Title: A Common Ancestral Origin of the Frequent and Widespread 2299delG USH2A Mutation
Abstract: Usher syndrome type IIa is an autosomal recessive disorder characterized by mild-to-severe hearing loss and progressive visual loss due to retinitis pigmentosa. The mutation that most commonly causes Usher syndrome type IIa is a 1-bp deletion, described as “2299delG,” in the USH2A gene. The mutation has been identified in several patients from northern and southern Europe and from North America, and it has been found in single patients from South America, South Africa, and China. Various studies have reported a range of frequencies (.16–.44) among patients with Usher syndrome, depending on the geographic origin of the patients. The 2299delG mutation may be the one that most frequently causes retinitis pigmentosa in humans. Given the high frequencies and the wide geographic distribution of the mutation, it was of interest to determine whether the mutation resulted from an ancestral mutational event or represented a mutational hotspot in the USH2A gene. Haplotype analysis was performed on DNA samples from 116 unrelated patients with Usher syndrome type IIa; the patients were from 14 countries and represented 148 2299delG alleles. On the basis of six single-nucleotide polymorphisms within the USH2A gene, 12 core haplotypes were observed in a panel of normal chromosomes. However, in our analysis, only one core haplotype was found to be associated with the 2299delG mutation. The data indicate that the widespread geographic distribution of the 2299delG mutation is the result of an ancestral mutation that has spread throughout Europe and into the New World as a result of migration. Usher syndrome type IIa is an autosomal recessive disorder characterized by mild-to-severe hearing loss and progressive visual loss due to retinitis pigmentosa. The mutation that most commonly causes Usher syndrome type IIa is a 1-bp deletion, described as “2299delG,” in the USH2A gene. The mutation has been identified in several patients from northern and southern Europe and from North America, and it has been found in single patients from South America, South Africa, and China. Various studies have reported a range of frequencies (.16–.44) among patients with Usher syndrome, depending on the geographic origin of the patients. The 2299delG mutation may be the one that most frequently causes retinitis pigmentosa in humans. Given the high frequencies and the wide geographic distribution of the mutation, it was of interest to determine whether the mutation resulted from an ancestral mutational event or represented a mutational hotspot in the USH2A gene. Haplotype analysis was performed on DNA samples from 116 unrelated patients with Usher syndrome type IIa; the patients were from 14 countries and represented 148 2299delG alleles. On the basis of six single-nucleotide polymorphisms within the USH2A gene, 12 core haplotypes were observed in a panel of normal chromosomes. However, in our analysis, only one core haplotype was found to be associated with the 2299delG mutation. The data indicate that the widespread geographic distribution of the 2299delG mutation is the result of an ancestral mutation that has spread throughout Europe and into the New World as a result of migration. The Usher syndromes (MIM 276900) are a group of recessively inherited disorders characterized by progressive visual loss caused by retinitis pigmentosa (RP) and by varying degrees of hearing impairment (which may occur in the presence or absence of vestibular dysfunction) (Smith et al. Smith et al., 1994Smith RJ Berlin CI Hejtmancik JF Keats BJ Kimberling WJ Lewis RA Möller CG Pelias MZ Tranebjærg L Clinical diagnosis of the Usher syndromes: Usher Syndrome Consortium.Am J Med Genet. 