定位Eyal~(bor/bor)基因型小鼠Eyal基因的遗传修饰基因
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摘要
鳃裂-耳-肾综合征(Branchio-oto-renal syndrome，BOR)是一种罕见的常染色体显性遗传性疾病，主要表现为鳃弓发育异常，听力障碍和泌尿系统畸形，在儿童中的发病率为1：40000，在深度聋儿中占2％。随着分子生物学的发展，人们已经定位和克隆出了BOR综合征的2个致病基因，EYA1基因和SIX1基因，他们分别是果蝇眼发育基因eya(Drosopbila eyes absent gene)和so(sine oculis)的同源基因。1999年KennethR．Johnson等人报道了C3H／HeJ小鼠基因自发突变出现了听力损失及兜圈(circling)的症状，病理检查发现该突变小鼠内耳发育畸形以及肾脏畸形或者缺失。通过对该突变小鼠的基因链锁定位发现突变位于1号常染色体Eya1基因，从而以该突变小鼠作为BOR综合征的动物模型，进行进一步研究发现这种突变小鼠的Eya1基因的7号内含子中插入了一个IAP(Intracisternal A Particle)。由于这种插入会导致Eya1基因转录的正常的mRNA量减少以及一些异常的mRNA的形成，从而导致由Eya1基因编码的蛋白量减少。当Eya1基因为纯合突变，其编码的蛋白量低于正常发育所需要的蛋白量，小鼠出现表型；当Eya1基因为杂和突变，其编码的蛋白量仍能满足正常发育的需要，小鼠则不出现表型，也正好解释了这种突变在该小鼠里的隐性遗传特征。
更进一步的研究发现，Eya1~(bor／bor)突变鼠的基因表现度，例如产后死亡率和听力障碍的程度，随着小鼠基因背景的不同而存在着很大的差异，所以人们推测在某些鼠种的基因组里存在着Eya1基因的遗传修饰基因。JenniferA．Floyd等人用C3HeB／FeJ小鼠与CAST／EiJ小鼠杂交，得到Eya1~(bor／bor)基因型的后代，再通过数量性状基因位点(Quantitative trait loci，QTL)分析，发现了一个调节基因Nxf1(nuclear export factor)可以减少带有Eya1~(bor／bor)基因型的杂交小鼠的死亡率和听力障碍程度，并且发现Nxf1基因是通过增加正常Eya1 mRNA的量来发挥作用。那么Eya1基因是否还存在其他的遗传修饰基因?其他的遗传修饰基因又如何对Eya1基因进行调节?
本课题以C3HeB／FeJ-Eya1~(bor/+)小鼠与C57BL／6J小鼠，CAST／EiJ小鼠，以及BALB／cJ小鼠杂交而得到的三种不同基因背景小鼠为研究对象，对各种杂交小鼠基因组的基因型进行检测。选择小鼠ABR值和耳蜗周数的变化作为BOR综合征表型的2个指标，运用QTL分析将基因型与表型进行关联分析，再建立小鼠同类系进行精确定位的方法来定位Eya1基因的遗传修饰基因。本课题为克隆Eya1~(bor／bor)基因的遗传修饰基因和研究各个基因的功能奠定基础，并且为研究人类综合征型耳聋的调节基因提供思路。本文共分五个部分。
第一部分C3HeB／FeJ-Eya1~(bor／+)小鼠与C57BL／6J小鼠，CAST／EiJ小鼠第二代杂交小鼠的培育
[目的]
培育具有不同基因背景的杂交小鼠，为研究Eya1基因的遗传修饰基因奠定基础。
[方法]
用C3HeB／FeJ-Eya1~(bor／+)小鼠分别与C57BL／6J小鼠，CAST／EiJ小鼠杂交得到两种不同基因背景的第一代杂交小鼠(F1)，再通过互交(intercross)得到第二代杂交小鼠(F2)，通过PCR检测所有F2的Eya1基因位点的基因型。
[结果]
C3HeB／FeJ-Eya1~(bor／+)×C57BL／6J F2共2255只，其中基因型为Eya1~(bor／bor)F2 267只，Eya1~(bor／+)F2 1342只，Eya1~(+／+)F2 646只；C3HeB／FeJ-Eya1~(bor／+)×CAST／EiJ F2共1000只，其中基因型为Eya1~(bor／bor)F2 178只，Eya1~(bor／+)F2 570只，Eya1~(+／+)F2 252只。
