Autosomal-Dominant Branchio-Otic (BO) SyndromeIs Not Allelic to the Branchio-Oto-Renal (BOR)Gene at 8q13

Shrawan Kumar,1* Henri A.M. Marres,2 Cor W.R.J. Cremers,2 and William J. Kimberling1

1Department of Genetics, Center for Hereditary and Communication Disorders, Boys Town National ResearchHospital, Omaha, Nebraska

2Department of Otorhinolaryngology, University Hospital Nijmegen, Nijmegen, The Netherlands

The manifestations of branchio-oto-renalsyndrome (BOR), Treacher Collins syn-drome, tricho-rhino-phalangeal syndrome,van der Woude syndrome, and Langer-Giedion syndrome are well-defined; theseconditions represent clinically and geneti-cally separate syndromes. Autosomal-dominant branchio-oto-renal syndromecomprises preauricular pits, branchialfistulas, hearing loss, and renal anomalies.However, several families have been de-scribed without one or more of these clinicalfindings. In some families, the phenotypicexpression is limited to branchial anoma-lies, preauricular pits, and hearing loss,with no renal dysplasia (branchio-otic orBO syndrome). In other families, branchialand renal anomalies occur without hearingimpairment. It is not known whether thevariable clinical manifestations are due tothe effect of a single gene or whether theserepresent different syndromes. We investi-gated BO syndrome in a large family to de-termine whether BOR and BO syndromesare allelic to each other. The genetic linkageanalysis provides evidence that BO syn-drome is not allelic to the BOR gene at 8q13.Am. J. Med. Genet. 76:395–401, 1998.© 1998 Wiley-Liss, Inc.

KEY WORDS: branchio-otic type syndrome;genetic linkage; chromosomearea 8q; separate locus

INTRODUCTION

Branchio-oto-renal syndrome (BOR) comprises pre-auricular pits, branchial fistulas, and hearing impair-ment, and was first reported by Heusinger [1864]. Sub-sequently, the hereditary nature of BOR syndrome wasdescribed [Martins, 1961; Hunter, 1974]. Melnick et al.[1976] defined the syndrome associated with renalanomalies and designated it as branchio-oto-renal syn-drome and concluded it was an autosomal-dominanttrait. The hearing impairment varies from mild to se-vere with or without associated anomalies. It may besensorineural, conductive, or mixed, and the type ofhearing loss may differ between two ears [Fraser et al.,1980]. Lateral cervical sinuses, cysts, or fistulas mayoccur. Renal anomalies such as polycystic, hypoplastic,or totally absent kidneys are common [Melnick et al.,1976; Fraser et al., 1980; Nevin, 1977; Widdershoven etal., 1983]; glomerular lesions and vesicoureteral refluxleading to renal failure have also been described [Du-mas et al., 1982].

The manifestations of branchiogenic syndrome varysignificantly from one family to another. Several fami-lies have been described with branchial anomalies, pre-auricular pits, and hearing loss with no evident renaldysplasia (branchio-oto, or BO syndrome) [Fourmanand Fourman, 1955; McLaurin et al., 1966; Wilder-vanck, 1962; Rowley, 1969; Bailleul et al., 1972; Bour-guet et al., 1966]. The phenotypic expression of thebranchial arch as well as audiologic and renal develop-ment can be quite variable, even within the same fam-ily. The complex clinical symptoms suggest the possi-bility of more than one gene involved in the develop-ment of BOR syndrome.

Until recently, it was not known whether the vari-able clinical manifestations such as branchio-oculo-facial (BOF), branchio-oto-uretral (BOU), branchio-otic(BO), and BOR syndromes are allelic or represent clini-cally different syndromes. The gene causing BOR syn-drome was mapped to chromosome band 8q13 [Smithet al., 1992; Kumar et al., 1992] with refined flankingmarkers [Kumar et al., 1994, 1996]. Recently, a candi-date gene EYA1 (human homologue of Drosophila eyeabsent gene) for BOR syndrome was identified by po-

Contract grant sponsor: NIH (NIDCD); Contract grant number:PO1 DC01813.

