Mapping Of Genetic Modifiers Of Eyal^bor/bor Mice

Branchio-oto-renal syndrome(BOR syndrome) is is a rare autosomal dominant diseasethat affects about 1 in 40,000 children,including 2% of profoundly deaf children.It wasdefined to include hearing loss,auricular malformations,branchial arch remnants,and renalanomalies.Two candidate genes of BOR syndrome were identified,EYA1 and SIX1,theyare homologous to genes involved in Drosopbila eye development,eye absent gene(eya),and sine oculis(so),respectively.Kenneth R.Johnson reported that a spontaneous mutationcausing deafness and circling behavior was discovered in a C3H/HeJ colony of mice atJackson Laboratory.Pathological analysis of mutant mice revealed gross morphologicalabnormalities of the inner ear,and also dysmorphic or missing kidneys.The phenotypewere mapped to mouse chromosome 1 near the position of the Eya1 gene.Because of thecoincident map position and the mutation's phenotypic similarity to human BOR syndrome,they use this kind of mutant mouse as an animal model of the human BOR syndrome.Molecular analysis of the Eya1 gene in mutant mice revealed the insertion of anintracisternal A particle(IAP) element in intron 7.The presence of the IAP insertion wasassociated with formation of additional aberrant transcripts.The hypomorphic nature of themutation may explain its recessive inheritance,if protein levels in homozygotes,they arebelow a critical threshold needed for normal developmental function,so the mutationcausing deafness and circling behavior. The phenotypic expression of Eya1^bor mutation was dramatically affected by thegenetic background,suggesting the existence of modifier quantitative trait loci(QTLs) inother strains.Specifically,postnatal survival and severity of hearing loss exhibitedstrain-related variation in expressivity.In a recent study,it was reported that nuclear exportfactor(Nxf1) is a modifier gene of Eyal^bor.Nxf1 suppressed the mortality and the deafnessin F2 Eya1^bor/bor mutants from a C3HeB/FeJ and CAST/EiJ intercross by increasing normalEya1 mRNA levels.In this study,We describe two QTLs and designate these loci as"modifier ofEya1-associated deafness"or Mead1 on chromosome 4 and Mead2 on chromosome 12.This paper is divided into five parts. Part 1 mouse crossesObjective:To produce two kinds of different genetic background Eya1^bor/bor F2 progeny foradvanced study.Methods:Eya1^bor/+ F1 progeny breeding pairs of C3HeB/FeJ-Eya1^bor/+ and C57BL/6J wereintercross to produce Eya1^bor/bor F2 progeny;another kind of genetic background Eya1^bor/borF2 was produced by intercrossing Eya1^bor/+ F1 progeny breeding pairs ofC3HeB/FeJ-Eya1^bor/+ and CAST/EiJ.Identify the Eya1 allele of F2 by PCR.Result:Of 2255 C3HeB/FeJ-Eya1^bor/+×C57BL/6J F2 mice generated,267 mice wereEya1^bor/bor (referred to F2-Eya1^bor/bor),1342 mice were Eya1^bor/+ and 646 mice were Eya1^+/+;of 1000 C3HeB/FeJ-Eya1^bor/+×CAST/EiJ F2 mice,178 mice were Eya1^bor/bor,570 micewere Eya1^bor/+,252 mice were Eya1^+/+.Conclusion:Some F2-Eya1^bor/bor behavior with circleing and head-bobbing,some not.The differentbehavior of F2-Eya1^bor/bor was shown that there must be some modifiers in C57BL/6J andCAST/EiJ mice genome. Part 2 Mapping of major genetic modifier loci of Eya1^bor/bor in twokinds of Eya1^bor/bor F2 progeny based on ABR thresholds andcochlear turnsObjective:To map genetic modifier loci of Eya1^bor/bor.Methods:DNA of two kinds of Eya1^bor/bor F2 was extracted from the external ear.98Dye-labeled SSLP markers were selected at～25-cm intervals across the mouse genome.Whole-genome analysis with the Dye-labeled SSLP makers.ABR thresholds werecategorized in 14 groups beginning with 0 for deafness and 1-13 as the subsequent groupsof thresholds ranging from 90 to 30 dB in 5-dB increments.Cochlear coiling in 89 of theF2-Eya1^bor/bor were examined.Cochlear turns were categorized at 1/4-turn increments.Thebetter ABR threshold or cochlear turn of two ears was used to represent to the mouse.MapManager QTXb20 was used to identify QTLs.QTL analysis using cochlear turns and ABRthresholds as quantitative trait was performed.Result:1.C3HeB/FeJ×c57BL/6J F2 52% (139/267) of the F2-Eya1^bor/borexhibited detectableresponses in at least one ear at thresholds varying from 35 to 90 dB.Two loci highlyassociated with the click ABR thresholds were identified.The first locus,Mead1,associatedwith marker D4Mit104 on chromosome 4,demonstrated a LOD score 12.0 and explained19% of the variation in hearing.The second locus,Mead2,associated with markerD12Mit283 