Branchio-oto-renal syndrome

� 2007 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 143A:1671–1678 (2007)

Research Review

Branchio-Oto-Renal Syndrome

Amit Kochhar,1 Stephanie M. Fischer,1 William J. Kimberling,2 and Richard J.H. Smith1*1Molecular Otolaryngology Research Laboratories, University of Iowa, Iowa City, Iowa

2Boys Town National Research Hospital, Omaha, Nebraska

Received 31 July 2006; Accepted 1 October 2006

Branchio-oto-renal syndrome, a phenotype consisting ofhearing loss, auricular malformations, branchial arch rem-nants, and renal anomalies is now recognized as one of themore common forms of autosomal dominant syndromichearing impairment. Three loci known to be associated withthe BOR phenotype have been identified and two genes thatact in a regulatory network have been cloned, EYA1 andSIX1. EYA1 and SIX1 are homologous to genes involved inDrosophila eye development, eyes absent gene (eya), andsine oculis (so), respectively. EYA1, a transcriptional co-activator has a conserved, 271-amino acid, C-terminal knownas the Eya Domain (ED). SIX1 has two highly conserveddomains; a homeodomain (HD) and a specific Six-domain(SD) whose products function as transcription factors withspecific DNA-binding activity that are crucial for protein–

protein interaction. To determine the molecular basis for theorgan defects that occur in BOR syndrome, many studieshave focused on the effects of mutations to EYA and effectsof mutations of the EYA-SIX regulatory system. However,over 60% of BOR syndrome patients do not have knownmutations in EYA1 and relatively little is known aboutmutations to SIX1. Further evaluation of SIX1 and its relatedtarget genes may provide a better understanding of thepathophysiology of BOR syndrome and offer greater clues tothe disease mechanisms. � 2007 Wiley-Liss, Inc.

Key words: branchio-otic syndrome 1; BOS1; BOS2; BOS3branchio-oto-renal syndrome; BOR syndrome; EYA1; hear-ing loss; syndromic hearing loss; SIX1

How to cite this article: Kochhar A, Fischer SM, Kimberling WJ, Smith RJH. 2007.Branchio-oto-renal syndrome. Am J Med Genet Part A 143A:1671–1678.

INTRODUCTION

The development of the vertebrate ear and periotictissues is directed by a series of tissue movementsand inductive interactions that promote spatialprogramming and cell differentiations [Noden andVan De Water, 1992]. The identification of genesinvolved in these processes is improving our under-standing of the complexity of ear development andthe molecular pathogenesis of certain forms ofgenetic hearing defects. Genetic hearing loss isdivided into two categories, non-syndromic (hearingimpairment occurring in isolation) and syndromic(hearing impairment in association with otherphysical anomalies). Over 400 forms of syndromicdeafness have been described in which hearing lossis an intrinsic part. Many of these syndromes can beclassified as early developmental defects, demon-strated by the abnormal formation of one or morecompartments (outer, middle, and inner) of the ear[Gorlin et al., 1995; Kalatzis et al., 1998].

In 1864, Heusinger first recognized an associationbetween hearing impairment, preauricular pits, andbranchial fistulae. However, it was not until a century

later that Melnick and Fraser first comprehensivelydescribed the specific phenotypes. Subsequently,branchio-oto-renal syndrome, BOR (OMIM 113650),was defined to include hearing loss, auricularmalformations, branchial arch remnants, and renalanomalies [Melnick et al., 1975; Fraser et al., 1978].

Many other clinical features—such as abnormal-ities of the face, palate, ureters, and bladder,dysfunction of the lacrimal system, otitis media,and shoulder abnormalities—have also been asso-ciated with BOR syndrome [Fitch and Sorolovitz,1976; Cremers and Fikkers-Van Noord, 1980; Preischet al., 1985; Heimler and Lieber, 1986; Pennie andMarres, 1992]. Observation of these other featureshas brought varied nomenclature to the disease.

Grant sponsor: NIH; Grant number: DC03544.*Correspondence to: Richard J.H. Smith, Department of Otolaryngol-

ogy-Head & Neck Surgery, Molecular Otolaryngology Research Labora-tories, 5270 CBRB, The University of Iowa, Iowa City, IA 52242.E-mail: [email protected]

DOI 10.1002/ajmg.a.31561

Examples include ear pits-deafness syndrome; pre-auricular pits, cervical fistulae, hearing loss syn-drome; branchio-oto-dysplasia syndrome; branchio-oto-ureteral syndrome; and branchio-oto-renal dys-plasia [Cremers and Fikkers-Van Noord, 1980; Fraseret al., 1983]. The name branchio-oto syndrome,BOS1, (OMIM 602588) has been used to describea similar combination to BOR syndrome withbranchial anomalies, preauricular pits and hearingloss, without associated renal findings [Marres andCremers, 1991; Kumar et al., 1998b; Stratakis et al.,1998].