[结论]
部分基因型为Eya1~(bor／bor)F2出现明程度不等地点头兜圈等症状，部分基因型为Eya1~(bor／bor)F2的症状并不明显，说明C57BL／6J小鼠和CAST／EiJ小鼠的某些基因位点对Eya1~(bor／bor)F2的耳部发育发挥了作用。
第二部分以基因型为Eya1~(bor／bor)的第二代杂交小鼠的ABR值和耳蜗周数的变化为指标初步定位Eya1基因的遗传修饰基因
[目的]
初步定位Eya1基因的遗传修饰基因位点。
[方法]
分别以267只和178只基因型为Eya1~(bor／bor)的第二代杂交小鼠为研究对象，剪取小鼠小块耳阔消化后提取小鼠基因组DNA，用染料标记的简单序列长度多态性标记(dye-labeled SSLP marker)对基因组DNA进行检测，以代表小鼠基因型，标记间距约为25cM，总共选取98个标记位点。将ABR值划分为14个等级，对每只小鼠进行ABR检测确定其听力等级。运用Map Manager QTXb20软件对每个标记位点与其听力变化的相关性进行QTL分析。解剖取出小鼠内耳，记录其耳蜗周数，运用MapManager QTXb20软件对每个标记位点与其耳蜗周数变化的相关性进行QTL分析。
[结果]
1．C3HeB／FeJ×C57BL／6J F2 52％(139／267)的F2-Eya1~(bor／bor)至少有一只耳朵的ABR值小于90db，其听力变化范围为35-90db。QTL分析得到2组标记与小鼠听力变化相关联，位于4号染色体上的标记D4Mit104与小鼠听力变化的相关性最强，其LOD值(logarithm ofthe odds oflinkage，LOD)为12．0，能解释其听力变化的19％。本课题组将该标记指示的位点命名为Mead1(Modifier of Eya1-associated deafness1)。位于12号染色体上的标记D12Mit283与小鼠听力变化的相关性强，其LOD值为12．1，能解释其听力变化的19％，该位点命名为Mead2。记录的89只F2的耳蜗周数变化范围从0周到1 3/4周，与耳蜗周数变化相关性强的两个标记也是D4Mit104和D12Mit283，其中D4Mit104的LOD值分别是15．8，能解释其耳蜗周数变化的58％。D12Mit283的LOD值是3．3，能解释耳蜗周数变化的16％。
2．C3HeB／FeJ×CAST／EiJ F2 86％(153／178)的F2-Eya1~(bor／bor)至少有一只耳朵的ABR值小于90db，其听力变化范围在90dB-30dB，QTL分析没有发现其听力变化与任何标记位点有明显的相关性。
[结论]
1．Mead1和Mead2两个位点内有基因对Eya1~(bor／bor)基因具有修饰作用，这两个位点可以提高Eya1~(bor／bor)基因型小鼠的听力并且可以促进小鼠耳蜗发育。
2．C3HeB／FeJ×CAST／EiJ F2可能有多种因子发挥调节作用或者以互交的方式得到的F2的表型与基因型的相关性不强，所以用回交的方式培育C3HeB／FeJ×CAST／EiJF2，再进行听力变化和耳蜗周数变化与小鼠基因型的相关性分析。
第三部分回交培育C3HeB／FeJ×CAST／EiJ第二代杂交小鼠N2定位Eya1基因的遗传修饰基因
[目的]
初步定位Eya1基因的遗传修饰基因位点。
[方法]
随机选取C3HeB／FeJ-Eya1~(bor／+)×CAST／EiJ小鼠第一代杂交小鼠F1 35只与C3HeB／FeJ-Eya1~(bor／+)小鼠回交得到Eya1~(bor／bor)基因型的第二代杂交小鼠N2，对N2-Eya1~(bor／bor)进行基因型的检测，听力等级的测定和记录耳蜗周数，QTL分析表型与基因型的相关性。方法同本文第二部分。
[结果]
1．C3HeB／FeJ×CAST／EiJ N2共909只，基因型为Eya1~(bor／bor)N2 179只，Eya1~(bor／+)N2470只，Eya1~(+／+)N2 260只。
