Spectrum of USH2A Mutations in Scandinavian Patients with Usher Syndrome Type II

Transcription

1 MUTATIO I BRIEF HUMA MUTATIO Mutation in Brief #995(2008) Online pectrum of UH2A Mutations in candinavian Patients with Usher yndrome Type II Bo reyer, Vigdis Brox 2, Lisbeth Tranebjærg -4, Thomas Rosenberg 5, Andrè M. adeghi 6, Claes Möller 6, 7, and Øivind ilssen, 2 * epartment of Medical Genetics, Institute of Clinical Medicine, University of Tromsø, O-907 Tromsø, orway; 2 University Hospital of orth-orway, O-908 Tromsø, orway; epartment of Audiology, H: Bispebjerg Hospital Copenhagen, K-2400 Copenhagen, enmark; 4 Wilhelm Johannsen Centre, Institute of Cellular and Molecular Medicine, IMCM, The Panum Institute, Copenhagen, K-2200 enmark; 5 Gordon orrie Centre for Genetic Eye iseases, Kennedy Institute-ational Eye Clinic, K-2900 Hellerup, enmark; 6 epartment of Audiology, ahlgrenska University Hospital, E-445 Göteborg, weden; 7 epartment of Audiology, wedish Institute of isability Research, E-7085 Örebro University Hospital, weden *Correspondence to: Øivind ilssen, epartment of Medical Genetics, Institute of Clinical Medicine, University of Tromsø, O-907 Tromsø, orway; Tel.: ; Oivind.ilssen@fagmed.uit.no Grant sponsor: orwegian Foundation for Health and Rehabilitation, anish ociety of the Blind, The Oticon Foundation; Grant number: 998/257 (FHR). Communicated by Andreas Gal Usher syndrome type II (UH2) is an autosomal recessive disorder, characterised by moderate to severe high-frequency hearing impairment, normal balance function and progressive visual impairment due to retinitis pigmentosa. Usher syndrome type IIa, the most common subtype, is defined by mutations in the UH2A gene encoding a short and a recently discovered long usherin isoform comprising 2 and 7 exons, respectively. More than 20 different disease-causing mutations have been reported, however, most of the previous reports concern mutations restricted to exons -2 of the UH2A gene. To explore the spectrum of UH2A disease-causing mutations among candinavian UH2 cases, patients from 8 unrelated families of which 27 previously had been found to carry mutations in exons -2 were subjected to extensive A sequence analysis of the full size UH2A gene. Altogether, 22 UH2A A sequence alterations were identified of which 57 were predicted to be disease-causing, 7 were considered to be of uncertain pathogenicity and 58 were predicted to be benign variants. Of 6 novel pathogenic UH2A mutations were located in exons 22-7, specific to the long isoform. UH2A mutations were identified in 89/8 (75.4%) families. In 79/89 (88.8%) of these families two pathogenic mutations were identified whereas in 0/89 (.2%) families the second mutation remained unidentified. In 5/8 (4.2%) families the UH phenotype could be explained by mutations in the UHA gene. The results presented here provide a comprehensive picture of the genetic aetiology of Usher syndrome type IIA in candinavia as it is known to date Wiley-Liss, Inc. KEY WOR: Usher syndrome; UH2A; usherin ITROUCTIO Usher syndrome (UH) is an autosomal recessive disorder comprising hearing impairment, retinitis pigmentosa (RP) and variable vestibular dysfunction. It is the most frequent cause of concurrent deafness and blindness and the prevalence in orthern Europe is estimated to range from. to 6.2 per 00,000 inhabitants (Marazita et al., 99; Received 6 April 2007; accepted revised manuscript 27 July WILEY-LI, IC. OI: 0.002/humu.9524

2 2 reyer et al. Rosenberg et al., 997; pandau and Rohrschneider, 2002, adeghi et al., 2004). The majority of Usher syndrome patients can be clinically classified into three subtypes (Kimberling and Möller, 995; Ahmed et al., 200). UH type I (UH, MIM# ) is characterised by profound prelingual sensorineural hearing loss, prepubertal onset of RP, and vestibular dysfunction. UH type II (UH2, MIM# 27690) is characterised by moderate to severe hearing impairment, onset of RP in the first or second decade of life and no vestibular impairment. UH type III (UH, MIM# ) is characterised by progressive postlingual hearing impairment, variable onset and severity of RP, with or without vestibular dysfunction. A minority of the Usher syndrome patients is clinically atypical deviating from the stringent classification. UH2 appears to be the most common clinical form of the disorder accounting for more than 50% of all Usher cases (Rosenberg et al., 997; pandau and Rohrschneider, 2002). UH is genetically heterogeneous and 7 distinct loci have been identified for UH (UHA-G), 4 for UH2 (UH2A-) and for UH (UHA) (Ahmed et al., 200; Ebermann et al., 2006). However, recently loci UHA and UH2B were withdrawn (reviewed in Kremer et al 2006). Among UH2 patients, mutations in the UH2A gene (MIM# ) seem to be the most frequent cause accounting for 74%-90% of the UH2 cases (Pieke ahl et al., 996; Weston et al., 2000). To date, more than 20 putative disease-causing mutations in the UH2A gene have been identified in patients with Usher syndrome type II (Eudy et al., 998; Liu et al., 999; Adato et al., 2000; reyer et al., 2000; Rivolta et al., 2000; Weston et al., 2000; Leroy et al., 200; ajera et al., 2002; Bernal et al., 200; Aller et al., 2004; Ouyang et al., 2004; Pennings et al., 2004; eyedahmadi et al., 2004; Maubaret et al., 2005; Aller et al., 2006; Cremers et al., 2007; Kaiserman et al., 2007; Baux et al, 2007). UH2A mutations have also been detected in patients with an atypical UH2 phenotype characterised by progressive hearing loss, variable vestibular dysfunction and RP (Liu et al., 999). Furthermore, putative pathogenic UH2A mutations have been identified in patients with nonsyndromic recessive RP and homozygously in two asymptomatic individuals (Rivolta et al., 2000; Bernal et al., 200). Mutation scanning of exons -2 in the UH2A gene in patients clinically diagnosed with Usher syndrome type II has revealed an unexpected problem. In a large fraction of patients the second disease allele remained unidentified. These cases accounted for 45%-6% of the patients in whom at least one UH2A mutation was identified (reyer et al., 2000; Pennings et al., 2004). The identification of additional 5 exons (van Wijk et al., 2004) as well