PREVALENCE

BOR syndrome is now recognized as one of themore common forms of autosomal dominant syn-dromic hearing impairment. It has an estimatedincidence of 1:40,000 and has been reported in 2% ofprofoundly deaf children [Fraser et al., 1980].Penetrance is high, but incomplete and variableexpressivity has been documented between andwithin families [Heimler and Lieber, 1986; Koniget al., 1994; Stratakis et al., 1998]. Early literaturementioned that the possibility of anticipation incertain families and also that a parent of origin effectwas implicated in the transmission of BOR syn-drome, however these were discounted [Chen et al.,1995]. Hearing impairment is found in over 70% to ashigh as 93% of affected persons, however, age ofonset may vary from early childhood to youngadulthood. The severity of hearing loss rangesfrom mild to profound and may be conductive,sensorineural, or mixed [Melnick et al., 1976; Fraseret al., 1978, 1980; Cremers and Fikkers-Van Noord,1980; Gimsing and Dyrmose, 1986; Chen et al., 1995].

THE BOR PHENOYPE

Melnick et al. [1975] described a father and three ofsix children with mixed hearing loss, cup-shapedpinnae, preauricular pits, branchial cleft fistulae, andbilateral renal anomalies of the collecting system.Successive reports focused heavily on the BORsyndrome phenotype.

Common characteristics included cup-shaped pin-nae, preauricular pits, branchial fistulae, and mildrenal anomalies; however, preauricular tags, lacrimalduct stenosis, renal aplasia or agenesis, a constrictedpalate, a deep overbite and a long, narrow face werealso seen (Fig. 1). Even more variation was observedby Fraser et al. [1980] after studying 133 affectedpatients in whom it was reported that the etiology ofhearing impairment ranged from conductive tosensorineural to mixed. This coupled with theobservation that renal anomalies could vary betweenmild and complete agenesis [Heimler and Lieber,1986; Ni et al., 1994], suggested to investigators thatBOR syndrome might be a heterogeneous group ofdiseases.

The diagnosis of BOR syndrome was traditionallybased on the presence of several different clinicalfindings, the relative importance of which was notknown. Chen et al. [1995] studied 45 patients with theclinical diagnosis of BOR syndrome and describedthe common phenotypic features as: hearing loss(93%), preauricular pits or tags (82%), renal anoma-lies (67%), branchial fistulae (49%), pinnae deformity(36%), and external auditory canal stenosis (29%).Chen et al. [1995] recommended that phenotypicanomalies occurring in >20% of affected patients beclassified as ‘‘major anomalies,’’ and those occurringin �20% of the affected patients be classified as‘‘minor anomalies’’ (Table I).

Chang et al. [2004] built on this work and per-formed the first study to analyze the BOR syndromephenotype based on genotypic data. Studying 106families with possible BOR syndrome, Chang et al.[2004] screened for EYA1 mutations and identifi-ed disease-causing mutations that segregated withthe BOR phenotype in 16 families. Based on the

TABLE I. Phenotypic Features of BOR Syndrome

Major anomalies,occurring >20% Minor anomalies, occurring <20%

Hearing loss Preauricular tagPreauricular pits Lacrimal duct aplasiaRenal anomalies Short palateBranchial fistulae RetrognathiaPinnae deformities Benign intracranial tumorExternal auditory Cleft palate

canal stenosis Congenital hip dysplasiaEuthyroid goiterFacial nerve paresisGustatory lacrimationNon-rotation of the gastrointestinal tractPancreatic duplication cystTemporoparietal linear nevus

Reprinted from J Comm Disor, 31, RJH Smith and C Schwartz, Branchio-Oto-Renal Syndrome, p. 414, copyright 1998, with permission from Elsevier.

FIG. 1. Typical facial appearance in BOR syndrome. Note the cup-shapedear deformity. Bilaterally symmetrical ear deformities may also be seen.(Reprinted from J Comm Disor, 31, RJH Smith and C Schwartz, Branchio-Oto-Renal Syndrome, p. 414, copyright 1998, with permission from Elsevier).