on chromosome 12,demonstrated a LOD score of 12.1 and explained 19% ofthe variation in hearing.89 F2-Eya1^bor/bor mice varied from agenesis to the normal 1 3/4 turns.Two loci highly associated with the cochlear turns were identified,they overlappedwith the hearing QTLs,Mead1 and Mead2.The first locus on 4 chromosome was linked tomarker D4Mit104 based on the cochlear coiling phenotype with a LOD=15.8 and explained56%of the variation.Similarly for the second locus,marker D12Mit283 was linked withcochlear coiling (LOD=3.3) and explained 16% of the variation.2.C3HeB/FeJ×CAST/EiJ F2 86% (153/178) of the F2-Eya1^bor/bor exhibited detectableresponses in at least one ear at thresholds varying from 30 to 90 dB,no statisticallysignificant linkage was detected.Conclusion:1.These data suggest that the same two genes within the Mead1 and Mead2 criticalregions modify hearing and cochlear coiling.2.We hypothesized that this finding was due to the presence of multiple modifierswith modest effects or insufficient power to detect linkage using an intercross strategy.Totest our hypothesis,we initiated a backcross strategy. Part 3 Mapping genetic modifier loci of Eya1^bor/bor inC3HeB/FeJ×CAST/EiJ N2Objective:To map genetic modifier loci of Eya1^bor/bor in C3HeB/FeJ×CAST/EiJ N2Methods:Eight breeding pairs between C3Fe-Eya1^bor/+and CAST mice were set up to generateF1 progeny;35 F1 Eya1^bor/+ were backcrossed to C3Fe-Eya^bor/+ mice to generate Eya1^bor/borhypomorphs (N2-Eya1^bor/bor).Whole-genome analysis with microsatellite markers wasperformed.ABR thresholds and cochlear turns were categorized,QTL analysis usingcochlear turns and ABR thresholds as quantitative trait was performed.Result:1.Of 909 C3HeB/FeJ×CAST/EiJ N2 mice generated,179 mice were Eya1^bor/bor,470mice were Eya1^bor/+,260 mice were Eya1^+/+.2.Of the 179 N2-Eya1^bor/bor mutants generated,62% (112/179) demonstated ABRthresholds ranging from 90 to 40 dB.Qnly 17% (19/112) of these 112 ABR-Positive micehad ABR thresholds of 50 dB or less,an obvious reduction from 44% seen in the intercross.Four loci were identified on chromosome 4,9,15 and 19,respectively (table1).For thechromosomes 4 locus,maker D4Mit149 showed the most significant LOD score of 8.4 andexplained 19% of the total variation seen in ABR thresholds (Fig.2a);For thechromosome 19 locus,marker D19Mit28 showed the most significant LOD score of 8.1and explained 19% of the total variation in ABR thresholds (Fig.2b).For the chromosome9 and chromosome 15,marker D9Mit126 and D15Mit242 showed LOD score of 2.4 and2.7,explained 6% and 7% respectively.89 cochlear turns of N2-Eya1^bor/bor were counted,three loci were identified on chromosomes 4,12,and 14,respectively.For the chromosome 4 locus,marker D4Mitl04 showed the highest LOD score of 4.5 and explained 21% of thetotal variation in the number of cochlear turns (Fig.2a);For the chromosome 12 locus,marker D12Mit218 showed the highest LOD score of 2.8 and explained 14% of thevariation in the number of cochlear turns (Fig.2c).Marker D14Mit174 showed the LODscore of 3.3,explained 16% of the variation in the number of cochlear turns.Conclusion:1.Marker D4Mit149 and marker D4Mit104 are located in chromosome 4,and they areadjacent markers.The overlap of this locus with the previously identified Mead1 locussuggests that the same gene or the same set of gene acts as suppressor in these two differentstrains.The gene or the same set of gene in this locus can modify hearing and cochlearcoiling.2.Marker D12Mit218 and marker D12Mit283 are located in chromosome 12,and theyare adjacent markers.The overlap of this locus with the previously identified Mead2 locussuggests that the same gene or the same set of gene in this locus can modify cochlearcoiling.3.Nxf1,a gene located in the vicinity of marker D19Mit28,has been identified as anEya1bor modifier and likely represents the Mead3 locus. Part 4 fine-mapping the critical regions of the Mead1 andMead2 in C3Fe.C57BL congenic strainsObjective:To fine-map the critical regions of Mead1 and Mead2 and to identify potentialcandidate genes in congenic strains.Methods:Developed congenic lines individual congenic strains on the C3He/FeJ backgroundwith the C57BL/6J allele covering the 95% confidence interval at the Mead1 and Mead2loci,respectively,by a marker-assisted breeding approach.Eya1^bor/+ F1 progeny frombreeding pairs of C3He/FeJ-Eya1^bor/+ and C57BL/6J and two or three mice with the leastamount of these heterozygous regions were chosen as breeders for the next generation.Thecongenic lines