as a novel alternatively spliced exon located after exon 70 (Adato et al., 2005) have provided obvious candidates for further mutation scanning in order to solve the incomplete molecular diagnosis of Usher syndrome type IIa. In fact, recently altogether 9 putative pathogenic mutations situated in exons were recently reported (van Wijk et al., 2004; Aller et al., 2006, Baux et al, 2007). In order to gain a better understanding of the molecular and genetic aetiology, and to improve the diagnostic tools for Usher syndrome type II we have investigated patients from 8 candinavian families by sequence analysis of the full size UH2A gene. Patients devoid of mutations in UH2A were subjected to UHA gene mutation analysis. ew data presented here in combination with results previously reported by our group have allowed us to provide a nearly complete spectrum of UH2A mutations in this large candinavian UH2 cohort. PATIET A METHO Altogether patients from 8 families diagnosed with Usher syndrome type II were included in this study. The families originated from enmark (48 families), orway (2 families) and weden (47 families). Patients from 87 families were subjected to first-time-molecular-investigation whereas families had previously undergone mutation analyses of exons -2 (reyer et al., 2000). Thirteen of these patients, 9 heterozygote and 4 mutation negative were subjected to mutation scanning of exons The remaining 8 patients were found to carry mutations on both alleles (reyer et al., 2000), and in order to present a complete spectrum of candinavian mutations these 8 patients were included in Table and in the statistical summaries presented here. The clinical diagnosis of Usher syndrome type II was based on established criteria (Kimberling and Möller, 995). The anish patients were diagnosed at The ational Eye Clinic for the Visually Impaired, Hellerup, enmark. everal ophthalmologists and audiologists have provided clinical information about the orwegian patients. wedish patients were diagnosed at the epartment of Audiology, ahlgrenska University Hospital, Göteborg, weden. ubjects were included in the study based on the following criteria: Clinical findings of congenital moderate to severe bilateral sensorineural hearing loss with normal vestibular function, and bilateral retinitis pigmentosa (RP) (night blindness, loss of peripheral visual fields and reduced or non-detectable

3 UH2A Mutations in candinavia electroretinogram). Auditory function was assessed by otoscopy, standard pure tone audiometry ( Hz) and tympanometry. In many cases the localization of hearing loss in the cochlea was confirmed by otoacoustic emissions (OAE) and auditory brainstem responses. Ophthalmological evaluations comprised funduscopy, visual acutity test, visual field test and electroretinography (ERG). Vestibular evaluation was performed in all wedish patients by bithermal-caloric test, rotatory test, computerized dynamic posturography and Romberg test. Written informed consent to allow genetic testing was obtained from all participants. This study was conducted according to the eclaration of Helsinki. Mutation analysis Genomic A extraction, intronic PCR primers, PCR amplification conditions and the A sequencing of UH2A were performed as previously described (reyer et al., 2000; Weston et al., 2000), except the use of Jumptart RETaq ReadyMix PCR Reaction Mix (igma) and a 00 automated sequencer unit (Applied Biosystems) for most of the reactions. Exons 22-7 were amplified by PCR and directly sequenced with the appropriate primers as previously described by van Wijk et al. (2004) except for primers used to amplify and/or to sequence exons 4, 4, 45, 47, 5, 58, 59, 60, 6, 64, 65, 68, 7 and 72 (ee supplementary Table ). Patients, in whom no UH2A mutation could be identified, were subjected to mutation analysis in the UHA gene according to Adato et al and Fields et al., equence variants were identified by the use of the eqcape software version 2. (Applied Biosystems) and by manual inspection of the electropherograms. canning for mutations were carried out until two mutations resulting directly or indirectly in termination codon were identified or when all exons were analysed. g.a numbering starts at nucleotide position in Human Refseq: gi: , T_02877, which represents the minus (-) strand of UH2A. For UH2A and UHA ca numbering corresponds to A in the ATG translation initiation codon in Refseqs. gi: , AY4857. and gi: , AF49577., respectively. Mutations of missense type were tested in orwegian control chromosomes. Haplotype analysis was carried out with P markers according to reyer et al., 200. atabases Usherin domain structure predictions was obtained from: For comparative protein alignments the following reference sequences were used: Human usherin protein refseq: gi: , P_99686.; og usherin: gi:740066, XP_ ; Rat usherin: gi: , XP_ ; Mouse usherin: gi: , AAZ264; Chicken usherin: gi: , XP_ Mutation nomenclature is according to REULT Molecular analysis of the UH2A gene in 8 unrelated candinavian patients revealed 22 sequence alterations of which 7 were new. Of the 22 sequence alterations identified 57, of which 6 were novel, were predicted to be disease-causing. even mutations were classified as of uncertain pathogenicity (UP) and 58 were predicted to be benign (Table ; A, B, C). Among the 57 putative pathogenic mutations 2 nonsense mutations, 8 missense mutations, 5 rearrangements and splice site mutation were identified. isease-causing UH2A mutations were identified in 89 families (75.4 %). In 79 families patients were either homozygous or compound heterozygous. In 2 families disease alleles were segregating. Patients from 0 families remained heterozygous after full-scale mutation analysis. Altogether 70 UH2A disease alleles were identified. The c.2299delg allele accounted for 0.6% all disease alleles detected whereas, on the other hand, 2/57 (56.%) different putative disease- causing mutations occurred in single families. In 29 families no UH2A mutations were detected, however, in 5 of these families we identified UHA gene mutations that likely explain their UH phenotype (Table 2). ummary of UH2A genotypes in 8 candinavian families is presented in Table.