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American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

phenotypic data from these families and a literaturereview, 40 families were identified segregating 33different EYA1 mutations. The most common phe-notypes included deafness (98.5%), preauricular pits(83.6%), branchial anomalies (68.5%), renal anoma-lies (38.2%), and external ear abnormalities (31.5%).This was the largest EYA1 screening performed todate and the families provided sufficient clinical datato adequately refine the BOR phenotype [Changet al., 2004].

Based on the genotypic data, Chang and collea-gues concluded that genetic testing for EYA1mutations should be performed in patients who fitthe following clinical criteria for BOR syndrome(Table II). This criteria include only persons whohave: (1) at least three major findings; (2) two majorfindings and at least two minor findings; (3) at leastone major finding and an affected first-degreerelative with BOR syndrome [Chang et al., 2004].

BOR GENETICS

The BOR gene was mapped to chromosome 8q12-22 in 1992 [Kumar et al., 1992; Smith et al., 1992].This localization was in accordance with an earlierreport by Haan et al. [1989] of a family with aninherited rearrangement of chromosome 8q, dirins(q24.11q13.3q21.13), which resulted in manifes-tations of both trichorhinophalangeal (TRP) andBOR syndromes. Location was refined in 1994, to thechromosome 8q13.3 interval between markersD8S553 and D8S286 by Ni et al. [1994] and Wanget al. [1994].

Identification of a second locus associated withBOR syndrome was achieved by Kumar et al. [1998a].A very large family in which some members wereaffected with BOS1 was studied. Genome-wideanalysis indicated that the disease gene in theseindividuals segregated in an autosomal dominantfashion, however, there was no evidence of linkageto markers in the chromosome 8q region [Kumaret al., 1998a]. An ongoing study with these indivi-duals provided conclusive evidence of geneticlinkage with markers on chromosome 1q31, thus

confirming previously hypothesized thoughts ofgenetic heterogeneity within BOR syndrome (BOS2OMIM 120502) [Kumar et al., 2000].

Abdelhak et al. [1997a] identified point mutationsand deletions in a novel gene called EYA1 in sevenpersons with the BOR syndrome phenotype. Expres-sion studies of EYA1 in the ear and kidney duringearly development confirmed that this gene isexpressed in the otic vesicle, and in both thesubsequent cochlear and vestibular neuroepithelia,as well as the condensing mesenchyme of thedeveloping kidney [Abdelhak et al., 1997a,b]. Sub-sequently, it was confirmed that haploinsufficiencyfor EYA1 resulted in BOR syndrome [Abdelhak et al.,1997a,b; Zhang et al., 2004].EYA1 is homologous to the Drosophila develop-

mental eyes absent gene or eya, which encodes atranscriptional co-activator required for eye mor-phogenesis [Bonini et al., 1993]. As a result ofalternative splicing, EYA1 is comprised of threeisoforms (a, b, c) and four transcript variants. Isoformb is the longest and consists of 16 coding exons thatextend over 156 kb. Two transcript variants ofisoform b encode various alternatively splicedtranscripts that differ only in their 5 prime regions[Abdelhak et al., 1997b; Chang et al., 2004]. Isoformsa and c each code for one transcript variant.EYA1 belongs to a novel gene family initially

defined as comprising twoothermembers,EYA2 andEYA3 [Abdelhak et al., 1997a]; the correspondingmurine orthologues, Eya2 and Eya3, have also beenisolated [Xu et al., 1997; Zimmerman et al., 1997]. Anadditional member, EYA4, and its murine orthologuewere identified in 1999 [Borsani et al., 1999]. The EYAfamily is characterized by a highly conserved, 271-amino acid, C-terminal called the eyaHR for eyahomologous region, also known as the Eya Domain(ED) [Abdelhak et al., 1997a; Xu et al., 1997].

Abdelhak et al. [1997b] hypothesized that themajority of EYA1 BOR-causing mutations occurredin exons belonging to, or adjacent to the ED in exons9–16. Mutations causing the BOR syndrome pheno-type were thought to be clustered within the ED andmutations outside were believed to lead to discreteunobserved phenotypes. With further investigation,a variety of large and small mutations, nonsense,missense, frameshift, aberrant splicing, and exonskipping were found within the ED in familiesaffected with BOR syndrome. Surprisingly, in about70% of persons with a BOR phenotype, EYA1mutations couldnot be found in the coding sequenceusing single-stranded conformational polymorph-ism analysis (SSCP) [Kumar et al., 1997–1998; Usamiet al., 1999; Rickard et al., 2000].