were generated after five or six backcrosses.All of the Eya1^bor/bor mice ABRthresholds were tested and all of the Eya1^bor/bor mice of congenic lines were examinedcochlear turns.Result:1.Suppression of the hearing loss in these congenic Eya1^bor/bor mice graduallydiminished and almost was lost eventually as the C57BL/6J genomic contribution wasreduced.2.Eya1^bor/bor mice consistently exhibited 1/2-3/4 cochlear turn,when heterozygous forC57BL/6J Mead1 allele between markers D4Mit149 and D4Mit193 (14 samples),and 1 to1 1/4 turns when homozygous for this same region (11 samples).3.Six percent(4/25) of the Eya1^bor/bor mice formed more than 1/4 cochlear turns whenheterozygous for the C57BL/6J Mead2 allele between markers D12Mit103 and D12Mit172and all of the Eya1^bor/bor mice formed 1/2-3/4 turn when homozygous for this region. Conclusion:1.These findings were consistent with a Mendelian semidominant inheritance pattern.Mead1 demonstrated complete penetrance;Mead2 demonstrated incomplete penetrance(16%) in heterozygous and complete penetrance in homozygous.2.By measuring cochlear turns in the Eya1^bor/bor congenic mice heterozygous forC57BL/6J Mead1 allele and using more genomic markers,we were able to narrow downthe Mead1 region to 5.4 cM between markers D4Mit227 and D4Mit171.3.By measuring cochlear turns in the Eya1^bor/bor mice homozygous for the C57BL/6JMead2 allele,we were able to narrow the critical region to 4.4 cM between markersD12Mit56 and D12Mit283. Part 5 confirmation of Mead1,Mead2 and Mead3 loci inC3Fe.CAST congenic strains and C3Fe.BALA congenic strainsObjective:To confirm Mead1,Mead2 and Mead3 in C3Fe.CAST congenic strains andC3Fe.BALA congenic strains and analysis the effects of the three loci.Methods:Developed congenic lines individual congenic strains on the C3He/FeJ backgroundwith the CAST/EiJ and BALA/cJ allele covering the 95% confidence interval at the Mead1and Mead2 loci,respectively,by a marker-assisted breeding approach.the same as the part4.The other processes are the same as the part4.Results:1.One C3Fe.CAST congenic line for the Mead1 locus,with a CAST region of 5-10cMlocated at the centromeric end of chromosome 4:13 C3Fe.CAST^Mead1/Mead1-Eya1^bor/bor miceand found 1/2-3/4 cochlear turns developed in all except one which a 1/4 turn was observed.This was in sharp contrast to our original finding in our C3Fe.B6^Mead1/Mead1-Eya1^bor/borcongenic line with 1-1/4 cochlear turns.2.One C3Fe.CAST congenic line for the Mead2 locus,with a CAST region ofapproximately 10 cM at the centromeric end of chromosome 12:4 C3Fe.CAST^Mead2/Mead2-Eya1^bor/bormice and found 1/2-3/4 cochlear turn in each,similar toC3Fe.B6^Mead2/Mead2-Eya1^bor/bor congenic line.3.Three independent C3Fe.CAST congenic lines(lines 501,504,505) for the Mead3locus.The CAST genomic regions in all three lines were almost identical due to arecombination hot spot.This 5-Cm(or 11Mbp) region is located at the centromeric end ofthe chromosome 19:All C3Fe.CAST^Mead3/Mead3-Eya1^bor/bor mice displayed a partial penetrance of the suppressed phenotype.In line 501,57%(8/14) of the mutants exhibitedrecognizable cochlear turns,ranging from 3/4 to 1 turn.In line 504 and 505,the penetranceand the range of cochlear turns were 44% (8/18),1/4-11/4 and 50% (8/16),1/4-1/2,respectively.4.One C3Fe.BALA congenic line for the Meadl locus,with a CAST region of5-10cM located at the centromeric end of chromosome 4:4 C3Fe.BALA^Mead1/Mead1-Eya1^bor/bor mice and found 1 1/2 cochlear turns,similar toC3Fe.B6^Mead1/Mead1-Eya1^bor/bor congenic line.5.Two C3Fe.BALA congenic line for the Mead2 locus,with a CAST region ofapproximately 10 cM at the centromeric end of chromosome 12:7 C3Fe.BALA congenicmice and found 1/2-1 3/4 cochlear turns,demonstrated a significant variability in the numberof cochlear turns.Conclusion:The alleles of different strains and different loci demonstrated different effect andpenetrance of the suppressed phenotype.Cochlear turns of C3Fe.CAST^Mead1/Mead1-Eya1^bor/bormice were quite different with the C3Fe.B6^Mead1/Mead1- Eya1^bor/bor mice suggested suppressorallelic heterogeneity in these two strains.The Mead1 and Mead2 loci are distinct from theMead3 locus in the pattern and the size of the cochlear agenesis suppressor effect.All threeprotective alleles(B6,BALA and CAST) of Mead1 and Mead2,when homozymous,arefully penetrance.In constrast,all three congenic strains of Mead3^CAST/CAST-Eya1^bor/bor miceexhibit partial penetrance type (～50%) and expressivity of the cochlear phenotype (1/4-11/2turns).