4 4 reyer et al. Table. pectrum of UH2A Mutations in candinavia A. Usherin truncating mutations ucleotide change onsense mutations c.00c>t a c.87c>t a c.779t>g b c.876c>t a b c.202c>t a c.2797c>t a c.298c>t c.88c>t a b c.92c>a c.4957c>t c.547g>t c.565a>t c.666c>t c.8557a>t c.920g>a c.0450c>t c.0684g>t c.28t>a c.46g>t c.864c>t b c.2954c>a c.822c>t c.4489c>g Exon no. EX2 EX2 EX4 EX EX2 EX EX4 EX8 EX8 EX24 EX27 EX28 EX4 EX42 EX46 EX5 EX54 EX58 EX59 EX6 EX6 EX64 EX66 Predicted effect p.r4x p.r6x p.l260x p.r626x p.q675x p.q9x p.q995x p.r295x p.x p.r65x p.e825x p.r885x p.q2206x p.r285x p.w040x p.r484x p.e562x p.y776x p.e806x p.w955x p.y48x p.r4608x p.480x Predicted pathology * * o. (%) of alleles 4 (2.4) 4 (2.4) (.8) (.8) 7 (4.) Patient origin / / / / // Ref. 2, 2, 2 4 eletions and insertions c.545_546delaa b c.98_92dupcagc a b f c.86_89duptaca c.965delt a c.2299delg a b c.2525dupt c.2878_2279delaa a g.98882_972870del c. 6795_6797delATA c.7574delc g.95096_952557delins2 c.9770dupa c.045delginsaa c.875_876delca c. 207_208delGG EX EX6 EX0 EX EX EX EX4 EX2-2 EX5 EX40 EX47 EX50 EX52 EX6 EX6 p.k82fs p.h08fs p.g64fs p.c655fs p.e767fs p.l84fs p.960fs EX2_EX2del p.e2265_y2266delins p.p2525fs EX47del p.257fs p.e449fs p.q959fs p.g440fs 4 (2.4) 52 (0.6) 6 (.5) 7 (4.) / // 5, 2, 6 7 plice-site mutation c.5776g>a IV28 EX28 splice defect

5 UH2A Mutations in candinavia 5 B. Missense mutations in UH2A ucleotide change Exon no. Predicted effect c.7a>g a EX2 p.a25t c.488g>a a EX p.c6y c.688g>a a c EX4 p.v20m c.802g>a b EX5 p.g268r c.907c>t EX6 p.r0c c.000c>t a EX6 p.r4w c.06a>c a EX6 p.46h c.055c>t b EX6 p.t52i c.44g>c EX8 p.e478 c.606t>c a EX9 p.c56r c.780c>t EX0 p.p595 c.9a>t EX p.644v c.27g>c a b c d EX2 p.g7r c.2276g>t a b EX p.c759f c.65c>t EX7 p.p22l c.4045t>c EX8 p.49p c.4457a>g a EX2 p.k486r c.474c>t EX22 p.l572f c.4994t>c EX25 p.t665i c.5270a>g d EX26 p.y757c c.6240g>t EX2 p.k2080 c.6257c>a EX2 p.t2086 c.67c>t EX2 p.t206i c.6506t>c EX4 p.t269i c.67a>c EX5 p.e228a c.6875g>a EX6 p.r2292h c.7685t>c d EX4 p.v2562a c.8624g>a EX4 p.r2875q c.8656c>t EX4 p.l2886f c.9262g>a EX47 p.e088k c.9296a>g EX47 p.099 c.94g>a EX47 p.t5a c.970a>g d EX47 p.r24g c.940g>a EX48 p.44 c.9595a>g EX49 p.99 c.022a>c EX52 p.e4a c.050c>a EX5 p.p504t c.056t>c EX5 p.w52r c.0769c>t EX55 p.p590l c.504c>t EX59 p.t85i c.602a>g EX60 p.m868v c.677c>a EX60 p.p89t c.26g>t EX62 p.4054i c.24c>t d e EX6 p.r45c c.2695c>g EX6 p.p422r c.297g>t EX6 p.v44l c.6c>t EX6 p.t449i c.460g>a EX6 p.y4487c c.776g>c EX6 p.q4592h c.870t>g EX64 p.f4624v c.484t>g EX66 p.l4795r c.509c>t EX70 p.r50w Predicted pathology UP() UP() UP()* 2 UP() UP()* 2 UP() Conservation in h/r/m/d/c species A-T/T/V/T/ C/C/C/C/C V-M/L/L/L/W G/G/G/G/G R/R/R/R/R R/R/R/R/R //// T/T/T/T/T E-/E/E/E/E C/C/C/C/C P-/P/P/P/P -V/V/V/V/Y G/G/G/G/G C/C/C/C/C P/P/P/P/P ///P/ K-R/R/R/R/ L-F/L/L/L/L T-I /T//I/I Y/H/H/Y/Y K-/R/R/K/T T-/T/T/T/T T-I /T/T/T/V T-I/L/T/T/- E-A/E/E/E/E R-H/H/H/H/Y V-A/A/A/V/I R-Q/L/L/L/L L-F/F/F/F/F E-K/E/E/E/E -//// T-A/I/I/T/A R/R/R/R/R -////E -//// E-A/A/A/A/A P/A/A/P/P W/W/W/W/W P-L/L/P/P/P T-I/A//A/ M-V/V/V/V/L P-T/P/P/P/P //// R-C/R/R/R/R P/P/P/P/P V-L/R/R/A/T T/T/T/T/T Y/Y/Y/Y/Y Q/R/R/R/R F-V/P/// L/L/L/L/L R-W/G/G/G/E o (%) of alleles 5 (.0) 2 (7.) 4 (2.4) Patient origin ///n /n // /n /n /n / ///n //n //n / /n //n ///n ///n /n ///n /n //n /n /n //n / //n //n ///n / /n /n ///n //n / /n /n /n Ref., 2, 5, 2, 5 2, 5, , 8, 5, 0, 0,, 2, 8 2 4

6 6 reyer et al. C. ilent mutations and unclassified intronic variants in UH2A ucleotide change Exon/Intron no. Amino acid or IV position 504G>A a 949C>A b d 49C>T a 209T>C 2256T>C 47A>G a 50C>A 7506G>A 76G>A 907A>T 946G>A 209C>T 262G>A 2666A>G 9A>G 440G>A 478A>G c t>c a c.44-28a>c c.287_2840delattg c.6444c>a a c.2677a>g c.575a>g a c.82-8g>t a c a>g a c.4252-_4252-6duptttc c a>c c.70-65c>t c c>a c a>g c.86820g>a EX EX6 EX8 EX2 EX EX20 EX25 EX40 EX6 EX6 EX6 EX62 EX6 EX6 EX6 EX6 EX67 IV IV6 IV7 IV9 IV2 IV5 IV7 IV8 IV9 IV20 IV8 IV42 IV4 IV4 68 R7 T47 70 H G67 P2502 E92 P969 L982 Y40 T4204 T4222 E497 R4480 K4906 IV-80T/C IV6-28A>C IV77_40delATTG IV94C/A IV27A>G IV55A/G IV7-8G>T IV8-44A/G IV9-_6dupTTTC IV20-92A>C IV8-65C/T IV4240C/A IV4-9A/G IV420G/A Predicted pathology UP() Complete list of UH2A sequence variants detected in candinavian Usher type II patients (A, B, C). Altogether 22 sequence alterations were identified. The 7 novel sequence variants are shown in bold. Fifty-seven mutations were predicted to be disease-causing of which 6 are novel. A; Usherin truncating mutations, B; UH2A missense mutation, C; UH2A silent mutations and unclassified intronic variants. // denotes enmark/orway/weden, respectively. All missense variants have been tested in a normal panel and n indicates that the sequence alteration has been found to be present in more than out of at least 00 orwegian normal chromosomes. UP denotes mutations of uncertain pathogenicity. h/r/m/d/c denotes human/rat/mouse/dog/chicken usherin orthologues, respectively. In A and B, percentages of mutant alleles were calculated according to a total number of 70 disease-alleles identified (including 2 families with disease alleles). a A variants previously identified in the candinavian patient material by