*Correspondence to: Dr. Shrawan Kumar, Department of Ge-netics, Boys Town National Research Hospital, 555 North 30thStreet, Omaha, NE 68131. E-mail: Kumar@boystown.org

Received 31 July 1997; Accepted 12 November 1997

American Journal of Medical Genetics 76:395–401 (1998)

© 1998 Wiley-Liss, Inc.

sitional cloning, and mutations were reported [Abdel-hak et al., 1997; Kumar et al., 1998]. The localization ofBOR syndrome in the human genome will help in de-fining the spectrum of defects as well as in resolvingthe issue of genetic heterogeneity/or allelic differencesbetween the various branchiogenic autosomal-dominant inherited syndromes.

We investigated a large family with branchio-oticsyndrome comprising deafness, preauricular sinus, ex-ternal ear anomaly, and commissural lip pits, but nocervical fistulas and renal anomalies. Our aim was todetermine whether this syndrome is the result of theeffect of same gene which causes branchio-oto-renalsyndrome. Our results provide evidence that BO syn-drome is not allelic to the BOR gene, localized to chro-mosome 8q13 region.

MATERIALS AND METHODSFamilies and Collection of Blood Samples

Blood samples were drawn from members of a largemultigeneration family with branchio-otic type syn-drome ascertained in the Netherlands [Marres andCremers, 1991; MIM 120502] (Fig. 1). The affectedrelatives have preauricular sinus (or cysts), commis-sural lip pits, an external ear anomaly, and hearingimpairment (Fig. 2). In total, 31 informative individu-als, 16 of whom were affected, are included in the ge-netic linkage analysis. The clinical details and the link-age results of the BOR families were reported earlier[Smith et al., 1992; Kumar et al., 1992; Cremers andFikkers-van Noord, 1980].

Clinical Evaluation

Clinical tests were performed on all living relativesshown in pedigree (Fig. 1). Each person underwent

general medical and detailed otorhinolaryngologicevaluations. Persons over age 3.5 years were testedaudiologically by pure tone audiometry. If this consis-tently showed conductive or mixed hearing loss, imped-ance audiometry was also performed. Renal examina-tion included blood and urine chemistry, renal ultraso-nography, and intravenous pyelography. Details of theclinical evaluation and history were described earlier[Marres and Cremers, 1991].

Polymorphic Markers

Individuals were typed using microsatellite polymor-phic markers from the chromosome 8q13 region. De-tails of the microsatellite markers investigated in thepresent study are given in Table I. Oligonucleotideprimers were synthesized in our laboratory, using aCruachem Automated DNA Synthesizer (Cruachem,Inc., Herndon, VA).

DNA Extraction

Genomic DNA was extracted (500 mg–2 mg) eitherfrom white blood cells or transformed lymphocytes bysodium dodecyl sulfate (SDS) lysis, proteinase K diges-tion, phenol/chloroform extraction, and ethanol precipi-tation using an Applied Biosystems Nucleic Acid Ex-tractor (Applied Biosystems, Inc., Foster City, CA).

PCR Amplification of Genomic DNA

PCR amplification of genomic DNA from BOR familyrelatives was performed in an automated thermocycler[Saiki et al., 1988]. Sample DNA reactions were carriedout in a volume of 25–100 ml. A standard reaction mix-ture contained the sample DNA (50–100 ng) to be am-plified, two primers (20–50 pmol of each primer), TaqDNA polymerase (1–4 units for each sample), 200 mMdGTP, dCTP, and dTTP, 2.5 mM dATP in a buffered

Fig. 1. BO family. The clinical findings are indicated as separate components for each individual in the pedigree. Blood samples were obtained fromthe individuals shown with an asterisk, and were included in genotyping and genetic linkage analysis.

396 Kumar et al.

Fig. 2. Manifestations of BOR and BO syndromes. A: Six-month-old girlwith BOR syndrome. Note presence of cup-ear, preauricular sinus (upperarrow), and cervical fistula (lower arrow). B: BO syndrome. Commissurallip pits are indicated with an arrow. C: BO syndrome. Preauricular sinus(arrow) in combination with flat helix.

Autosomal-Dominant Branchio-Otic Syndrome 397

solution (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 1.5 mMMgCl2, and 0.1% Triton X-100), and 1 mCi 32P-dATP at800 Ci/mmol for each sample. Samples were overlaidwith mineral oil (Sigma Chemical Co., St. Louis, MO),and the template DNA was first denatured by heating,followed by polymerase chain reaction (PCR). Multi-plex PCR typing of markers on chromosome 8q13 wascarried out in the following way. The PCR amplifica-tion was performed in a 25-ml volume for each sample,and reactions contained 50 ng of genomic DNA tem-plate, 20 ng each of the 2–4 different sets of oligo-nucleotide primers, 1 mCi 32p at 800 Ci/mmol, and 0.5units of Taq DNA polymerase. DNA was amplified us-ing synthetic oligonucleotide primers through 26 tem-perature cycles consisting of 1 min at 94°C (denatur-ation), 2 min at 55°C (annealing), and 2 min at 72°C(extension), and the last extension was lengthened to 5min.