2．62％(112／179)的N2至少有一只耳朵的ABR值小于90 dB，其听力变化范围在90 dB-40 dB。根据第二部分的结果，44％(67／153)F2的ABR值(?)50 dB，而只有17％N2 ABR值(?)50 dB。通过QTL分析共发现4组标记与之关联，标记D4Mit149与N2的听力变化关联性最强，LOD值为8．4，可解释听力变化的19％，该位点与Mead1位点重合；标记D19Mit28与其也有很强的关联性，LOD值为8．1，可解释听力变化的19％；标记D9Mit126和D15Mit242也与听力变化相关联，LOD值分别为2．4和2．7，分别解释听力变化的6％和7％。记录89只N2耳蜗周数，发现3组标记与耳蜗周数相关联，标记D4Mit104与耳蜗周数的关联性最强，LOD值为4．5，可解释耳蜗周数变化的21％，该位点与Mead1位点重合；标记D12Mit218与其关联的LOD值为2．8，可解释耳蜗周数变化的14％，该位点与Mead2位点重合；标记D14Mit174与其关联的LOD值为3．3，可解释耳蜗周数变化的16％。
[结论]
1．标记D4Mit149和标记D4Mit104是4号染色体上相邻的两个遗传学标记，说明C57BL-／6J和CAST／EiJ这两个不同鼠种在Mead1位置有相同或者同系列基因对Eya1基因发挥调节作用法，该位点能提高小鼠听力和促进耳蜗发育。
2．标记D12Mit218和标记D12Mit283是12号染色体上相邻的两个遗传学标记，说明C57BL／6J和CAST／EiJ这两个不同鼠种在Mead2位置有相同或者同系列基因对Eya1基因发挥调节作用，其作用主要是促进耳蜗发育。
3．命名标记D19Mit28所指代的位置为Mead3，已经报道的Eya1调节基因Nxf1位于该范围内。
第四部分建立同类系进一步精确定位遗传修饰基因位点Mead1和Mead2位置
[目的]
分别建立以C3HeB／FeJ小鼠的基因组DNA为基因背景，在Mead1和Mead2位点被C57BL／6J小鼠基因替代的同类系各一个，进一步缩小位点Mead1和Mead2的范围
[方法]
Mead1位点为C57BL／6J的C3Fe．B6同类系小鼠的培育：C3HeB／FeJ×C57BL／6J F1与C3HeB／FeJ-Eya1~(bor／+)回交，再将得到的基因型为Eya1~(bor／+)N2用染料标记的简单序列长度多态性标记进行基因组DNA检测，选择2—3只基因型为Eya1~(bor／+)，Mead1位点为杂合位点，而其他位点为杂合位点数目最少的雄性小鼠为种鼠，与其父代回交进行下一代的繁殖，同类系在经过五代繁殖之后建立。本课题选择第五代同类系小鼠作为试验对象。Mead2位点为C57BL／6J的C3Fe．B6同类系小鼠的培育方法同上。对所有Eya1~(bor／bor)基因型小鼠均进行基因组DNA检测，并计取所有的同类系小鼠的耳蜗周数。
[结果]
1．对每一代Eya1~(bor／bor)杂交小鼠进行听力检测发现，随着C57BL／6J小鼠基因在杂交小鼠基因组内的减少，Mead1和Mead2位点抑制Eya1~(bor／bor)基因型杂交小鼠听力障碍的功能逐渐减弱。
2．当Eya1~(bor／bor)杂交小鼠在标记D4Mit149和D4Mit193(Mead1位点内)之间为C57BL／6J和C3HeB／FeJ等位基因杂合时，Eya1~(bor／bor)基因型小鼠(14只)持续存在有1/2-3/4圈耳蜗；当其在该位置为C57BL／6J等位基因纯合时，Eya1~(bor／bor)基因型小鼠(11只)持续存在有1-1 1/4圈耳蜗。
3．当Eya1~(bor／bor)杂交小鼠在标记D12Mit103和D12Mit172(Mead2位点内)之间为C57BL／6J和C3HeB／FeJ等位基因杂合时，只有6％(4／25)Eya1~(bor／bor)基因型杂合小鼠持续存在有多于1/4圈耳蜗，其余没有耳蜗发育；当其在该位置为C57BL／6J等位基因纯合时，所有Eya1~(bor／bor)基因型小鼠持续存在有1／2-3／4圈耳蜗。
[结论]
1．Mead1和Mead2位点均进行孟德尔半显性遗传方式遗传，Mead1位点具有完全外显性；Mead2位点杂合时表现为不完全外显性，其外显率为16％；位点为纯和时，则表现为完全外显性。
