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The USH1C 216G a Mutation and the 9-Repeat VNTR(T,T) Allele Are in Complete Linkage Disequilibrium in the Acadian Population

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The USH1C 216G a Mutation and the 9-Repeat VNTR(T,T) Allele Are in Complete Linkage Disequilibrium in the Acadian Population Human Genetics DOI 10.1007/s00439-001-0653-7 Short Report The USH1C 216G A mutation and the 9-repeat VNTR(t,t) allele are in complete linkage disequilibrium in the Acadian population Sevtap Savas · Ben Frischhertz · Mary Z. Pelias · Mark A. Batzer · Prescott L. Deininger · Bronya J. B. Keats( ) S. Savas · M.Z. Pelias · B.J.B. Keats Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA B. Frischhertz · P.L. Deininger Tulane Cancer Center, Department of Environmental Health Sciences, New Orleans, La., USA M.A. Batzer Department of Biological Sciences, Biological Computation and Visualization Center, Louisiana State University, Baton Rouge, La., USA E-mail: [email protected] Phone: +1-504-5686150 Fax: +1-504-5688500 Received: 25 May 2001 / Accepted: 31 October 2001 / Published online: Electronic database information: URLs for the data in this article are as follows: Primer3 software, http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi Abstract. Recently, mutations in USH1C were shown to be associated with Usher syndrome type IC, and a mutation (216G A) in exon 3 was identified in an Acadian family. In addition, a 45-bp variable number of tandem repeat (VNTR) polymorphism was found in intron 5 of USH1C. Polymerase chain reaction amplification of the VNTR region and restriction enzyme analysis of exon 3 of USH1C showed that, of 44 Acadian patients, 43 were homozygous for both the 216G A mutation and nine repeats of the VNTR, with a "t" nucleotide replacing a "g" nucleotide at the 8th position of both the eighth and ninth copies of the repeat, viz., 9VNTR(t,t). The remaining Acadian patient was reported to be a compound heterozygote for 216G A/9VNTR(t,t) and 238-239insC, a USH1C mutation that has been found in other populations. These data demonstrate that the 9VNTR(t,t) allele is in complete linkage disequilibrium with the 216G A mutation in the Acadian - 1 - population. Among 82 Acadian controls, one was heterozygous for 216G A/9VNTR(t,t). The 238-239insC mutation was not found in Acadian controls. Analysis of 340 non-Acadian normal samples showed the presence of a 9-repeat VNTR allele in one Hispanic sample. This individual had neither the 216G A mutation nor the Acadian VNTR(t,t) structure. These results suggest that the 216G A mutation and the 9VNTR(t,t) allele are restricted to the Acadians and are in complete linkage disequilibrium. Introduction Usher syndrome type IC (USH1C), an autosomal recessive disorder characterized by profound hearing impairment, early onset retinitis pigmentosa, and vestibular dysfunction, was recently shown to be caused by mutations in USH1C, a gene encoding a PDZ domain-containing protein named harmonin (Bitner-Glindzicz et al. 2000; Verpy et al. 2000). In Acadian USH1C patients, two molecular events specific to the Acadian population were found: (1) the 216G A mutation in exon 3 (Bitner-Glindzicz et al. 2000) and (2) a 9-repeat VNTR allele with an unusual structure in intron 5 (Verpy et al. 2000). The VNTR was shown to have a "t" nucleotide in the 8th position of the last copy of the repeat and a "g" nucleotide at this position in the preceding copies (VNTR(t)). In contrast, the 9-repeat VNTR allele associated with USH1C in Acadians had a "t" at the 8th position in both the eighth and ninth copies of the repeat, viz., 9VNTR(t,t). A single Acadian patient was reported to be a compound heterozygote for the Acadian 216G A mutation and a second mutation (238-239insC) that has been found in other populations (Bitner-Glindzicz et al. 2000; Verpy et al. 2000; Zwaeneopel et al. 2001). In this study, we have analyzed the frequency and association of the USH1C 216G A mutation and 9VNTR(t,t) allele in Acadians and in non-Acadian populations. Methods and materials The VNTR locus was genotyped by using the following primers: VNTR-F: 5’-ACCTTTTCACGGGGATCA-3’, and VNTR-R: 5’-GATCGTCCGGATCAATGG-3’. Polymerase chain reaction (PCR) amplification and restriction digestion analysis of the 216G A mutation was performed as explained in Verpy et al. (2000) and Bitner-Glindzicz et al. (2000), respectively. To distinguish between the VNTR(t,t) and VNTR(t) alleles, we designed an allele-specific primer (Wu et al. 1989) and paired it with the VNTR-F primer in a PCR. The sequence of the allele-specific primer was: VNTR-ARMS: 5’-GGAGGGCGGAGGAGCAGGT-3’. Samples were screened for the 238-239insC mutation by PCR amplification (Verpy et al. 2000) followed by sequencing with the BigDye Terminator Cycle Sequencing Reaction Kit (PE Applied Biosystems, Foster