Five microliters of polymerase chain reaction prod-uct of each sample were mixed with 10 ml of gel loadingdye buffer containing formamide (98% deionized form-amide, 10 mM EDTA, pH 8.0, 0.025% xylene cyanol FF,and 0.025% bromophenol blue), and samples were de-natured by boiling for 3 min before loading. In eachlane, 4–5 ml of denatured sample were loaded, and elec-trophoresis was carried out in 6% denaturing poly-acrylamide DNA sequencing gel (FB-Seq-3545, FisherBiotech, Pittsburgh, PA), containing urea, in 1 × TBE

buffer at 70 W. The urea was removed after electropho-resis in 5% acetic acid and 15% methanol. The gel wasdried and autoradiographed overnight on KodakXAR-5 film (Eastman Kodak Co., Rochester, NY).

Data Analysis

Linkage analysis was performed on a personal com-puter using the LINKAGE program, version 5.1 [Lath-rop et al., 1985; Ott, 1985]. Pairwise lod scores werecalculated using MLINK and ILINK options of LINK-AGE package. Only genotypes that were distinct inPCR results for each individual were included in thelinkage analysis, and dominant mode of inheritancewith full penetrance was assumed. No inferred geno-types were included in the statistical analysis.

RESULTS AND DISCUSSIONThe 8q13 microsatellite markers, both from Gene-

thon and CEPH, earlier reported to be linked with BORfamilies [Smith et al., 1992; Kumar et al., 1992], weretyped in affected and unaffected individuals from alarge BO family (Table I). The results of two-point link-age analysis with BO are provided in Table II. A lodscore of −2.0 significantly excludes the disease locusfrom the marker at the recombination fraction indi-cated, whereas a lod score of 3 and above is an indica-tion in favor of linkage. Being logarithmic, a lod score of+1 means a 10 to 1 chance in favor of linkage, +2 is 100

TABLE I. Description of Markers Used in Genetic Linkage Analysis

Locussymbol

Cytogenetic maplocation

Oligonucleotidesfor PCR amplification

Numberof alleles

Productsize in bp

Nature ofpolymorphism

D8S165 8q11–q13 5’-ACAAGAGCACATTTAGTCAG-3’ 7 CA repeat(MFD 117) 5’-AGCTTCATTTTTCCCTCTAG-3’ 138–152D8S285 8 5’-GCATCACACAGAATCTTTG-3’ 8 CA repeat

5’-ATGGGTTTATGGCCTTTAC-3’ 108–124PENK 8q23–q24 5’-TAATAAAGGAGCCAGCTATG-3’ 5 CA repeat(MFD 31) 5’-ACATCTGATGTAAATGCAAGT-3’ 75–83D8S166 8 5’-GATTGTGTCATTGCACTCCA-3’ 10 CA repeat(MFD 159) 5’-ACAAGGAAGTTCCTTTTTGG-3’ 110–132D8S260 8 5’-AGGCTTGCCAGATAAGGTTG-3’ 10 CA repeat

5’-GCTGAAGGCTGTTCTATGGA-3’ 187–213D8S510 8 5’-CTGGCCCACCAGTCTT-3’ 4 CA repeat

5’-GGCAACGCATGGACTA-3’ 217–223D8S553 8 5’-TGAAACCTTGTCTAAAACACAC-3’ 10 CA repeat

5’-GTCGTCTGATGCAATACAGATA-3’ 221–253D8S543 8 5’-TGGTGTCATTGCTTTCTAGTCT 7 CA repeat

5’-TGCACAGGTGAGTAAATTTGTAA-3’ 116–140D8S530 8 5’-GCAGGGTTGGGTGATG-3’ 10 CA repeat

5’-GCTTGGGCTCAGAGGC-3’ 201–227D8S279 8 5’-AAACACAGGTCTGTAGGATTTTAGT-3’ 10 CA repeat

5’-GTGTCAGGTCGGGGTG-3’ 229–257D8S164 8q13–q22 5’-GATCATGTGAGTTAATACTTTAAT-3’ 14 CA repeat(MFD 104) 5’-TCAGCTGCCTGTATTACTCA-3’ 165–199D8S167 8q22-qter 5’-TTGTTCCTTTTCATGGCTGA-3’ 14 CA repeat(MFD 185) 5’-CAACTTATATATATTCCATGGC-3’ 105–135D8S286 8 5’-GCTGTTTATTTGCCCATGT-3’ 7 CA repeat