1994; 50: 32-38Crossref PubMed Scopus (259) Google Scholar). The syndromes constitute the most frequent cause of acquired deaf-blindness, with a prevalence of 4.4:100,000 in the United States (Boughman et al. Boughman et al., 1983Boughman JA Vernon M Shaver KA Usher syndrome: definition and estimate of prevalence from two high-risk populations.J Chronic Dis. 1983; 36: 595-603Abstract Full Text PDF PubMed Scopus (272) Google Scholar) and 2.2:100,000 in Denmark (Rosenberg et al. Rosenberg et al., 1997Rosenberg T Haim M Hauch AM Parving A The prevalence of Usher syndrome and other retinal dystrophy-hearing impairment associations.Clin Genet. 1997; 51: 314-321Crossref PubMed Scopus (130) Google Scholar). Usher syndrome has been subcategorized into three distinct clinical types and has been linked to 10 distinct loci, USH1A–1F, USH2A–2C, and USH3 (Hereditary Hearing Loss Home page). Usher type II probably accounts for more than half of all Usher cases. Since the time of the initial identification of mutations in the USH2A gene, which are responsible for Usher syndrome type IIa and nonsyndromic retinitis pigmentosa (RP) (Eudy et al. Eudy et al., 1998Eudy JD Weston MD Yao S Hoover DM Rehm HL Ma-Edmonds M Yan D Ahmad I Cheng JJ Ayuso C Cremers C Davenport S Möller C Talmadge CB Beisel KW Tamayo M Morton CC Swaroop A Kimberling WJ Sumegi J Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.Science. 1998; 280: 1753-1757Crossref PubMed Scopus (288) Google Scholar; Rivolta et al. Rivolta et al., 2000Rivolta C Sweklo EA Berson EL Dryja TP Missense mutation in the USH2A gene: association with recessive retinitis pigmentosa without hearing loss.Am J Hum Genet. 2000; 66: 1975-1978Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), a total of 31 distinct disease-causing mutations have been reported (Adato et al. Adato et al., 2000Adato A Weston MD Berry A Kimberling WJ Bonne-Tamir A Three novel mutations and twelve polymorphisms identified in the USH2A gene in Israeli USH2 families.Hum Mutat. 2000; 15: 388Crossref PubMed Scopus (31) Google Scholar; Dreyer et al. Dreyer et al., 2000Dreyer B Tranebjærg L Rosenberg T Weston MD Kimberling WJ Nilssen Ø Identification of novel USH2A mutations: implications for the structure of USH2A protein.Eur J Hum Genet. 2000; 8: 500-506Crossref PubMed Scopus (65) Google Scholar; Weston et al. Weston et al., 2000Weston MD Eudy JD Fujita S Yao S Usami S Cremers C Greenburg J Ramesar R Martini A Möller C Smith RJ Sumegi J Kimberling WJ Genomic structure and identification of novel mutations in Usherin, the gene responsible for Usher syndrome type IIa.Am J Hum Genet. 2000; 66: 1199-1210Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Most of the mutations are private. However, the 2299delG mutation, in exon 13, stands out because of its high frequency (.16–.44) across various studies (Beneyto et al. Beneyto et al., 2000Beneyto MM Cuevas JM Millan JM Espinos C Mateu E Gonzalez-Cabo P Baiget M Domenech M Bernal S Ayuso C Garcia-Sandoval B Trujillo MJ Borrego S Antinolo G Carballo M Najera C Prevalence of 2314delG mutation in Spanish patients with Usher syndrome type II (USH2).Ophthalmic Genet. 2000; 21: 123-128Crossref PubMed Google Scholar; Dreyer et al. Dreyer et al., 2000Dreyer B Tranebjærg L Rosenberg T Weston MD Kimberling WJ Nilssen Ø Identification of novel USH2A mutations: implications for the structure of USH2A protein.Eur J Hum Genet. 2000; 8: 500-506Crossref PubMed Scopus (65) Google Scholar; Liu et al. Liu et al., 1999Liu X-Z Hope C Liang CY Zou JM Xu LR Cole T Mueller RF Bundey S Nance W Steel KP Brown SDM A mutation (2314delG) in the Usher syndrome type IIA gene: high prevalence and phenotypic variation.Am J Hum Genet. 