2．通过对建立同类系过程中Mead1位点为等位基因杂合的Eya1~(bor／bor)基因型小鼠的耳蜗与Mead1位点内更多的遗传标记位点关联，将Mead1位点缩小到标记D4Mit227和D4Mit171之间，大小为5．4cM。
3．通过对建立同类系过程中Mead2位点为等位基因纯和的Eya1~(bor／bor)基因型小鼠的耳蜗与Mead2位点内更多的标记位点的关联，将Mead2位点缩小到标记D12Mit56和D12Mit283之间，大小为4．4cM。
第五部分建立C3Fe．CAST和C3Fe．BALA同类系验证Mead1，Mead2和Mead3位点
[目的]
通过建立C3Fe．CAST和C3Fe．BALA两种同类系以验证并分析Mead1，Mead2和Mead3位点的作用。
[方法]
建立C3Fe．CAST~(mead1／mead1)同类系1个，C3Fe．CAST~(mead2／mead2)同类系1个，C3Fe．CAST~(mead3／mead3)同类系3个；建立C3Fe．BALA~(mead1／mead1)同类系1个，C3Fe．CAST~(mead2／mead2)同类系2个。方法同第四部分。
[结果]
1．C3Fe．CAST~(mead1／mead1)同类系：13只C3Fe．CAST~(Mead1／Mead1)-Eya1~(bor／bor)小鼠都有耳蜗发育，范围为1／2-3／4周，一只为1／4周，与C3Fe．B6~(Mead1／Mead1)-Eya1~(bor／bor)小鼠耳蜗周数为1-11／4周结果不同。
2．C3Fe．CAST~(mead2／mead2)同类系：4只C3Fe．CAST~(Mead2／Mead2)-Eya1~(bor／bor)小鼠均有耳蜗发育，范围为1／2-3／4周，与C3Fe．B6~(Mead2／Mead2)-Eya1~(bor／bor)小鼠耳蜗周数基本相同。
3．3个C3Fe．CAST~(mead3／mead3)同类系：3个同类系表现出不同的外显率，同类系501得到C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠14只，其中只有8只(57％)小鼠有耳蜗发育，范围为3／4-1周；同类系504最后得到的18只C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠中8只(44％)有耳蜗发育，范围为1／4-1 1／4周；同类系505最后得到16只C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠中8只(50％)有耳蜗发育，范围为1／4-1／2周。
4．C3Fe．BALA~(mead1／mead1)同类系(Mead1位点距小鼠4号染色体着丝粒5-10cM)：C3Fe．BALA~(Mead1／Mead1)-Eya1~(bor／bor)小鼠4只都有接近1 1/2周耳蜗发育，与C3Fe．B6~(Mead1／Mead1)-Eya1~(bor／bor)小鼠耳蜗周数为1-1 1/4周结果相近。
5．2个C3Fe．BALA~(mead2／mead2)同类系(Mead2位点距小鼠12号染色体着丝粒约10cM)：C3Fe．BALA~(Mead2／Mead2)-Eya1~(bor／bor)小鼠共7只均见耳蜗发育，范围为1／2-1 3／4，变化范围较大，与C3Fe．B6~(Mead2／Mead2)-Eya1~(bor／bor)小鼠耳蜗周数不同。
[结论]
各个种属和各个位点的保护性等位基因的外显性和抑制作用有不同。这些不同表现在C3Fe．CAST~(Mead1／Mead1)-Eya1~(bor／bor)小鼠和C3Fe．B6~(Mead1／Mead1)-Eya1~(bor／bor)小鼠耳蜗周数不同。这说明CAST／EiJ和C57BL／6这两个鼠种在Mead1位点的发挥抑制性的作用不同。Mead1和Mead2位点对突变小鼠耳蜗发育不全的抑制作用与Mead3位点有很大不同。当三种保护性等位基因(CAST／EiJ，C57BL／6J，BALA／cJ)在Mead1和Mead2位点纯合时，对耳蜗发育不全的抑制作用表现出完全外显性；而在Mead3位置，C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠存在不完全外显性(50％)，以及广泛的耳蜗周数变化。