City, Calif.) and analyzed on an ABI PRIZM 377 DNA Sequencer (PE Applied Biosystems). The primers were designed by using Primer3 software (Rozen et al. 1998). Results and discussion Of the 44 Acadian USH1C patients analyzed, 43 were homozygous for both the 216G A mutation and the 9VNTR(t,t) allele. The remaining Acadian patient was a compound heterozygote for the 238-239insC and 216G A mutations, as previously described by Verpy et al. (2000). This patient was also heterozygous for the 9VNTR(t,t) allele. The results for these 44 patients demonstrate complete linkage disequilibrium between the 216G A mutation and the 9VNTR(t,t) allele in the Acadian population. - 2 - Analysis of both the VNTR locus and the 216G A mutation in 126 unrelated controls (82 Acadian, 44 European) showed that one Acadian individual was heterozygous for 216G A/9VNTR(t,t). None of these samples had the 216G A mutation but not the 9VNTR(t,t) allele, or conversely, the 9VNTR(t,t) allele without the 216G A mutation. A subset (n=69) of the Acadian controls was also screened for the 238-239insC mutation, and none had this mutation. These results, together with our finding of only one copy of the 238-239insC mutation among Acadian patients, suggest that this mutation is rare in Acadians and was probably introduced into this population very recently. Among the 340 unrelated controls who came from various populations and who were genotyped for the VNTR locus, viz., 65 African, 39 African-American, 71 Asian, 117 European (the 44 stated above plus an additional 73), and 48 Hispanic, one Hispanic sample was heterozygous for a 9-repeat VNTR allele. This individual did not have the 216G A mutation, and allele-specific amplification demonstrated that the structure of the 9-repeat VNTR was not 9VNTR(t,t) as shown in Fig. 1. Thus, the 9-repeat VNTR allele is rare in populations around the world, and the 9VNTR(t,t) allele is solely found in Acadians. - 3 - Fig. 1. A Schematic representation of the amplification recovery mutation system (ARMS) applied in this study. For simplicity, only 9VNTR(t,t) and 3-repeat VNTR(t) structures are depicted in this scheme, where each box represents the 45-bp repeating unit of the VNTR in USH1C. Above the boxes, the 8th nucleotide (either a "g" or a "t") in the repeating unit is shown. Long and small arrows Amplification primers VNTR-F and VNTR-ARMS, respectively. This PCR reaction is expected to yield a 117-bp product from a template containing a VNTR(t) structure, and 162-bp and 117-bp products from the 9VNTR(t,t) Acadian allele. B Gel analysis of ARMS PCR product. Lane 1 Negative PCR control, lane 2 Hispanic sample, lane 3 Acadian USH1C carrier, lane 4 Acadian USH1C patient, lane 5 control sample, lane 6 molecular weight standard, arrow position of the 100-bp fragment in the molecular weight standard (1 kb Plus DNA ladder, Life Technologies, Md., USA). Note that since the 117-bp product is also amplified from the 9VNTR(t,t) Acadian USH1C allele, the 117-bp product predominates in quantity over the 162-bp product in carriers of this allele The haplotype at markers surrounding USH1C and the 216G A/9VNTR(t,t) allele shows significant linkage disequilibrium over more than 6 cM (Keats et al. 1994; Nouri et al. 1994), suggesting that both the 216G A mutation and the 9VNTR(t,t) allele arose fairly recently in the Acadian population. Taking this information together with the absence of any chromosomes with the 216G A mutation but not the 9VNTR(t,t) allele, or vice versa, we conclude that the two events occurred at about the same time. However, it remains to be clarified whether the Acadian-specific 216G A mutation and the 9VNTR(t,t) allele arose simultaneously, and also whether the 9VNTR(t,t) allele contributes to the USH1C phenotype in Acadian patients. Acknowledgements. We are grateful to the Acadian families for their participation in this study. We thank San San Ng for helping with sequencing analysis. This work was supported by grants from the Foundation Fighting Blindness and the Louisiana Board of Regents Health Excellence Fund. - 4 - References Bitner-Glindzicz M, Lindley KJ, Rutland P, Blaydon D, Smith VV, Milla PJ, Hussain K, Furth-Lavi J, Cosgrove KE, Shepherd RM, Barnes PD, O’Brien RE, Farndon PA, Sowden J, Liu X-Z, Scanlan MJ, Malcolm S, Dunne MJ, Aynsley-Green A, Glaser B (2000) A recessive contiguous gene deletion syndrome causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene. Nat Genet 26:56-60 Keats BJB, Nouri N, Pelias MZ, Deininger PL, Litt M (1994) Tightly linked flanking microsatellite markers for the Usher syndrome type I locus on the short arm of chromosome 11. Am J Hum Genet 54:681-686 Nouri N, Risch NJ, Pelias MZ, Litt M, Keats BJB (1994) Predicting the age of the mutation for Usher syndrome type I in the Acadian population.
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