1999; 64: 1221-1225Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar; Weston et al. Weston et al., 2000Weston MD Eudy JD Fujita S Yao S Usami S Cremers C Greenburg J Ramesar R Martini A Möller C Smith RJ Sumegi J Kimberling WJ Genomic structure and identification of novel mutations in Usherin, the gene responsible for Usher syndrome type IIa.Am J Hum Genet. 2000; 66: 1199-1210Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The mutation has been identified in patients from northern and southern Europe and North America and in single patients from South America, South Africa, and China. Given the high frequencies of the 2299delG mutation among patients with Usher type IIa and the mutation's wide geographic distribution, it was of interest to investigate whether the 2299delG alleles have a common origin or represent a hotspot in the USH2A gene. We therefore analyzed DNA samples obtained from 116 unrelated patients with Usher type II, of whom 32 were homoallelic and 84 were heteroallelic for the 2299delG mutation. The patients originated in 14 countries: the United States (43 patients), Denmark (22 patients), The Netherlands (17 patients), United Kingdom (10 patients), Spain (6 patients), Sweden (6 patients), Norway (4 patients), Belgium (2 patients), Canada (1 patient), China (1 patient), Colombia (1 patient), France (1 patient), Germany (1 patient), and South Africa (1 patient); all the United States patients were of European ancestry, except for one African American. To establish the haplotype for the 2299delG alleles, six single-nucleotide polymorphisms (SNPs) within the USH2A gene, exons 2–21, were selected (Dreyer et al. Dreyer et al., 2000Dreyer B Tranebjærg L Rosenberg T Weston MD Kimberling WJ Nilssen Ø Identification of novel USH2A mutations: implications for the structure of USH2A protein.Eur J Hum Genet. 2000; 8: 500-506Crossref PubMed Scopus (65) Google Scholar). Two SNPs (IVS15+35A/G and IVS17−8T/G) were in noncoding sequence. Four SNPs were in coding sequence, of which two were synonymous (504G/A and 1419C/T) and two were nonsynonymous (373A/G and 4457A/G). In addition, three flanking markers, AFM144XF2, AFM268ZD1, and AFM143XF10, which span ∼460 kb of the USH2A locus, were included (fig. 1). The allele frequencies of these SNPs were established in normal chromosomes from Norwegian nuclear families (n=108), Spanish control chromosomes (n=94), and in normal chromosomes from parents of patients with Usher syndrome (n=49). There were no significant differences in allele frequencies among the three control panels (data not shown). SNP analysis was performed by PCR amplification in a standard reaction mixture, as described by Dreyer et al. (Dreyer et al., 2000Dreyer B Tranebjærg L Rosenberg T Weston MD Kimberling WJ Nilssen Ø Identification of novel USH2A mutations: implications for the structure of USH2A protein.Eur J Hum Genet. 2000; 8: 500-506Crossref PubMed Scopus (65) Google Scholar), except IVS15+35A/G, which was detected by allele-specific PCR using primers (reverse: 5′-GCAGTCCCCTGTATGATGATGC-3′; reverse: 5′-ATGCAGTCCCCTGTATGATGGTGT-3′; and forward: 5′-AAGCCGTCTTACTCTACAATGCT-3′). The products were 321 bp and 323 bp for IVS15+35G and IVS15+35A, respectively. Fluorescence-labeled reverse primers were used, and products were analyzed on a 377-ABI sequencer (Applied Biosystems). The underlined mismatches were introduced to increase the specificity of the PCR. Genotyping for the three markers (AFM144XF2, AFM268ZD1, and AFM143XF10) was performed by use of information from the Genome Database. The determination of haplotype phase was based either on homozygosity or on the analysis of parental samples, which could be used fully or partially to determine the phase. A complete description of the USH2A haplotypes for the 148 USH2A alleles is presented in table 1.Table 1Haplotypes Associated with the 