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 crosses
Objective:
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/bor)F2 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 turns
Objective:
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/bor)exhibited 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 N2
Objective:
To map genetic modifier loci of Eya1~(bor/bor) in C3HeB/FeJ×CAST/EiJ N2
Methods:
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/bor)hypomorphs (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 strains
Objective:
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 strains
Objective:
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 part
4.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/bor)congenic 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/bor)mice 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/bor)mice 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).
更进一步的研究发现，Eya1~(bor／bor)突变鼠的基因表现度，例如产后死亡率和听力障碍的程度，随着小鼠基因背景的不同而存在着很大的差异，所以人们推测在某些鼠种的基因组里存在着Eya1基因的遗传修饰基因。JenniferA．Floyd等人用C3HeB／FeJ小鼠与CAST／EiJ小鼠杂交，得到Eya1~(bor／bor)基因型的后代，再通过数量性状基因位点(Quantitative trait loci，QTL)分析，发现了一个调节基因Nxf1(nuclear export factor)可以减少带有Eya1~(bor／bor)基因型的杂交小鼠的死亡率和听力障碍程度，并且发现Nxf1基因是通过增加正常Eya1 mRNA的量来发挥作用。那么Eya1基因是否还存在其他的遗传修饰基因?其他的遗传修饰基因又如何对Eya1基因进行调节?
本课题以C3HeB／FeJ-Eya1~(bor/+)小鼠与C57BL／6J小鼠，CAST／EiJ小鼠，以及BALB／cJ小鼠杂交而得到的三种不同基因背景小鼠为研究对象，对各种杂交小鼠基因组的基因型进行检测。选择小鼠ABR值和耳蜗周数的变化作为BOR综合征表型的2个指标，运用QTL分析将基因型与表型进行关联分析，再建立小鼠同类系进行精确定位的方法来定位Eya1基因的遗传修饰基因。本课题为克隆Eya1~(bor／bor)基因的遗传修饰基因和研究各个基因的功能奠定基础，并且为研究人类综合征型耳聋的调节基因提供思路。本文共分五个部分。
第一部分C3HeB／FeJ-Eya1~(bor／+)小鼠与C57BL／6J小鼠，CAST／EiJ小鼠第二代杂交小鼠的培育
[目的]
培育具有不同基因背景的杂交小鼠，为研究Eya1基因的遗传修饰基因奠定基础。
[方法]
用C3HeB／FeJ-Eya1~(bor／+)小鼠分别与C57BL／6J小鼠，CAST／EiJ小鼠杂交得到两种不同基因背景的第一代杂交小鼠(F1)，再通过互交(intercross)得到第二代杂交小鼠(F2)，通过PCR检测所有F2的Eya1基因位点的基因型。