us (). b Mutations previously reported as pathogenic in other populations. c Benign mutations previously reported as pathogenic, by us (). d Mutations of uncertain pathogenicity were denoted UP. In most cases these mutations were regarded as non-pathogenic in this work; UP(-). g.a numbering starts at nucleotide position in Human Refseq: gi: , T_02877 which represents the minus (-) strand of UH2A. For ca numbering corresponds to A in the ATG translation initiation codon in Refseqs. gi: , AY4857. for UH2A and gi: , AF for UHA. e Occurs in cis with nonsense mutations. f According to new nomenclature guidelines ( HGV.org./mutnomen/.) mutation c.92-22inscagc (, 2) was renamed to c.98-92dupcagc. *Missense mutations occurring in cis. References:. reyer et al., 2000; 2. Weston et al., 2000;. ovel mutations identified in this study; 4. van Wijk et al., 2004; 5. eyedahmadi et al., 2004; 6. Eudy et al., 998; 7. Baux et al, 2007; 8. Adato et al., 2000; 9. Cremers et al., 2007; 0. ajera et al., 2002;. Rivolta et al., 2000; 2. Kaiserman et al., 2007;. Aller et al., 2006; 4. Pennings et al., Ref 4, 2 2, 8, 2, 8, 8

7 UH2A Mutations in candinavia 7 Usherin truncating mutations As displayed in Table A, a total of 9 mutations of which 25 were novel were predicted, directly or indirectly, to cause premature termination of usherin translation or causing deletion of one or more amino acid residue(s). These were 2 nonsense mutations, 5 rearrangements (deletions, insertions, duplications and indels) and splice site mutation. Out of 70 different candinavian disease alleles detected 29 (75.9%) belonged to this class. onsense mutations accounted for 48 alleles (28.2%), rearrangements (deletions, insertions and indels) for 80 alleles (47%) and a splice site mutation for allele (0.59%). onsense mutations were distributed evenly throughout the UH2A gene and 4 were located in exons 22-7 specific to the long usherin isoform. Most nonsense mutations were rare, each of them representing less than 2% of all disease alleles. Exceptions were c.87c>t (p.r6x), c.876c>t (p.r626x) and c.864c>t (p.w955x) that were found on 4, 4 and 7 chromosomes, respectively. In agreement with Cremers et al (2007) who recently described p.w955x to be common among European and American UH2 patients, p.w955x was the only nonsense mutation detected in patients from all three candinavian countries. onsense mutation c.298c>t (p.q9x) was detected in two anish families, however curiously, in one of the families the 9X allele coexisted in cis with another nonsense mutation, c.202c>t (p.q675x). Fifteen truncating rearrangements were identified of which 9 were novel. Twelve of these mutations were small frame-shifting deletions or insertions of to 4 nucleotides (c.545_546delaa, c.98_92dupcagc, c.86_89duptaca, c.965delt, c.2299delg, c.2525dupt, c.2878_2279delaa, c.7574delc, c.9770dupa, c.045delginsaa, c.875_876delca and c.207_208delgg). The common bp deletion c.2299delg was detected on 52 chromosomes accounting for 0.6% of all disease alleles identified. This was the only mutation in this class that was detected in families from all candinavian countries. One in-frame deletion was identified, c.6795_6797delata that results in the replacement of amino acids E2265 and Y2266 with Two novel large deletions were discovered that constitute 7.% of all disease alleles detected. Patients from 5 families of wedish origin, homozygous and 4 compound heterozygous, contained mutation g.98882_972870del resulting in a deletion of 9009 nucleotides that removes exons 22-2 (Ex22_2del). imilarly, patients from 5 families of orwegian origin, 2 homozygous and compound heterozygous, contained mutation g.95096_952557delins2 resulting from a deletion of 669 nucleotides followed by insertion of 2 nucleotides. The deletion breakpoints reside in introns 46 and 47 and, hence, this deletion/insertion mutation removes exon 47 (Ex47del). One single splice site mutation, c.5776g>a (IV28G>A), accounting for one disease allele was detected in a orwegian family. This particular mutation was predicted to disrupt a splice donor site and thereby compromising the splicing of exon 28. Putative disease-causing UH2A missense mutations Altogether 52 missense variants were detected (Table B). These were categorized into three classes according to predicted pathogenicity. Missense mutations were classified as putative disease-causing when they remained undetected among at least 00 normal control chromosomes. Including UP variant (c.970g>a) p.r24g (Table B) eighteen missense mutations, of which were novel, were predicted to be disease-causing. Fifteen missense mutations were rare and found to be present only in l or 2 families. Three mutations c.056t>c (p.w52r), c.802g>a (p.g268r) and c.06a>c (p.46h) were detected on 4, 5, and 2 chromosomes, respectively. Only p.46h was detected in patients from all three candinavian countries. Haplotype analysis using 6 P markers covering exons 2-2 (reyer et al., 200) indicate that the p.46h expressing allele, c.06c, is associated with a core haplotype that has a frequency of <0.0 in candinavia (results not shown). eventeen of the putative pathogenic missense mutations affected amino acid residues that were fully conserved between human, rat, mouse, dog and chicken usherin orthlogues. The one exception was c.776g>c (p.q4592h) which was found in one patient in combination with p.w955x. Whereas, glutamine at position 4592 is conserved in chimpanzee - the rat, mouse, dog and chicken usherin orthologues contain arginine in this position. Except for c.907c>t (p.r0c), which was found in an heterozygous state, putative disease-causing missense mutations co-occurred with truncating mutations in 29 families and in combination with another missense mutation in 5 families. Out of novel missense mutations 9 reside in exons 22-7 affecting residues specific to the long usherin isoform.