2299delG Alleles in 116 Unrelated Patients of Geographically Diverse OriginsGenotype atCountry and Patient NumberAFM144 XF2373A/G504G/A1419C/TIVS15+35A/GIVS17−8T/G4457A/GAFM268ZD1AFM143XF10United States: 791aPatient was African American.116AGCATA250111 229116AGCATA250111126AGCATA250111 1143126AGCATA250111126AGCATA250111 2140116AGCATA250111116AGCATA250111 2720126AGCATA250111126AGCATA250111 52116AGCATA250113 296118/126AGCATA250111 427116AGCATA250111 560116/126AGCATA250111/115 685126AGCATA250111 747116AGCATA250111 777116/126AGCATA250111/115 795116AG…ATA250111 869116AGCATA250119 876116/126AGCATA250109/111 225126AGCATA250111/113 927116AGCATA250119 1052116/126AGCATA250107/111 1121116/126AGCATA250/254111 1147126AGCATA250111/113 1280116/126AGCATA250/254111 1299116/126AGCATA250111 1340116/126AGCAT…250111/113 1479116AGCATA250111 2456116/126AGCATA250111 2463126AGCATA250… 2499126AGCATA250115 2502118AGCATA250111/113 2505126AGCATA250115 2517116/118AGCATA250… 2572126AGCATA250107 2579126AGCATA250111 2580126AGCATA250111 2584126AGCATA250113/115 2725126AGCATA250/252113 2728126AGCATA250111 1155116/126AG/ACATA250111/115 2455126AG/ACATA250111/113 1870116/126AGCA/GTA250/252107/111 2041118AG…A/GTA/G250… 2561116/126AG/AC/TA/GT/GA/G250111/113 2573126AGC/TA/GT/GA/G250111 2625116A/GG/ACA/GTA/G250/256111Denmark: D2116AGCATA250113116AGCATA250113 D4116AGCATA250111126AGCATA250111 D13116AGCATA250113116AGCATA250113 D18126AGCATA250111126AGCATA250113 D19126AGCATA250111126AGCATA250111 D40116AGCATA250111/113126AGCATA250 D44116AGCATA250111116AGCATA250113 D5126AGCATA250113 D7126AGCATA250111 D8116/126AGCATA250107/113 D10126AGCATA250111/113 D15116AGCATA250113 D16116/126AGCATA250111 D22116/126AGCATA250111 D24116AGCATA250111/113 D39116/118AGCATA250111/115 D32116/126AGCA/GTA/G…111/115 D11116/126A/GG/AC/TATA250111/115 D17118/126AG/AC/TA/GTA250107/111 D25116AGC/TA/GTA/G250109/113 D31116AGCA/GT/GA/G250113/115 D23116/126AGC/TA/GT/GA/G250111The Netherlands: 1003126AGCATA250115126AGCATA250115 1011126AGCATA250111126AGCATA250111 1370126AGCATA250111126AGCATA250113 1374126AGCATA250115126AGCATA250115 1404126AGCATA250113126AGCATA250115 1298126AGCATA250115 1375126AGCATA250111 1386128AGCATA250111 1387126AGCATA250113 1415126AGCATA250115 1044116AGCATA250111 1238126AGCATA250113 1420118/126AGCATA250111 776126AGCATA250107/113 1008126AGCATA250111/115 1368126AGCATA250111/113 956126A/GG/ACATA/G250111United Kingdom:bSamples of genomic DNA of the patients in the United Kingdom were provided by Dr. Xue Zhong Liu. UK1126AGCATA250111126AGCATA250111 UK2116AGCATA250111126AGCATA250111 UK5116AGCATA250111126AGCATA250111 UK7116/126AGCATA250111/113 UK3116/126AGCA/G…A/G…107/111 UK4116/126AGCA…A…113 UK6126A/GG/ACATA…111 UK8116/126A/GG/AC/TA/GT/GA/G…107/111 UK10116/126AG/ACATA…111/115 UK11cPatient was Chinese.116/126A/GG/A…A/G………… UK12116/126AG/ACATA……Spain: SP1126AGCATA250113126AGCATA250113 SP2116AGCATA250111116AGCATA250111 SP3116AGCATA250111116AGCATA250115 SP5126AGCATA250111/115 1076126AGCATA250111 SP4116/118A/GG/AC/TA/GT/GA/G246/252113Sweden: S1116AGCATA250113126AGCATA250113 S2116AGCATA250111126AGCATA250111 S3116AGCATA250113126AGCATA250113 S4116AG/ACATA250111/115 S5116A/GG/AC/TATA250111/113 S6116/126A/GG/AC/TATA250113Norway: N4116AGCATA250111126AGCATA250111 N5126AGCATA250111126AGCATA250111 N25116/126AGCATA250111/113 N15116/126AGCA/GT/GA/G250109/111Belgium: 1862116AGCATA250111116AGCATA250111 1863126AGCA/GT/GA/G250115Canada: 1158128AG/ACATA250111Colombia: 1150116AGCATA250111126AGCATA250111France: 1223116AGCATA250111116AGCATA250113Germany: 1294116/118A/GG/AC/TA/GT/GA/G250…South Africa: 1128126AGCATA250111126AGCATA250111Note.—Genotyping was incomplete in some patients because of a lack of DNA material.a Patient was African American.b Samples of genomic DNA of the patients in the United Kingdom were provided by Dr. Xue Zhong Liu.c Patient was Chinese. Open table in a new tab Note.