[结果]
C3HeB／FeJ-Eya1~(bor／+)×C57BL／6J F2共2255只，其中基因型为Eya1~(bor／bor)F2 267只，Eya1~(bor／+)F2 1342只，Eya1~(+／+)F2 646只；C3HeB／FeJ-Eya1~(bor／+)×CAST／EiJ F2共1000只，其中基因型为Eya1~(bor／bor)F2 178只，Eya1~(bor／+)F2 570只，Eya1~(+／+)F2 252只。
[结论]
部分基因型为Eya1~(bor／bor)F2出现明程度不等地点头兜圈等症状，部分基因型为Eya1~(bor／bor)F2的症状并不明显，说明C57BL／6J小鼠和CAST／EiJ小鼠的某些基因位点对Eya1~(bor／bor)F2的耳部发育发挥了作用。
第二部分以基因型为Eya1~(bor／bor)的第二代杂交小鼠的ABR值和耳蜗周数的变化为指标初步定位Eya1基因的遗传修饰基因
[目的]
初步定位Eya1基因的遗传修饰基因位点。
[方法]
分别以267只和178只基因型为Eya1~(bor／bor)的第二代杂交小鼠为研究对象，剪取小鼠小块耳阔消化后提取小鼠基因组DNA，用染料标记的简单序列长度多态性标记(dye-labeled SSLP marker)对基因组DNA进行检测，以代表小鼠基因型，标记间距约为25cM，总共选取98个标记位点。将ABR值划分为14个等级，对每只小鼠进行ABR检测确定其听力等级。运用Map Manager QTXb20软件对每个标记位点与其听力变化的相关性进行QTL分析。解剖取出小鼠内耳，记录其耳蜗周数，运用MapManager QTXb20软件对每个标记位点与其耳蜗周数变化的相关性进行QTL分析。
[结果]
1．C3HeB／FeJ×C57BL／6J F2 52％(139／267)的F2-Eya1~(bor／bor)至少有一只耳朵的ABR值小于90db，其听力变化范围为35-90db。QTL分析得到2组标记与小鼠听力变化相关联，位于4号染色体上的标记D4Mit104与小鼠听力变化的相关性最强，其LOD值(logarithm ofthe odds oflinkage，LOD)为12．0，能解释其听力变化的19％。本课题组将该标记指示的位点命名为Mead1(Modifier of Eya1-associated deafness1)。位于12号染色体上的标记D12Mit283与小鼠听力变化的相关性强，其LOD值为12．1，能解释其听力变化的19％，该位点命名为Mead2。记录的89只F2的耳蜗周数变化范围从0周到1 3/4周，与耳蜗周数变化相关性强的两个标记也是D4Mit104和D12Mit283，其中D4Mit104的LOD值分别是15．8，能解释其耳蜗周数变化的58％。D12Mit283的LOD值是3．3，能解释耳蜗周数变化的16％。
2．C3HeB／FeJ×CAST／EiJ F2 86％(153／178)的F2-Eya1~(bor／bor)至少有一只耳朵的ABR值小于90db，其听力变化范围在90dB-30dB，QTL分析没有发现其听力变化与任何标记位点有明显的相关性。
[结论]
1．Mead1和Mead2两个位点内有基因对Eya1~(bor／bor)基因具有修饰作用，这两个位点可以提高Eya1~(bor／bor)基因型小鼠的听力并且可以促进小鼠耳蜗发育。
2．C3HeB／FeJ×CAST／EiJ F2可能有多种因子发挥调节作用或者以互交的方式得到的F2的表型与基因型的相关性不强，所以用回交的方式培育C3HeB／FeJ×CAST／EiJF2，再进行听力变化和耳蜗周数变化与小鼠基因型的相关性分析。
第三部分回交培育C3HeB／FeJ×CAST／EiJ第二代杂交小鼠N2定位Eya1基因的遗传修饰基因
[目的]
初步定位Eya1基因的遗传修饰基因位点。
[方法]
随机选取C3HeB／FeJ-Eya1~(bor／+)×CAST／EiJ小鼠第一代杂交小鼠F1 35只与C3HeB／FeJ-Eya1~(bor／+)小鼠回交得到Eya1~(bor／bor)基因型的第二代杂交小鼠N2，对N2-Eya1~(bor／bor)进行基因型的检测，听力等级的测定和记录耳蜗周数，QTL分析表型与基因型的相关性。方法同本文第二部分。
