American Journal of Medical Genetics 110:397–399 (2002)

Research Letter

Structural and Mutational Analysis of Antiquitinas a Candidate Gene for Meniere Disease

To the Editor:

Meniere disease (MD) has been defined as an idio-pathic disorder of the inner ear characterized by thetriad of tinnitus, vertigo, and sensorineural hearingloss [Andrews and Honrubia, 1996]. This triad may beaccompanied by any of a range of secondary symptomsthat include nausea, vomiting, diarrhea, and nystag-mus [Fagan, 1998]. At the physiological level, MDpatients present with endolymphatic hydrops, anaccumulation of endolymph in the inner ear that leadsto swelling of the vestibular labyrinth.

Epidemiological surveys conducted among culturallydiverse populations report the prevalence of MD asranging between 1 in 5,000 [Shojaku and Watanabe,1997] and 1 in 500 [Wladislavosky-Waserman et al.,1984]. Approximately 90% of MD cases are sporadic[Morrison et al., 1994; Morrison, 1995]. Such cases maylack a hereditary basis or may involve a polygeniccomponent that is difficult to pinpoint, and whichmanifests as MD only in the presence of a particularenvironmental trigger. The remaining 10% of MD casesare genetic. Birgerson et al. [1987] cited family pedigreedata (N¼91) as evidence of familial MD. Clear auto-somal dominant inheritance and the phenomenon ofanticipation has been observed in some extended pe-digrees [Oliviera and Braga, 1992;Morrison, 1995]. Thebases for both the sporadic and inherited forms of MDremain undefined.

Despite knowledge of themode of inheritance and thephenomenon of anticipation, MD has yet to be mappedgenetically. We therefore initiated a candidate geneanalysis to approach the genetic basis of MD. The genewe chose to study in MD patients, antiquitin (ATQ1),was originally isolated and shown to have substantialsimilarity with the pea protein 26g [Lee et al., 1994].Overall, antiquitin and 26g are 60% identical, withsome amino acid stretches showing over 80% identity

[Lee et al., 1994]. The 26g protein in peas and relatedproteins in other plant species are utilized to counteractwater stress [Guerrero et al., 1990]. Antiquitin alsoshows similarity to aldehyde dehydrogenases. In viewof the striking similarities between the antiquitin and26g proteins, it is possible that antiquitin may alsofunction as a regulator of fluid balance. Antiquitin wasalso cloned from a fetal cochlear cDNA library andshown to be expressed in a variety of fetal tissuesincluding ovary, eye, heart, kidney, inner ear, andcochlear outer hair cells [Skvorak et al., 1997].

Nine patients from eight families were diagnosedbased on the primary criteria of hearing loss and at-tacks of vertigo. Tinnitus was prevalent in the patients,as were secondary symptoms including nausea, vomit-ing, incontinence, and drop attacks.

Figure 1 shows the pedigrees of the eight familialcases of MD used in this study. Familial cases had onefirst-degree relative who also suffered symptoms ofMD.There was no sex bias on transmission. In six of thefamilies, transmission over at least two generationswas observed. In the seventh family, the child (labeledwith a question mark) had symptoms but had not beenformally diagnosed. The eighth family only had affectedsiblings. These findings are in agreement with previousstudies suggesting an autosomal dominant mode ofinheritance. One patient was affected bilaterally andthe other eight were affected unilaterally. The age atwhich diagnosis was made varied from 15 to 62 yearsof age.

In order to screen the ATQ1 gene for mutations, wefirst characterized the intron/exon structure of thegene. Bacterial artificial chromosome (BAC) clonesencompassing the gene (256O16 and 31M3) wereisolated and used to determine the gene structure bya combination of methods. DNA sequencing was per-formed on three types of template: the BAC clonesthemselves, plasmid subclones of the BAC isolatedby hybridization with either the ATQ1 cDNA or exon-specific oligonucleotides, and long-range polymerasechain reaction (PCR) products between exons. Thegene was found to have 18 exons ranging in size from42 base pairs (bp) (exon 10) to 352 bp (exon 1). TheATQ1 coding region is 1,533 bp in length. Transcriptionappears to be initiated 244 bp upstream of the putativestart codon; thus, the 50 untranslated region is 244 bp inlength. The 30 untranslated region is 215 bp in length.Consequently, the ATQ1 cDNA is 1,992 bp in length.