— Genotyping was incomplete in some patients because of a lack of DNA material. In all the 2299delG alleles for which the phase could be determined (116 of 148), the 2299delG mutation was associated with one core haplotype, A-G-C-A-T-A. In the remaining 32 alleles, other haplotypes for the 2299delG allele would be formally possible; however, the A-G-C-A-T-A core haplotype could not be excluded. Except in one Spanish patient (SP4), the core haplotype could be extended on the 3′ side, to span 325 kb (A-G-C-A-T-A-250), including the marker AFM268ZD1. Twelve different USH2A SNP haplotypes were observed in a panel of normal chromosomes from Norwegian nuclear families (n=108); four major haplotypes (1–4) had frequencies of .60, .13, .06, and .03, and eight derivative haplotypes showed frequencies <.03. These haplotype frequencies were not significantly different from those observed in chromosomes from parents of patients with Usher syndrome (n=49) (table 2).Table 2Frequencies of 2299delG and Normal HaplotypesGenotype ataGenotypes are given according to SNPs (frequencies).Frequencies ofHaplotype373A/G (.76/.24)504G/A (.72/.28)1419C/T (.78/.22)IVS15+35A/G (.66/.34)IVS17−8T/G (.76/.24)4457A/G (.64/.36)Normal Control Alleles (n = 108)Normal Parental Alleles (n = 49)2299delG Alleles (n= 116)1AGCATA.60.531.00bStatistical significance of the association was measured by the χ2 test (χ2 = 57.2, P < .001).2GATGGG.13cThe frequency of haplotype 2 is higher than expected on the basis of the individual allele frequencies and illustrates a situation of strong linkage disequilibrium, (χ2 = 42.0, P < .001)..22c3AGCGGG.06.024GACATA.05.045–12dEight haplotypes with frequencies <.03.……………….16.19a Genotypes are given according to SNPs (frequencies).b Statistical significance of the association was measured by the χ2 test (χ2 = 57.2, P < .001).c The frequency of haplotype 2 is higher than expected on the basis of the individual allele frequencies and illustrates a situation of strong linkage disequilibrium, (χ2 = 42.0, P < .001).d Eight haplotypes with frequencies <.03. Open table in a new tab Because of the wide geographic distribution of the mutation and because of the haplotype heterogeneity at the microsatellite markers (AFM144XF2, AFM268ZD1, and AFM143XF10) that flank the USH2A locus, it had been previously suggested that the 2299delG mutation did not arise in a common ancestor (Eudy et al. Eudy et al., 1998Eudy JD Weston MD Yao S Hoover DM Rehm HL Ma-Edmonds M Yan D Ahmad I Cheng JJ Ayuso C Cremers C Davenport S Möller C Talmadge CB Beisel KW Tamayo M Morton CC Swaroop A Kimberling WJ Sumegi J Mutation of a gene encoding a protein with extracellular matrix motifs in Usher syndrome type IIa.Science. 1998; 280: 1753-1757Crossref PubMed Scopus (288) Google Scholar; Liu et al. Liu et al., 1999Liu X-Z Hope C Liang CY Zou JM Xu LR Cole T Mueller RF Bundey S Nance W Steel KP Brown SDM A mutation (2314delG) in the Usher syndrome type IIA gene: high prevalence and phenotypic variation.Am J Hum Genet. 1999; 64: 1221-1225Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In our analysis, however, all 116 2299delG chromosomes for which the phase was known were found to be in complete association with one specific core haplotype, which was designated “USH2A haplotype 1.” Statistical significance of the association was demonstrated using the χ2 test (χ2=57.2; P<.001). The data strongly indicate that the 2299delG alleles originated from an ancestral mutational event. The variation at the flanking markers, especially the AFM144XF2 and AFM143XF10, among the 2299delG chromosomes probably reflects the generally high mutation rate (10−3 to 10−4 per locus per generation) at microsatellite sites (Weber and Wong Weber and Wong, 1993Weber JL Wong C Mutation of human short tandem repeats.Hum Mol Genet. 