8 8 reyer et al. UH2A mutations of uncertain pathogenicity Although absent from orwegian normal control chromosomes 6 missense mutations, and silent mutation, were of questionable pathogenicity (Tables B and C). We chose a conservative approach in our predictions and for the following reasons we have categorized 6 out of 7 of these sequence alterations as non-disease-causing: Mutations c.5270a>g (p.y757c) and c.970a>g (p.r24g) occurred only in cis. In contrast to p.y757, p.r24 is conserved in mouse, rat, dog and chicken orthologues. Thus, we suggest p.r24g to be diseasecausing and p.y757c to be benign. Mutation c.27g>c (p.g7r) was previously reported by us to be pathogenic (reyer et al., 2000). However since then, ajera et al (2002) have demonstrated that this mutation occurs in panish normal controls (ajera et al., 2002) and therefore p.g7r was regarded as benign in this work. Mutation c.7685t>c (p.v2562a) was identified in one family of wedish origin, however, p.v2562 in human is p.a2562 in mouse and rat and p.i2562 in chicken, hence, we considered this mutation to be nonpathogenic. Mutation c.24c>t (p.r45c) was previously reported to be of uncertain pathogenicity by van Wijk et al (2004). In this work p.r45c was found to coexist with nonsense mutations p.r626x and p.r65x in one patient and with p.r626x in another. Expectedly, segregation analysis demonstrated p.45c to be in cis with p.626x and therefore it was predicted to be benign. Mutation c.4045t>c (p.49p) results in the replacement of serine with proline at amino acid position p.49. og usherin harbours proline at this residue and, hence, we considered p.49p not to be pathogenic. Finally, silent mutation c.949c>a (p.r7r) was previously suggested to be a putative splice site mutation (Pennings et al., 2004). However, since this suggestion was based on in silico predictions rather than functional studies we chose to consider c.949c>a as non-pathogenic in this work. on-pathogenic UH2A missense variants Twenty-nine missense variants were considered to be clearly non-pathogenic. Primarily this was based on the fact that they were detected among normal control chromosomes (Table B). One exception was c.688g>a (p.v20m) previously reported by us to be disease-causing (reyer et al., 2000). However, as was the case for most non-pathogenic variants p.v20m was found to co-occur in patients who carried two additional mutations that were both leading, directly or indirectly, to premature stop codons. ine out of 29 variant amino acid residues were conserved among mouse, rat, dog and chicken usherin orthologues (p.e478, p.p595, p.l572f, p.t2086, p.e228a, p.l2886f, p.e088k, p.e4a, and p.p89t). Twenty variant amino acid residues were not conserved and for 6 of these variants the affected residue was replaced with an amino acid that occurred in an usherin orthologue from another species (p.k486r, p.r2292h, p.099, p.t5a, p.p590l and p.m868v). ine benign nucleotide substitutions reported here were recently identified in panish normal controls (Aller et al., 2006) (Table B). ilent mutations and unclassified intronic variants As shown in Table C seventeen silent mutations, of which are novel, were detected in the UH2A coding sequence. one of these alterations were expected to compromise the expression of the UH2A gene. As discussed above this also include c.949c>a (p.r7r) that has been suggested to create a novel splice site (Pennings et al., 2004). Furthermore, 4 intronic sequence variants that were presumed not to conflict with the splicing process were identified (Table C). Mutations in the UHA gene Usher syndrome type III displays phenotypic variability (ess et al., 200) and has been shown to mimic both UH and UH2 (Pennings et al., 200). Thus, in patients where no pathogenic mutation could be identified in UH2A, A sequence analysis of the UHA gene was performed. Four different pathogenic mutations were indeed identified (Table 2). Two orwegian patients were homozygous for the c.528t>g (p.y76x) mutation (Finn_major) (Joensuu et al., 200), one patient was compound heterozygous for the p.y76x and the deletion/insertion mutation 49delCAGGinsTGTCCAAT (Fields et al., 2002) and one was heterozygous for p.y76x with the other disease-causing mutation left unidentified. Two novel UHA mutations were detected. One wedish patient was found to be homozygous for missense mutation c.t>c (p.05p) and a anish patient of Turkish origin was found to be homozygous for the truncating mutation c.8dela (p.m6fs).