Vervoort et al. [2002] reported results attained frommutation screening of the coding region of EYA1 in apanel of families using SSCP and direct sequencinganalysis. Interestingly, only one point mutation infive probands was detected. However, complex

TABLE II. Appropriate Phenotypic Criteria for EYA1 Testing in BORSyndrome*

Major criteria Minor criteria

Branchial anomalies External ear anomaliesDeafness Middle ear anomaliesPreauricular pits Inner ear anomaliesRenal anomalies Preauricular tags

Other: facial asymmetry, palateabnormalities

*Affected individuals must have at least three major criteria; two major criteriaand at least two minor criteria; or one major criteria and an affected first-degreerelative meeting criteria for BOR.[Chang et al. [2004]. Reprinted with permission of Wiley-Liss, Inc., a subsidiaryof John Wiley & Sons, Inc.]

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rearrangements, such as inversions or large dele-tions, were discovered in the other four patientsusing Southern blot analysis. These data suggestedthat SSCP and direct sequencing, commonly usedmethods of detecting mutations in EYA1, could notdetect more complex rearrangements of EYA1[Vervoort et al., 2002].

Through the use of a semi-quantitative PCR-basedscreen, Chang et al. [2004] identified complexrearrangement mutations in patients who wereoriginally negative for EYA1 coding sequence muta-tions. In 18% of these patients, new EYA1 mutationswere identified outside the coding sequence. In fact,Chang et al. [2004] reported complex mutationsinvolving every exon, as well as point mutations andsmall insertion/deletions involving every exonexcept 1–3 were found within the EYA1 gene. Thediscovery of new mutations randomly scatteredthroughout the EYA1 gene countered the clusteringhypothesis proposed by Abdelhak et al. [1997a][Chang et al., 2004].

As of June 2006, 117 different mutations of EYA1hadbeen associatedwithBOR syndrome.A full list ofthe mutations is available on the Pendred/BOR webpage (www.medicine.uiowa.edu/pendredandbor/).Included are 61 frameshift or nonsense mutations, 20missense mutations, 24 splice site mutations, and 12complex mutations that involve large deletions orchromosomal rearrangements (Fig. 2).

Ruf et al. [2003] performed a genome wide searchfor linkage in a large pedigree with BOS1, in whichlinkage to the EYA1 locus on chromosome 8q13 hadbeen excluded. This resulted in another new locus(BOS3 OMIM 608389) for BOR/BO syndromemapped to human chromosome 14q23.1-q24.3 [Ruf

et al., 2003]. Within the 33-megabase critical geneticinterval, SIX1, SIX4, and SIX6 genes were located.The mammalian Six gene family is consists of sixmembers (Six1-6)which share twohighly conserveddomains; a homeodomain (HD) and a specific Six-domain (SD) whose products function as tran-scription factors with specific DNA-binding activitythat are crucial for protein–protein interaction[Kawakami et al., 1996, 2000; Chen et al., 1997;Pignoni et al., 1997]. In addition to the eye, SIX genesare widely co-expressed with EYA and otherregulatory genes in many tissues in mammalianorganogenesis including the developing branchialarch, ear, and kidney [Oliver et al., 1995a,b; Xu et al.,1997, 1999, 2002; Zheng et al., 2003]. Owing to thefact that they are known to act within a geneticnetwork of genes, Ruf et al. [2003] believed thesegenes represented excellent candidate genes forBOS3.

In 2004, Ruf confirmed that SIX1, in addition toEYA1, was a BOR syndrome causing gene. Throughthe introduction of three different SIX1 mutationsfound in human kindreds into murine models,Ruf and colleagues demonstrated that the SIX1mutations led to deficiencies in both protein–proteinand protein–DNA interactions. All three humanSIX1 mutations were also crucial for murine Eya1–Six1 interactions, and the two mutations locatedwithin the HD region proved to be essential forspecific murine Six1-DNA binding [Ruf et al.,2004]. With the conclusion that SIX1 mutationscause BOR syndrome by disruption of EYA1-SIX1-DNA complexes, additional complexity wasfound within the pathway responsible for BORsyndrome.

FIG. 2. Diagram of all 117 BOR mutations. (Pendred/BOR web page. www.medicine.uiowa.edu/pendredandbor/) [Color figure can be viewed in the online issuewhich is available at www.interscience.wiley.com.]