1993; 2: 1123-1128Crossref PubMed Scopus (1298) Google Scholar). The fact that the 2299delG-associated haplotype is also the most frequent in the unaffected population suggests that the genetic distance between these two groups is low. All but two of the patients in this study are European or of European descent. It is tempting to speculate that the 2299delG mutation results from an old mutational event that happened to arise on the most common haplotype in the present European genetic background and that was spread, through migration and subsequent founder effects, throughout Europe. Furthermore, the 2299delG mutation was probably brought to the New World in recent times, because it occurs in countries with a history of European immigration. This definitely applies to the United States and to the singleton patients in South Africa and Canada. However, two notable exceptions are the American patient of African ancestry (patient 791) and the patient of Chinese ancestry (UK11, for whom the phase could not be determined). Both patients are heterozygous for the 2299delG mutation. A DNA sample from a Chinese patient who has been reported to be homozygous for the 2299delG mutation (Liu et al. Liu et al., 1999Liu X-Z Hope C Liang CY Zou JM Xu LR Cole T Mueller RF Bundey S Nance W Steel KP Brown SDM A mutation (2314delG) in the Usher syndrome type IIA gene: high prevalence and phenotypic variation.Am J Hum Genet. 1999; 64: 1221-1225Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) was not available for this study. Because no one has reported studies of 2299delG mutation frequencies in non-European populations, it remains to be elucidated whether 2299delG is present in these two patients as a result of recent ethnic admixture or is present in the gene pool of other populations such as those of Africa and China. In summary, our data provide the first molecular evidence that the frequent and widespread 2299delG mutation results from an ancient mutation of common origin rather than from multiple recurrent mutational events on a common haplotype. Although all the 2299delG alleles studied contain a conserved SNP core haplotype that spans >250 kb, the haplotype varies widely, and the association between the 2299delG mutation and the haplotypes of the flanking microsatellite markers appears to be random. Regarding the physical distances between these markers, this variability cannot be explained by recombination alone (fig. 1). Thus, the above data clearly demonstrate that, because of their relatively high mutation frequency, microsatellites must be applied in combination with SNP markers when studying the origin and distribution of mutation. This work has been supported by Norwegian Foundation for Health and Rehabilitation grant 1998/257 (B.D., Ø.N., and L.T.) and a grant from the Danish Support Foundation for the Blind (T.R.). The study was approved by the Regional Research Ethics Committee in Tromsø. We would like to thank Dr. Xue Zhong Liu, for providing DNA samples from UK/China, and Dr. Carmen Ayuso, for providing Spanish DNA control samples. Erratum et al.The American Journal of Human GeneticsOctober, 2001In BriefIn the July 2001 issue of the Journal, in the report “A Common Ancestral Origin of the Frequent and Widespread 2299delG USH2A Mutation,” by Dreyer et al. (69:228–234), the list of authors' names should have included Cor W. R. J. Cremers and Xue-Zhong Liu. The corrected list of the article's authors is as follows: Bo Dreyer, Lisbeth Tranebjærg, Vigdis Brox, Thomas Rosenberg, Claes Möller, Magdalena Beneyto, Michael D. Weston, William J. Kimberling, Cor W. R. J. Cremers, Xue-Zhong Liu, and Øivind Nilssen. Full-Text PDF Open Archive