Michael Lynch and Trevor L. Cameron contributed equally tothis publication.

*Correspondence to: Dr. Susan M. Forrest, Murdoch ChildrensResearch Institute, Royal Children’s Hospital, Parkville, Victoria,3052, Australia. E-mail: forrest@cryptic.rch.unimelb.edu.au

Received 17 October 2000; Accepted 10 March 2002

DOI 10.1002/ajmg.10494

� 2002 Wiley-Liss, Inc.

Primers were designed to amplify each exon of ATQ1with its flanking intronic sequence by PCR from thegenomic DNA of MD patients. One affected individualfrom each of the eight families was analyzed, as was oneaffected individual without affected relatives and twounaffected control individuals. The resulting PCR pro-ducts were screened for mutations by single-strandedconformational polymorphism analysis (SSCP). SSCPwas performed at room temperature or at 48C with andwithout 10% glycerol. Details of the intron/exon boun-daries, the sequences determined, and the primers usedare available on request.

Two single bp substitutions were detected in the exon14 PCR product. The first was a C to T transition inintron 13, 27 bp 50 of exon 14 (IVS13-27C/T). The secondchange was an A to C transversion at nucleotide 1475(A1475C), which changes the predicted amino acid fromlysine to glutamine (K411Q).

A number of control individuals were analyzed toascertain whether these changes were present in thegeneral population. Exon 14 was amplified in normal

individuals and the presence of the intronic C or Tnucleotide was assayed by digestion with FokI, whichcuts if the C nucleotide is present. Five heterozygoteswere found in 20 control individuals, indicating afrequency of 12.5% for the C allele. The A or C atnucleotide 1475 within exon 14 was detected bydigestion with Bst NI, which cuts if the C is present.Four heterozygotes were found among 20 control indi-viduals, which indicated a frequency of 10% for theC allele.

As these changes are present in unaffected indivi-duals, as well as in affected individuals, they are poly-morphisms and unlikely to be disease causing.

SOUTHERN BLOTS TO LOOK FOR GENOMICREARRANGEMENTS OF ATQ1

It is possible that the mutations underpinning fami-lialMD take the form of one ormore genomic rearrange-ments of the ATQ1 gene. Consequently, a southern blotwas performed with HindIII and PstI digested DNAfrom affected patients, and was probed with a full-length ATQ1 cDNA clone. Analysis was complicated bythe presence of several pseudogenes; however, the band-ing pattern of ATQ1 did not vary amongst the affectedindividuals (data not shown).

ANALYSIS OF ATQ1 BAC CLONES FORTRINUCLEOTIDE REPEAT SEQUENCES

Anticipation, the onset of a disease phenotype at anearlier age and with greater severity from generation togeneration, has been shown to occur in cases of familialMD [Oliviera and Braga, 1992; Morrison, 1995]. Sinceanticipation is known at the genetic level to be causedby trinucleotide repeat expansions, EcoRI digests ofDNA from theATQ1 containing BACswere probed with(ATG)10, (CAG)10, (CCT)10, (CTT)10, and (TGG)10 oligo-nucleotides (results not shown). Since BACs 256O16and 31M3 contain the entire coding region of the geneand showed no hybridization to the oligonucleotidesused, we concluded that no triplet repeats of significantlength lie within the ATQ1 gene.