9 UH2A Mutations in candinavia 9 Table 2. Mutations in UHA ucleotide change Exon no Amino acid position Predicted pathology c.528t>g 2 p.y76x 49delCAGGinsTGTCCAAT p.50fs 8delA* p.m6fs c.t>c 2 p.05p References * Identified in anish patient of Turkish origin. References:. Joensuu et al., 200; 2. Fields et al., 200;. This work. 2 ICUIO ince the identification of the UH2A gene (Eudy et al., 998) several research groups have scanned the UH2A gene in UH2 patients from various ethnic backgrounds (Eudy et al., 998; Liu et al., 999; Adato et al., 2000; reyer et al., 2000; Rivolta et al., 2000; Weston et al., 2000; Leroy et al., 200; ajera et al., 2002; Bernal et al., 200; Aller et al., 2004; Ouyang et al., 2004; Pennings et al., 2004; eyedahmadi et al., 2004; Maubaret et al., 2005; Aller et al., 2006; Cremers et al., 2007; Kaiserman et al., 2007; Baux et al., 2007). uch investigations are a prerequisite in order to provide an accurate and unambiguous molecular diagnosis for patients with Usher syndrome type II. However, most of these studies have in common that the mutation analysis has been restricted to exons -2 and, hence, the mutation detection rate is relatively low, but most importantly, a large fraction of the UH2 patients studied have been diagnosed as heterozygous with one disease allele left unidentified. In the present work we have addressed this problem by performing an extensive A sequence analysis of all 7 UH2A exons in a patient material that included 8 unrelated patients of candinavian origin. Out of 22 sequence alterations identified in candinavian UH2 patients 57 (46.7%) were predicted to be disease-causing. Twenty-five mutations reside in exons -2 whereas 2 reside in exons Out of 57 putative disease-causing mutations identified only 2 mutations (2%) have been reported in patients from other countries such as pain, Israel, etherland, UK, France and UA. This phenomenon can be explained by the fact that of the 57 mutations identified 2 (56.%) were private occurring only in single families and, moreover, (22.8%) additional mutations were observed in no more than 2 families. Putative disease-causing mutations were identified in probands from 89 unrelated families. Probands from 0 families remained heterozygous after extensive mutation analysis. Assuming that heterozygous patients suffer from UH2A 68 disease alleles were identified among a total of 78 (94.4%). Two mutations were found to be frequent among candinavian UH2A alleles. Mutation c.2299delg accounts for 0.6% whereas missense mutation p.46h accounts for 7.% of all disease alleles detected. In particular, c.2299delg was frequent among anish UH2A alleles accounting for 47.5%. In accordance with our previous report showing that c.2299delg alleles have a common ancestral origin (reyer at al, 200) the high frequency among anish families is likely to result from a strong founder effect. Likewise, the pronounced frequency of the c.06g allele (p.46h) among candinavian patients likely results from a founder effect, as it appeared to be associated with a specific core haplotype (results not shown). Thirty-nine usherin truncating mutations were identified. Out of 5 novel nonsense mutations discovered 4 were predicted only to affect the expression of the long usherin isoform. Likewise, 8 out of 9 novel rearrangements (deletion, insertion, indel mutations) reside in exons that are specific to the long usherin isoform. This include 2 large deletions, g.98882_972870del and g.95096_952557delins2, that remove exons 22-2 and exon 47 respectively. ingle base-pair substitutions in splice junctions have been proposed to constitute 0-5% of all mutations causing human inherited disease (Krawczak et al., 992; Krawczak et al., 2007). Remarkably, however, among 57 pathogenic mutations identified here only, c.5776g>a affecting the invariant 5 donor site of intron 28, was predicted to affect pre-mra splicing. Altogether 52 nucleotide substitutions were identified that caused amino acid replacements. Twenty-nine variants detected among normal controls were classified as benign. Twenty-three missense variants not observed

10 0 reyer et al. among normal control chromosomes were classified either as putative pathogenic or as of uncertain pathogenicity. ine out of 29 (%) benign mutations affected residues that were conserved among rat, mouse, dog and chicken orthologues. In contrast, among putative pathogenic missense mutations, 7 out of 8 (94.4%) affected residues were conserved among these usherin orthologues. The missense mutations resulted in major changes in the side chain properties of the corresponding amino acid residues with regard to charge, hydropathy and/or bulkiness. Three missense mutations corresponding to p.c6y, p.g268r and p.r0c, affect the thrombospondin-type laminin G domain (TP-LG). Three mutations corresponding to p.r4w, p.46h and p.t52i affect the laminin domain (L). Two mutations corresponding to p.c56r and p.c759f affect laminin-egf-like (LE) domains and 5, respectively. The remaining 9 missense mutations affect residues located in the long isoform. Mutant residues p.p22l, p.p504t, p.w52r, p.p422r, p.t449i, p.q4292h and p.l4795r were predicted to affect fibronectin type III (FnIII) repeats 2, 8, 8, 25, 28, 29 and 0, respectively, whereas p.r24g was predicted to be located between FnIII repeats 7 and 8, p.4054i between 2 and 24 and, finally, p.y449c between FnIII repeats 26 and 27. Whether, these missense mutations cause usherin misfolding or if they abolish binding to interacting protein partners will have to await investigation until an usherin structure becomes available. ix missense variants were not observed among normal controls but were considered to be of uncertain pathogenicity, either because they were detected in normal controls from another population (p.g7r), because they