[结果]
1．C3HeB／FeJ×CAST／EiJ N2共909只，基因型为Eya1~(bor／bor)N2 179只，Eya1~(bor／+)N2470只，Eya1~(+／+)N2 260只。
2．62％(112／179)的N2至少有一只耳朵的ABR值小于90 dB，其听力变化范围在90 dB-40 dB。根据第二部分的结果，44％(67／153)F2的ABR值(?)50 dB，而只有17％N2 ABR值(?)50 dB。通过QTL分析共发现4组标记与之关联，标记D4Mit149与N2的听力变化关联性最强，LOD值为8．4，可解释听力变化的19％，该位点与Mead1位点重合；标记D19Mit28与其也有很强的关联性，LOD值为8．1，可解释听力变化的19％；标记D9Mit126和D15Mit242也与听力变化相关联，LOD值分别为2．4和2．7，分别解释听力变化的6％和7％。记录89只N2耳蜗周数，发现3组标记与耳蜗周数相关联，标记D4Mit104与耳蜗周数的关联性最强，LOD值为4．5，可解释耳蜗周数变化的21％，该位点与Mead1位点重合；标记D12Mit218与其关联的LOD值为2．8，可解释耳蜗周数变化的14％，该位点与Mead2位点重合；标记D14Mit174与其关联的LOD值为3．3，可解释耳蜗周数变化的16％。
[结论]
1．标记D4Mit149和标记D4Mit104是4号染色体上相邻的两个遗传学标记，说明C57BL-／6J和CAST／EiJ这两个不同鼠种在Mead1位置有相同或者同系列基因对Eya1基因发挥调节作用法，该位点能提高小鼠听力和促进耳蜗发育。
2．标记D12Mit218和标记D12Mit283是12号染色体上相邻的两个遗传学标记，说明C57BL／6J和CAST／EiJ这两个不同鼠种在Mead2位置有相同或者同系列基因对Eya1基因发挥调节作用，其作用主要是促进耳蜗发育。
3．命名标记D19Mit28所指代的位置为Mead3，已经报道的Eya1调节基因Nxf1位于该范围内。
第四部分建立同类系进一步精确定位遗传修饰基因位点Mead1和Mead2位置
[目的]
分别建立以C3HeB／FeJ小鼠的基因组DNA为基因背景，在Mead1和Mead2位点被C57BL／6J小鼠基因替代的同类系各一个，进一步缩小位点Mead1和Mead2的范围
[方法]
Mead1位点为C57BL／6J的C3Fe．B6同类系小鼠的培育：C3HeB／FeJ×C57BL／6J F1与C3HeB／FeJ-Eya1~(bor／+)回交，再将得到的基因型为Eya1~(bor／+)N2用染料标记的简单序列长度多态性标记进行基因组DNA检测，选择2—3只基因型为Eya1~(bor／+)，Mead1位点为杂合位点，而其他位点为杂合位点数目最少的雄性小鼠为种鼠，与其父代回交进行下一代的繁殖，同类系在经过五代繁殖之后建立。本课题选择第五代同类系小鼠作为试验对象。Mead2位点为C57BL／6J的C3Fe．B6同类系小鼠的培育方法同上。对所有Eya1~(bor／bor)基因型小鼠均进行基因组DNA检测，并计取所有的同类系小鼠的耳蜗周数。
[结果]
1．对每一代Eya1~(bor／bor)杂交小鼠进行听力检测发现，随着C57BL／6J小鼠基因在杂交小鼠基因组内的减少，Mead1和Mead2位点抑制Eya1~(bor／bor)基因型杂交小鼠听力障碍的功能逐渐减弱。
2．当Eya1~(bor／bor)杂交小鼠在标记D4Mit149和D4Mit193(Mead1位点内)之间为C57BL／6J和C3HeB／FeJ等位基因杂合时，Eya1~(bor／bor)基因型小鼠(14只)持续存在有1/2-3/4圈耳蜗；当其在该位置为C57BL／6J等位基因纯合时，Eya1~(bor／bor)基因型小鼠(11只)持续存在有1-1 1/4圈耳蜗。