That no mutations were detected in ATQ1 in theeight familial cases suggests that ATQ1 is not thecausative gene underpinning MD. The possibility re-mains that MD is genetically heterogeneous and thatmany different genes are responsible for different casesof MD. Thus, the absence of causative mutations inATQ1 in our subset of families does not necessarily ruleout ATQ1 as a gene that contributes to MD per se.Analysis of complex traits involves either linkage anal-ysis or association studies [Lander and Schork, 1994].Should it be resolved that MD is genetically complex,the novel polymorphisms we have identified in thecandidate gene under study will be essential tools forany such analysis.

REFERENCES

Andrews JC, Honrubia V. 1996. Meniere’s disease. In: Baloh RW, HalmagyiGM, editors. Disorders of the vestibular system. New York: OxfordUniversity Press.

Fig. 1. Pedigrees describing eight cases of familial MD used in thisstudy. Affected individuals are indicated by filled circles/boxes. Diagonallines indicate deceased individuals. Individuals labeled with questionmarks presented with symptoms closely resembling MD but have yet to bediagnosed clinically. Arrows indicate the individual used primarily forDNA analysis. Numbers in brackets beneath affected individuals indicatethe age at which diagnosis of MD was made.

398 Lynch et al.

Birgerson L, Gustavson KH, Stahle J. 1987. Familial Meniere’s disease:a genetic investigation. Am J Otol 8:323–326.

Fagan PA. 1998. Diagnosis and treatment of Meniere’s disease. Mod MedAust 41:61–73.

Guerrero FD, Jones JT, Mullet JE. 1990. Turgor-responsive gene transcrip-tion and RNA levels increase rapidly when pea shoots are wilted.Sequence and expression of three inducible genes. Plant Mol Biol 15:11–26.

Lander ES, Schork NJ. 1994. Genetic dissection of complex traits [publish-ed erratum appears in Science 1994;266:353]. Science 265:2037–2048.

Lee P, KuhlW, Gelbart T, Kamimura T,West C, Beutler E. 1994. Homologybetween a human protein and a protein of the green garden pea.Genomics 21:371–378.

Morrison AW. 1995. Anticipation inMeniere’s disease. J Laryngol Otol 109:499–502.

Morrison AW,Mowbray JF,Williamson R, Sheeka S, Sodha N, Koskinen N.1994. On genetic and environmental factors in Meniere’s disease. AmJ Otol 15:35–39.

Oliviera CA, Braga AM. 1992. Meniere’s syndrome inherited as anautosomal dominant trait. Ann Otol Rhinol Laryngol 101:590–594.

Shojaku H,Watanabe Y. 1997. The prevalence of definite cases of Meniere’sdisease in the Hida and Nishikubiki districts of central Japan: a surveyof relatively isolated areas of medical care. Acta Otolaryngol Suppl528:94–96.

Skvorak AB, Robertson NG, Yin Y, Weremowicz S, Her H, Bieber FR,Beisel KW, Lynch ED, Beier DR, Morton CC. 1997. An ancientconserved gene expressed in the human inner ear: identification,expression analysis, and chromosomal mapping of human and mouseantiquitin (ATQ1). Genomics 46:191–199.

Wladislavosky-Waserman P, Facer GW, Mokri B, Kurland LT. 1984.Meniere’s disease: a 30-year epidemiologic and clinical study inRochester, MN, 1951–1980. Laryngoscope 94:1098–1102.

Michael LynchTrevor L. CameronMelanie KnightTak Yue KwokPaul ThomasMurdoch Childrens Research InstituteRoyal Children’s HospitalParkville, Victoria, Australia

Susan M. Forrest*Murdoch Childrens Research InstituteRoyal Children’s HospitalParkville, Victoria, AustraliaDepartment of PaediatricsUniversity of MelbourneRoyal Children’s HospitalParkville, Victoria, Australia

Anne B. Skvorak GierschDepartment of Medical GeneticsUniversity of WashingtonSeattle, Washington

Robert J.S. BriggsMercy Private HospitalEast Melbourne Victoria, Australia

Brian C. PymanRingwood Private HospitalRingwood East Victoria, Australia

Research Letter 399