5’-GCATGAAACTGTCACTGAGA-3’ 220–238D8S84 8 5’-CGAAAGTTCAGAGATTTGCA-3’ 8 CA repeat

5’-ACATTAGGATTAGCTGTGGA-3’ 181–195D8S275 8 5’-AAATCGCTAGAAAATGTCCA-3’ 8 CA repeat

5’-TCACACCTGGGAATTAGAAG-3’ 139–157D8S88 8 5’-TCCAGCAGAGAAAGGGTTAT-3’ 10 CA repeat

5’-GGCAAAGAGAACTCATCAGA-3’ 76–100D8S199 8q22–qter 5’-CCTTCTTTTTCTGCTCTGCT-3’ 11 CA repeat(MFD 177) 5’-AGTCACAGAGTAAATGATGG-3’ 204–230

398 Kumar et al.

to 1, +3 is 1,000 to 1, and so on. Markers such asD8S285, PENK, D8S166, D8S260, and D8S553 gavepositive lod scores in BOR families [Smith et al., 1992;Kumar et al., 1992], but yielded negative lod scoreswith BO, indicating no linkage to 8q13 markers. Fur-ther analyses in our BO family with regard to severalother polymorphic markers in the vicinity of the BORlocus showed no evidence of linkage (Table II). On theother hand, gene analyses excluding the localization ofthe BO gene from the BOR region suggest that the BOgene is not allelic to BOR.

It is evident from other studies that the phenotypicexpression of the branchial arch, and audiologic andrenal development, can be quite variable even withinthe same family [Heimler and Lieber, 1986]. As statedbefore, the nature of the intra- and interfamilial vari-ability of expression is unknown. The fact that manyclinical features cluster in families suggests the possi-bility that there is a gene at another locus segregatingin some families which produces a distinct genetic entity.

There are also reports of overlap of hemifacial micro-somia (HFM) and branchio-oto-renal syndrome inwhich both syndromes present similar clinical expres-sions such as malformed auricles, preauricular ap-pendages and/or pits, hearing loss, branchial cleft car-tilage, and facial paresis [Rollnick and Kaye, 1985;Cote and O’Regan, 1982; Fraser et al., 1978; Melnick etal., 1976; Legius et al., 1990]. Several anomalies com-mon to both branchio-oculo-facial (BOF) syndromeand BOR have also been reported [Legius et al., 1990].The overlapping manifestations in the different disor-ders, associated with branchial anomalies, are pre-sented in Table III. Haan et al. [1989] reported a familywith an inherited rearrangement of chromosome 8q inindividuals presenting with TRPS (tricho-rhino-phalangeal syndrome) and BO syndrome. This chromo-some rearrangement has further helped in mappingand cloning the BOR gene. This finding implies thatmany families presenting clinical signs such as BO andBR types are not necessarily displaying a different syn-drome but rather represent different forms of BOR syn-drome [Vincent et al., 1997]. The family we describe

here possibly has atypical BO syndrome and is notlinked to the 8q13 region. There are many other fami-lies with branchial anomalies, which cannot be mappedon 8q13 due to small size, who were screened for mu-tation in the EYA1 (BOR) gene. No mutations havebeen found (results not presented). It is possible thatthe genetic heterogeneity associated with branchialanomalies may not be as great as the diverse clinicalanomalies might indicate. However, once this gene iscloned and characterized, it will open a door to screenmany putative BOR families and will provide betterdefinition of the syndromes associated with branchialanomalies and its genetic entity.