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EYA1 EXPRESSION AND ITS ROLE INTHE DEVELOPMENTAL PATHOGENESIS

OF BOR SYNDROME

Kalatzis et al. [1998] have reported a detailed studyof the temporal and spatial pattern of expression ofEya1 in the murine ear and kidney development.Branchial arch anomalies, such as fistulas, sinuses,and cysts are correlated with expression of Eya1 inthe branchial arch apparatus of mice at embryonicday (E) 10.5, however, expression of Eya1 in outerand middle ear is not evident until slightly later atE13.5 [Kalatzis et al., 1998]. Eya1 is found in themesenchyme adjacent to the first branchial cleft thatgives rise to the external auditory canal, themesenchyme of the auricular hillocks, the primordiaof the middle ear ossicles, and in the epithelium ofthe tubotympanic recess. The presence of Eya1expression in these locations is consistent with theexternal auditory canal and middle ear ossicleanomalies that are associated with BOR syndrome[Kalatzis et al., 1998].Eya1 is also expressed in the ventromedial wall of

the otic placode, consistent with cochlear anomaliesand sensorineural hearing loss in BOR syndrome.Interestingly, Eya1 expression is observed in thedifferentiating hair and supporting cells of thesensory epithelia, as well as the associated ganglia,and persists after differentiation takes place, suggest-ing that Eya1 may be essential for the differentiationand/or survival of these inner ear cell populations[Kalatzis et al., 1998].

In the murine kidney, Eya1 expression is limited tothe developing metanephros where it is restrictedto the condensing mesenchymal cells. Due to thereciprocal epithelial–mesenchymal interactions alsorequired to direct kidney morphogenesis [Clapp andAbrahamson, 1994], this single site of expressionis believed to account for both the excretory andcollecting system anomalies of BOR syndrome[Kalatzis et al., 1998].

In a mouse mutant with targeted deletion of Eya1,Xu et al. [1999] found that heterozygotes (Eya1þ/�)have conductive hearing loss and renal abnormal-ities similar to BOR syndrome. Eya1 homozygosity incontrast, was lethal as Eya1�/� mice lacked both earsand kidneys as well as the expression of downstreamgenes Six1 and Fgf3 [Xu et al., 1999].

SIX1 EXPRESSION AND ITS ROLE IN THEDEVELOPMENTAL PATHOGENESIS

OF BOR SYNDROME

Prior to reports by Ruf et al. [2004] that mutations inSIX1 also led to BOR syndrome, several reportsconfirmed the importance of Six1 and its function inthe development of the mammalian auditory systemand kidney. Consistent with these reports was theobservation that the Six1þ/� mouse mutant had a

phenotype similar to that found in BOR syndrome[Laclef et al., 2003; Xu et al., 2003; Zheng et al., 2003].

Expression studies during early inner ear develop-ment of mice indicate that Six1 is co-expressed withEya1 and is first detected in the ventral region of oticpit at E8.75. However, Six1 is excluded from thedorsal sensory epithelium that ultimately developsinto the semicircular canals [Zheng et al., 2003]. ByE9.5, Six1 is restricted to the middle and ventral oticvesicle within which, respectively, the vestibular andauditory epithelia form. At E15.5, Six1 is expressed inall sensory epithelia of the inner ear and in thevestibuloacoustic ganglion [Zheng et al., 2003].

During early organogenesis of the mammaliankidney, Six1 expression overlaps Eya1 and is firstseen in the uninduced metanephric mesenchyme(E10.5) and in the induced mesenchyme aroundthe ureteric bud (E11.5). From E17.5 to P0, Six1expression is restricted to a subpopulation ofcollecting tubule epithelial cells. Molecularly, Six1is not required for Eya1 expression in the otic epi-thelium, however it is required for normal expressionof Fgf3, Fgf10, Bmp4, Gata3, and Nkx5.1, consistentwith the role specification in the early otic vesicle[Zheng et al., 2003].

Almost all heterozygous Six1 mutants (Six1þ/�)studied by Zheng et al. [2003] demonstrated hearingloss as determined by ABR threshold measurements.Analysis of the sectioned ears demonstrated a failureof ossicles to transmit sound from the tympanicmembrane to the ovalwindow, thus impeding soundtransmission through the middle ear. Interestingly inhomozygous mutants (Six1�/�) the entire auditorysystem including the outer, middle, and inner ears ismalformed [Zheng et al., 2003].

Approximately 15% of Six1þ/� mice display renaldefects, including renal hypoplasia and bilateralagenesis. Renal abnormalities associated with Six1demonstrate low penetrance, consistent with thewide variability found in BOR syndrome phenotypes[Xu et al., 2003; Ruf et al., 2004]. Noteworthy has beenthe finding that in early kidney development, the lossof Six1 leads to failure of ureteric bud invasion intothe mesenchyme and subsequent apoptosis of themesenchyme [Xu et al., 2003].

