shows bilateral mild to moderate sensorineural hearing loss with severe hearing loss at higher frequencies. Anterior segment photographs of the right (R) and the left (L) eye show a dense lens opacification with posterior synechiae on the right and a cloudy cornea on the left eye. b The mother of family member 6 (family member 5) is a 48-year-old carrier female and presents with normal hearing threshold and progressive visual deterioration. Fundus photographs of the right (R) and the left (L) eye reveal pigmentary changes in the lower nasal quadrant after a resolved serous retinal detachment in the left eye. c Family member 4, the father of the proband, is a healthy 52-year-old male with normal hearing threshold and no visible fundus abnormalities.
dB –10 0 10 20 30 40 50 60 70 80 90 100 110 120
Parzefall/Lucas/Ritter/Ludwig/Ramsebner/ Frohne/Schöfer/Hengstschläger/Frei
Color version available online
Norrie disease Late-onset mild retinal phenotype
1*
2
4*
a
3
5*
6* hemi c.277T
hemi c.277C
het c.277T/C
Cys
Arg
Cys Arg
b
Leader peptide
Cysteine knot domain 133 aa aa 93
Human Rat Opossum Mouse Dog Horse Cattle Rhesus monkey Pig
Fig. 2. a The family contains 3 males with
Norrie disease (family members 2, 3 and 6) and a female carrier with a late-onset mild retinal phenotype (family member 5). Individuals indicated with an asterisk were available for this study. b Mutational analysis of the NDP gene in the family identified a c.277T>C missense mutation transmitted by the carrier mother. c The mature norrin protein contains a leader peptide and a cysteine knot domain. c.277T>C leads to an amino acid exchange at residue 93 of norrin [p.(Cys93Arg)]. The residue 93 is a highly conserved amino acid across several species. d The p.(Cys93Arg) mutation is predicted to cause a conformational change of the loop structure connecting the β2- and β3-strands of the mature norrin protein. WT = Wild type.
Novel Missense NDP Mutation in Norrie Disease
c
Variable
Average WT
Conserved p.(Cys93Arg)
d
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
207
previously in families with Norrie mutations and has been attributed to clinical mosaicism caused by stochastic X chromosomal inactivation [Kondo et al., 2007; Yamada et al., 2001]. The progressive introduction of routine molecular genetic testing for hereditary diseases is becoming a powerful tool in the diagnosis and therapy of Norrie disease. As in many congenital disorders, adaptation to congenital or early-onset blindness was less problematic for these patients than adjustment to the later-onset, slowly progressive hearing loss. For this reason, the proband (family member 6) sought medical advice when hearing loss occurred later in adolescence. A recent study of 59 patients affected with Norrie syndrome reported the manifestation of depression in nearly all cognitively normal Norrie disease patients at the onset of hearing loss in that cohort [Smith et al., 2012]. Our patient (family member 6) initially received the diagnosis of idiopathic sensorineural hearing loss and underwent recurrent standard treatment including repeated high-dose intravenous corticoids and pentoxifyllin without effect. It is essential for the otolaryngologist to be aware of this disease when being consulted by affected individuals and to take into consideration genetic testing. There has been a recent report that blindness could be effectively treated in a preterm neonate with Norrie syndrome within the first few days after birth (at a gestational age of 37 weeks) with laser photocoagulation therapy [Chow et al., 2010]. This patient was tested genetically after amniocentesis due to occurrence of Norrie disease in the family. This report supports a further series of cases of successful vitrectomy before 12 months of age in 14 boys with Norrie disease. In 7 of the 14 probands light perception acuity could be maintained by this treatment method [Walsh et al.,
2010]. These reports underline that genetic diagnosis is crucial for potential treatment options in families with Norrie disease. Norrie disease is a rare genetic disorder and has been reported in several populations worldwide. Here, we report the first case of Norrie disease in the Austrian population. Since the catchment area of our clinic includes approximately 3 million patients and our institutes are the only tertiary medical settings offering genetic testing in this area, we estimate that the prevalence in the Austrian population is similar to the prevalence of below 1/1,000,000 inhabitants found in previously reported populations.
Conclusion
In summary, we report data regarding the prevalence of Norrie disease in the Austrian population and report a novel missense mutation within the highly conserved cysteine knot domain of norrin. This is the first case of Norrie disease in Austria and a rare case of a disease-affected female carrier.
Acknowledgments We wish to thank the participating family members and Oliver Brandau for genetic counseling of the patients. This project was supported by funds of the Oesterreichische Nationalbank (Anniversary Fund, project No. AP14058ONB).
Disclosure Statement The authors declare no conflict of interest.
References Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR: A method and server for predicting damaging missense mutations. Nat Methods 2010;7:248–249. Allen RC, Russell SR, Streb LM, Alsheikheh A, Stone EM: Phenotypic heterogeneity associated with a novel mutation (Gly112Glu) in the Norrie disease protein. Eye (Lond) 2006; 20: 234–241. Arnold K, Bordoli L, Kopp J, Schwede T: The Swiss-model workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006;22:195–201. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N: Consurf 2010: calculating evolutionary
208
conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res 2010;38:W529–W533. Berger W, Meindl A, van de Pol TJ, Cremers FP, Ropers HH, Doerner C, Monaco A, Bergen AA, Lebo R, Warburg M, et al: Isolation of a candidate gene for Norrie disease by positional cloning. Nat Genet 1992a;1:199–203. Berger W, van de Pol D, Bachner D, Oerlemans F, Winkens H, Hameister H, Wieringa B, Hendriks W, Ropers HH: An animal model for Norrie disease (ND): gene targeting of the mouse ND gene. Hum Mol Genet 1996;5:51–59. Berger W, van de Pol D, Warburg M, Gal A, Bleeker-Wagemakers L, de Silva H, Meindl A, Meitinger T, Cremers F, Ropers HH: Muta-
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
tions in the candidate gene for Norrie disease. Hum Mol Genet 1992b;1:461–465. Chow CC, Kiernan DF, Chau FY, Blair MP, Ticho BH, Galasso JM, Shapiro MJ: Laser photocoagulation at birth prevents blindness in Norrie’s disease diagnosed using amniocentesis. Ophthalmology 2010;117:2402–2406. Halpin C, Owen G, Gutierrez-Espeleta GA, Sims K, Rehm HL: Audiologic features of Norrie disease. Ann Otol Rhinol Laryngol 2005;114:533–538. Kondo H, Qin M, Kusaka S, Tahira T, Hasebe H, Hayashi H, Uchio E, Hayashi K: Novel mutations in Norrie disease gene in Japanese patients with Norrie disease and familial exudative vitreoretinopathy. Invest Ophthalmol Vis Sci 2007;48:1276–1282.
Parzefall/Lucas/Ritter/Ludwig/Ramsebner/ Frohne/Schöfer/Hengstschläger/Frei
Meindl A, Berger W, Meitinger T, van de Pol D, Achatz H, Dorner C, Haasemann M, Hellebrand H, Gal A, Cremers F, et al: Norrie disease is caused by mutations in an extracellular protein resembling C-terminal globular domain of mucins. Nat Genet 1992;2:139–143. Meitinger T, Meindl A, Bork P, Rost B, Sander C, Haasemann M, Murken J: Molecular modelling of the Norrie disease protein predicts a cystine knot growth factor tertiary structure. Nat Genet 1993;5:376–380. Parving A, Warburg M: Audiological findings in Norrie’s disease. Audiology 1977;16:124–131. Perez-Vilar J, Hill RL: Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix. J Biol Chem 1997;272:33410–33415.
Novel Missense NDP Mutation in Norrie Disease
Rehm HL, Zhang DS, Brown MC, Burgess B, Halpin C, Berger W, Morton CC, Corey DP, Chen ZY: Vascular defects and sensorineural deafness in a mouse model of Norrie disease. J Neurosci 2002;22:4286–4292. Richter M, Gottanka J, May CA, Welge-Lussen U, Berger W, Lutjen-Drecoll E: Retinal vasculature changes in Norrie disease mice. Invest Ophthalmol Vis Sci 1998;39:2450–2457. Rozen S, Skaletsky H: Primer3 on the www for general users and for biologist programmers. Methods Mol Biol 2000;132:365–386. Sims KB, de la Chapelle A, Norio R, Sankila EM, Hsu YP, Rinehart WB, Corey TJ, Ozelius L, Powell JF, Bruns G, et al: Monoamine oxidase deficiency in males with an X chromosome deletion. Neuron 1989;2:1069–1076. Smith SE, Mullen TE, Graham D, Sims KB, Rehm HL: Norrie disease: extraocular clinical manifestations in 56 patients. Am J Med Genet A 2012;158A:1909–1917. Walsh MK, Drenser KA, Capone A Jr, Trese MT: Early vitrectomy effective for Norrie disease. Arch Ophthalmol 2010;128:456–460.
Warburg M: Norrie’s disease: a new hereditary bilateral pseudo-tumor of the retina. Acta Ophthalmol 1961;39:757–772. Xu Q, Wang Y, Dabdoub A, Smallwood PM, Williams J, Woods C, Kelley MW, Jiang L, Tasman W, Zhang K, Nathans J: Vascular development in the retina and inner ear: control by norrin and frizzled-4, a high-affinity ligandreceptor pair. Cell 2004;116:883–895. Yamada K, Limprasert P, Ratanasukon M, Tengtrisorn S, Yingchareonpukdee J, Vasiknanonte P, Kitaoka T, Ghadami M, Niikawa N, Kishino T: Two Thai families with Norrie disease (ND): association of two novel missense mutations with severe ND phenotype, seizures, and a manifesting carrier. Am J Med Genet 2001a;100:52–55.
Audiol Neurotol 2014;19:203–209 DOI: 10.1159/000358866
209
Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