existed in cis with a nonsense mutation (p.r45c) or another missense mutation (p.y757c and p.r24g), or because the mutations introduce new amino acids that were identical to the corresponding residue in usherin orthologues from other species (p.v2562a and p.49p). We favour the idea that, except p.r24g, these mutations are non-disease-causing. However, the classification of p.g7r still remains somewhat elusive as peptide competition experiments have shown the p.7r variant to abolish usherin collagen IV interaction (Bhattacharya et al., 2004). van Wijk et al (2004) reported alternative splicing of exons 42, and 62. Only skipping of exon 42 led to interruption of the reading frame. However, the functional significance of these splice forms was questioned (van Wijk et al., 2004). Here we report the identification of mutations c.8557a>t (p.r285x), c.045delginsaa and c.26g>a (p.4054i), corresponding to sequence alterations in exons 42, 52 and 62, respectively. These mutations clearly demonstrate the functional importance of these exons in the maintenance of vision and hearing. Also the question remained whether two alleles with mutations in exons 22-7 could lead to Usher syndrome or causing non-syndromic hearing loss or RP and, moreover, whether other organs would be involved such as heart and kidney, both expressing the long usherin isoform (van Wijk et al., 2004). Among the unrelated candinavian UH2A patients 5 contained 2 putative disease-causing mutations that were predicted to affect the long usherin isoform. even patients were homozygous or compound heterozygous for 2 truncating mutations whereas 8 patients were compound heterozygous containing one truncating and one missense mutation. All 5 patients displayed an Usher type II phenotype, hence, these results demonstrate that the combination of 2 mutations affecting the long usherin isoform is sufficient to cause classical Usher syndrome type II with no additional symptoms. Patients with 2 missense mutations affecting the long isoform were not observed, thus, as of to date the above conclusion is valid only for truncating mutations and for the combination of truncating and missense mutations. Furthermore, since p.c759f has been found homozygously present in patients with RP but also in nonsymptomatic individuals it was speculated if the mutation c.2276g>t, causing p.c759f, is in linkage disequilibrium (L) with a so-far-unknown mutation in the exons (van Wijk et al., 2004). Complete A sequencing analysis of the 7 usherin coding exons in 2 patients, both compound heterozygous and harboring c.2276g>t on one allele, failed to disclose other pathogenic mutations in cis. This may indicate that p.c759f is disease-causing by itself or that c.2276g>t might be in L with an UH2A regulatory mutation located outside the coding region. Alternatively, the variable phenotype displayed by c.2276g>t might be caused by the effect of modifier genes. In 5 families including 7 patients pathogenic mutations in the UHA gene were identified. Thus, mutant UHA alleles constitute a small but significant proportion (4.2%) of the disease alleles detected. A similar finding was recently reported from an extensive, chip-based, analysis by Cremers et al (2007) who identified UHA mutations in patients originally classified as UH2. These results are in accordance with the findings of Pennings et al (200) and ess et al (200) who pointed to the diagnostic confusion that may occur between these two Usher syndrome subtypes. The p.y76x causing mutation (Finn_major) (Joensuu et al., 200) was found in orwegian patients. This probably reflects the migration history from Finland to orway. A similar migration pattern has seen

11 UH2A Mutations in candinavia for the AGU-Fin mutation causing aspartylglucosaminuria (Tollersrud et al., 994). Two novel UHA mutations were identified. Besides the truncating mutation c.8dela (p.m6fs), a missense mutation c.t>c causing p.05p was found to potentially affect a putative outside-inside trans-membrane segment of the UHA protein (clarin-). Patients from 0 families remained heterozygous upon extensive UH2A mutation scanning. In general, direct sequencing of coding exons provides a high detection rate. However, mutations such as large deletions and duplications as well as regulatory mutations located outside coding exons might have been missed. The two large deletions reported here were identified solely because they appeared homozygously in our patient material. Hence, such deletions might be underreported since they are not likely to be discovered by conventional PCR based methods. Interestingly in this respect, large deletions in PCH5 were recently reported to be a significant cause of UHF syndrome (Le Guédard et al., 2007). To improve UH2A diagnostics quantitative detection techniques such as multiplex ligation dependent probe amplification (MLPA) or direct quantitative PCR (qpcr) should be implemented. In 24 of the UH2 families no mutations were identified. When disregarding families with UHA mutations this number corresponds to 2.2% (24/) of total. The possibility exists that at least some of these families harbor obscure UH2A mutations that escape our detection methods. However, the ratio of affected families with pathogenic mutation versus affected families with 2 pathogenic mutations is low (0.2). Thus, given that there is a binomial distribution (a 2 2ab b 2 =) of the fractions of families with 2 mutations (a 2 ); with mutation (2ab); and with no mutation detected (b 2 ) we expect that most, if not all, true UH2A patients have been identified in this study (b 2 =0.0027). Hence, we suggest that the remaining 24 (2.2%) families may carry mutations in other genes of which the most obvious candidate would be the VLGR gene (UH2C) (Weston et al., 2004). In addition, mutations in the CH2 (UH) gene