In the compound heterozygote mouse mutant,Eyaþ/�/Six1þ/�, renal hypoplasia is observed about75% of the time. Xu et al. [2003] believed that renalhypoplasia is secondary to reduced inductionbetween the ureteric bud and metanephric tissue inthe peripheral nephrogenic zone caused by thecompromised interaction between Eya1 and Six1.

THE EYA-SIX REGULATORYSYSTEM AND BOR SYNDROME

To determine the molecular basis for the organdefects that occur in BOR syndrome, several studiesfocused on the effects of mutations of the EYA-SIX



regulatory system [Buller et al., 2001; Ozaki et al.,2002; Mutsuddi et al., 2005]. ED mutations specifi-cally identified by Buller et al. [2001] and Ozaki et al.[2002], disrupt the Eya1-Six complex and reduceexpressionof downstream targets, thus triggering theBORphenotype. Interestingly, noneof themutationsthey studied prevented in vitro nuclear translocationof the Eya1 protein by Six1 or Six2. However, Eya1-Six1 nuclear complex formation was decreased innumerous cell lines. Eya1-Six2 nuclear complexformation was not affected, an important point, sinceEya1 is co-expressed with Six1 in branchial arch andotic development and with both Six1 and Six2 duringearly kidney induction [Buller et al., 2001]. Accordingto Ozaki et al. [2002], in vitro BOR-type mutations inthe ED also cause defective co-activator function inEya1 when in contact with a Six-responsive gene(myogenin).

Building on this work, Mutsuddi et al. [2005] soughtto identify the impact of ED mutations on thephosphatase activity, transcriptional capability, andprotein–protein interactions of EYA and raised thehypothesis that the effects of BOR-type mutationsmay differ from target gene to target gene. In vivoanalysis showed that ED mutations impaired thecatalytic activity (i.e., dephosphorylation) of the EYAprotein, suggesting that loss of phosphatase activitymay contribute to impaired EYA activity and in turnthe BOR phenotype. Interestingly, they were unableto replicate the results of Buller et al. [2001] or Ozakiet al. [2002] when performing in vivo analysis on thetranscriptional capability or protein–protein interac-tions of EYA protein. In contrast to the in vitromyogenin promoter-based studies performed byOzaki et al. [2002], Mutsuddi et al. [2005] found thatBOR type genes retained significant, albeit reduced,transcriptional activity in vivo. An explanation forthis observation may be that in vivo EYA recruitsdifferent sets of interacting proteins to the targetpromoters, subsequently leading to abnormal, butstill present, transcriptional output. A better under-standing of the pathophysiology of BOR syndromemay rest on further examination of various mutationprofiles within the EYA-SIX transcription factor andhow these mutations affect downstream target genes[Mutsuddi et al., 2005].

FURTHER RESEARCH

Ando et al., [2006] identified over 300 Six1 targetgenes involved in renal development. Many of thegenes included in this register are also important tootologic development, and the Molecular Otolaryn-gology Research Lab (MORL, University of Iowa;Iowa City, Iowa) has identified a number of targetgenes for evaluation due to a high likelihood ofinvolvement in the pathogenesis of BOR syndrome[MORL unpublished data, 2006]. Investigations

are also underway to identify patients with BORsyndrome who have mutations in SIX1 and the huntfor new genes that may be implicated in the BORphenotype is ongoing. Recent reports have also ledto examination of a possibile dosage affect betweenEya1 and known target genes that may contribute tovariations in phenotype [Sajithlal et al., 2005].

Though extensive research has been performed toelucidate the effect of mutations of EYA1 on thepathogenesis of BOR syndrome, relatively little isknown about the effects of mutations to the SIX1gene. Over 60% of BOR syndrome patients do nothave known mutations in EYA1, thus, SIX1 offersanother avenue for study. Evaluation of SIX1 and itsrelated target genes may provide greater clues to thedisease mechanisms involved in BOR syndrome.Furthermore, Ruf et al. [2004] believe that thedetermination of a genotype/phenotype relation-ship caused by these mutations may represent theunderlying diagnosis in many of persons who areaffected with congenital abnormalities of the urinarytract, deafness, and branchial arch anomalies.

ACKNOWLEDGMENTS

We are indebted to the families who made thiswork possible. This research was supported in partby NIH grant DC03544 (RJHS) and the Doris DukeClinical Research Fellowship (AK).

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