In most BOR-affected individuals, auricular deformi-ties, preauricular pits, ossicular malformations andcervical sinuses, cysts, or fistulas occur together, indi-cating a common embryologic origin in the develop-ment of the branchial arch apparatus. Maldevelopmentof branchial arches 1 and 2 must affect the auditorysystem, leading to hearing impairment. However, renalanomalies obviously result from a distinctly differentpathogenesis. It has been argued that since the mal-formations of BOR syndrome comprise several organswhich are embryologically unrelated, the disease iscaused by the combined effect of two or more genes,each being responsible for lesions in one or more organs[Gimsing and Dyrmose, 1986]. Another hypothesis sug-gests that the different organs involved in causing thedisease are under the control of the same inducing ororganizing mechanism, so that a single gene disturbingthis mechanism would have a multiorgan effect [Mel-nick, 1979]. Since the BOR gene is mapped to chromo-some 8q, it is now evident that branchial, auditory, andrenal anomalies are the result of a single gene defect.However, families having either branchial and hearingimpairment (BO) or branchial and renal defect (BR)are not necessarily due to the same gene affecting threeorgans, as is true for BOR.

ACKNOWLEDGMENTSWe thank Kathleen Brennan, Karen Deffenbache,

Tom Fowler, Denise Hoover, Phil Kelly, Judy Kenyon,

TABLE II. Two-Point Lod Scores of Different Markers on Chromosome 8q Region With BO*

Recombination fraction u

0.0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

D8S165 −~ −1.913 −1.340 −1.010 −0.775 −0.585 −0.421 −0.281 −0.164D8S285 −~ −2.564 −1.119 −0.439 −0.079 0.106 0.181 0.186 0.147PENK −~ −2.949 −1.747 −1.065 −0.619 −0.231 −0.126 −0.010 0.042D8S166 −~ −5.023 −2.771 −1.627 −0.938 −0.498 −0.215 −0.042 0.043D8S260 −~ −3.550 −1.999 −1.207 −0.733 −0.434 −0.245 −0.127 −0.054D8S510 −~ −2.693 −2.072 −1.613 −1.205 −0.864 −0.592 −0.380 −0.216D8S553 −~ −3.141 −1.409 −0.572 −0.115 0.124 0.216 0.203 0.120D8S543 −~ −1.256 −0.330 0.074 0.262 0.324 0.305 0.234 0.141D8S530 −~ −1.787 −0.170 0.543 0.868 0.963 0.896 0.707 0.427D8S279 −~ −2.306 −0.645 0.117 0.492 0.642 0.636 0.514 0.308D8S164 −~ −3.066 −1.124 −0.209 0.266 0.486 0.528 0.439 0.257D8S286 −~ −1.721 −0.382 0.179 0.409 0.451 0.380 0.249 0.113D8S84 −~ −1.914 −0.827 −0.283 0.011 0.149 0.181 0.144 0.075D8S275 −~ −3.556 −1.530 −0.553 −0.030 0.228 0.307 0.260 0.143D8S88 −~ −4.559 −2.497 −1.451 −0.830 −0.449 −0.221 −0.095 −0.034D8S199 −~ −1.228 −0.081 0.425 0.656 0.726 0.682 0.550 0.351

*Lod scores were calculated assuming u male 4 u female.

Autosomal-Dominant Branchio-Otic Syndrome 399

Larry Overbeck, and Michael D. Weston for their helpwith manuscript preparation, figures, and genotyping.This study was supported by grant PO1 DC01813 fromthe National Institutes of Health (NIDCD).

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TABLE III. Overlapping Manifestations of the Branchial Arch Syndromes*

BORsyndrome

BOsyndrome

Oto-facial-cervical

syndromeBOU

syndromeBOF

syndrome HMFPresentfamily

Pinna malformation + + − + + + +Preauricular sinus + + + + + + +Ear appendage + − − + − + +Aural atresia + − − + − + −High-arched or cleft

palate+ − − − + − −

Nasal deformity − − + − + − −Hearing loss + + + + + + +Cervical fistulas + + + − − − −Renal anomalies + − + + − − −Duplication of ureters − − − + − − −Shoulder anomaly − − + − − − −Commissural lip pits − − − − − − +Vestibular hypofunction + − − − − + −Upper lip deformity − − − − + − −Coloboma − − − − + − −Reference Cremers et al.,

1981;Melnick et al.,

1976;Fraser et al.,

1978

Melnick et al.,1978

Fara et al.,1967

Fraser et al.,1983

Fujimoto,1987

Gorlin et al.,1995

Marres andCremer,1991

*+, common feature of syndrome; −, very rare feature.

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