have been associated not only with Usher syndrome type I, but has also been detected in patients with Usher syndrome type II phenotype (Astuto et al., 2002) and, moreover, recently several patients displaying UH2 phenotype were found to harbor mutations in MYO7A, either heterozygously or compound heterozygously with mutations in the UH2A gene (Cremers et al., 2007). The fraction of unrelated UH2 patients in whom no UH2A mutations were detected appears to be somewhat higher among wedish families (5/46, 2.6%) as compared to anish (7/47, 4.9%) and orwegian families (/20, 5%). This might be due to a difference in the genetic aetiology of UH2 phenotype, such as to a larger extent the involvement of other UH loci among wedish UH2 families (see Table ). Table. ummary of UH2A Genotypes in 8 candinavian Families umber of families Compound heterozygous Homozygous Heterozygous egative enmark orway weden candinavia * 6* 6* 29* 5 * Indicates,, and 5 families with UHA mutations among anish, orwegian, wedish and candinavian UH2 families, respectively. Five missense mutations classified as UP(-) were considered non-disease-causing in the above summaries. The results reported here will facilitate future molecular diagnosis of Usher syndrome type IIa. However, considering the large size of the UH2A gene, the wide spectrum of UH2A mutations and the fact that a large fraction of mutations seem to be private create significant practical problems. For instance, of the 57 diseasecausing mutations reported here 45 (~79%) occur in or 2 families only. A similar pattern of allelic heterogeneity was observed in panish and French patients and, noteworthy, when comparing the populations there is only a modest degree overlap in the mutational spectrum. The 4 panish and 9 out of 20 French mutations specific to

12 2 reyer et al. the long usherin isoform (Aller et al., 2006, Baux et al., 2007) were all different from those reported here in the candinavian patients. Although most previous studies have been restricted to exons -2, allelic heterogeneity has also been observed in other populations. Thus, even with high through-put technology, mutation scanning of UH2 patients will be a formidable task. The recent establishment of a microarray based detection system for UH2 causing mutations is promising (Cremers et al., 2007). However, such a strategy will rely heavily on data from many different populations and, hence, the contribution from multiple research groups. Furthermore, efforts must be made to investigate the spectrum of large deletions and duplications. According to the data presented here such large rearrangements may potentially account for as much as ~% of all independent UH2A alleles in candinavia. REFERECE Adato A, Weston M, Berry A, Kimberling WJ, Bonne-Tamir A Three novel mutations and twelve polymorphisms identified in the UH2A gene in Israeli UH2 families. Hum Mutat 5:88. Adato A, Vreugde, Joensuu T, Avidan, Hamalainen R, Belenkiy O, Olender T, Bonne-Tamir B, Ben-Asher E, Espinos C, Millan JM, Lehesjoki AE, Flannery J, Avraham KB, Pietrokovski, ankila EM, Beckmann J, Lancet UHA transcripts encode clarin-, a four-transmembrane-domain protein with a possible role in sensory synapses. Eur J Hum Genet 0:9-50. Adato A, Lefervre G, elprat B, Michel V, Michalski, Chardeboux, Weil, El-Amraoui A, Petit C Usherin, the defective protein in Usher syndrome type IIa, is likely to be a component of the interstereocilia ankle links in the inner ear sensory cells. Hum Mol Genet 4: Ahmed ZM, Riazuddin, Wilcox ER The molecular genetics of Usher syndrome. Clin Genet 6: Aller E, ajera C, Millan JM, Oltra J, Perez-Garrigues H, Vilela C, avea A. Beneyto, M Genetic analysis of c.2299delg and C759F mutations (UH2A) in patients with visual and/or auditory impairments. Eur J Hum Genet 2: Aller E, Jaijo T, Benyoto M, ajera C, Oltra, Ayuso C, Baiget M, Carballo M, Antonolo G, Valverde, Moreno F, Viela C Collado, Perez-Garrigues H, avea A, Millan JM Identification of 4 novel mutations in the long isoform of UH2A in panish patients with Usher syndrome type II. J Med Genet 4:e55. Astuto LM, Bork JM, Weston M, Askew JW, Fields RR, Orten, J, Ohliger J, Riazuddin, Morell RJ, Khan, Kremer H, van Hauwe P, Moller CG, Cremers, CW, Ayuso C, Heckenlively JR, Rohrschneider K, pandau U, Greenberg J, Ramesar R, Reardon W, Bitoun P, Millan J, Legge R, Friedman TB, Kimberling WJ CH2 mutation and phenotype heterogeneity: a profile of 07 diverse families with Usher syndrome and nonsyndromic deafness. Am J Hum Genet 7: Baux, Larrieu L, Blanchet C, Hamal C, afouane B, Vielle A, Gilbert-ussardier, Holder M, Calvas P, Philip, Edery P, Bonneau, Clausters M, Malcolm Roux A-F Molecular and in silico analysis of the full-length isoform of Usherin identify new pathogenic alleles in Usher Type II patients. Hum Mut 28: Bernal, Ayuso C, Antinolo G, Gimenez A, Borrego, Trujillo MJ, Marcos I, Calaf M, el Rio E, Baiget M Mutations in UH2A in panish patients with autosomal recessive retinitis pigmentosa: high prevalence and phenotypic variation. J Med Genet 40:e8. Bhattacharya G, Kalluri R, Orten J, Kimberling WJ, Cosgrove A domain-specific usherin/collagen IV interaction may be required for stable integration into the basement membrane superstructure. J Cell ci 7: Cremers FP, Kimberling WJ, Kulm M, de Brouwer A, van Wijk E, Te Brinke H, Cremers CW, Hoefsloot LH, Banfi, imonelli F, Fleischhauer JC, Berger W, Kelley PM, Haralambous E, Bitner-Glindzicz M, Webster AR, aihan Z, ebaere E, Leroy BP, ilvestri G et al evelopment of a genotyping. Microarray for Usher syndrome. J Med Genet 44:5-60. reyer B, Tranebjaerg L, Rosenberg T, Weston M., Kimberling WJ, ilssen Ø Identification of novel UH2A mutations: implications for the structure of UH2A protein. Eur J Hum Genet 8:

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