3．当Eya1~(bor／bor)杂交小鼠在标记D12Mit103和D12Mit172(Mead2位点内)之间为C57BL／6J和C3HeB／FeJ等位基因杂合时，只有6％(4／25)Eya1~(bor／bor)基因型杂合小鼠持续存在有多于1/4圈耳蜗，其余没有耳蜗发育；当其在该位置为C57BL／6J等位基因纯合时，所有Eya1~(bor／bor)基因型小鼠持续存在有1／2-3／4圈耳蜗。
[结论]
1．Mead1和Mead2位点均进行孟德尔半显性遗传方式遗传，Mead1位点具有完全外显性；Mead2位点杂合时表现为不完全外显性，其外显率为16％；位点为纯和时，则表现为完全外显性。
2．通过对建立同类系过程中Mead1位点为等位基因杂合的Eya1~(bor／bor)基因型小鼠的耳蜗与Mead1位点内更多的遗传标记位点关联，将Mead1位点缩小到标记D4Mit227和D4Mit171之间，大小为5．4cM。
3．通过对建立同类系过程中Mead2位点为等位基因纯和的Eya1~(bor／bor)基因型小鼠的耳蜗与Mead2位点内更多的标记位点的关联，将Mead2位点缩小到标记D12Mit56和D12Mit283之间，大小为4．4cM。
第五部分建立C3Fe．CAST和C3Fe．BALA同类系验证Mead1，Mead2和Mead3位点
[目的]
通过建立C3Fe．CAST和C3Fe．BALA两种同类系以验证并分析Mead1，Mead2和Mead3位点的作用。
[方法]
建立C3Fe．CAST~(mead1／mead1)同类系1个，C3Fe．CAST~(mead2／mead2)同类系1个，C3Fe．CAST~(mead3／mead3)同类系3个；建立C3Fe．BALA~(mead1／mead1)同类系1个，C3Fe．CAST~(mead2／mead2)同类系2个。方法同第四部分。
[结果]
1．C3Fe．CAST~(mead1／mead1)同类系：13只C3Fe．CAST~(Mead1／Mead1)-Eya1~(bor／bor)小鼠都有耳蜗发育，范围为1／2-3／4周，一只为1／4周，与C3Fe．B6~(Mead1／Mead1)-Eya1~(bor／bor)小鼠耳蜗周数为1-11／4周结果不同。
2．C3Fe．CAST~(mead2／mead2)同类系：4只C3Fe．CAST~(Mead2／Mead2)-Eya1~(bor／bor)小鼠均有耳蜗发育，范围为1／2-3／4周，与C3Fe．B6~(Mead2／Mead2)-Eya1~(bor／bor)小鼠耳蜗周数基本相同。
3．3个C3Fe．CAST~(mead3／mead3)同类系：3个同类系表现出不同的外显率，同类系501得到C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠14只，其中只有8只(57％)小鼠有耳蜗发育，范围为3／4-1周；同类系504最后得到的18只C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠中8只(44％)有耳蜗发育，范围为1／4-1 1／4周；同类系505最后得到16只C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠中8只(50％)有耳蜗发育，范围为1／4-1／2周。
4．C3Fe．BALA~(mead1／mead1)同类系(Mead1位点距小鼠4号染色体着丝粒5-10cM)：C3Fe．BALA~(Mead1／Mead1)-Eya1~(bor／bor)小鼠4只都有接近1 1/2周耳蜗发育，与C3Fe．B6~(Mead1／Mead1)-Eya1~(bor／bor)小鼠耳蜗周数为1-1 1/4周结果相近。
5．2个C3Fe．BALA~(mead2／mead2)同类系(Mead2位点距小鼠12号染色体着丝粒约10cM)：C3Fe．BALA~(Mead2／Mead2)-Eya1~(bor／bor)小鼠共7只均见耳蜗发育，范围为1／2-1 3／4，变化范围较大，与C3Fe．B6~(Mead2／Mead2)-Eya1~(bor／bor)小鼠耳蜗周数不同。
[结论]
各个种属和各个位点的保护性等位基因的外显性和抑制作用有不同。这些不同表现在C3Fe．CAST~(Mead1／Mead1)-Eya1~(bor／bor)小鼠和C3Fe．B6~(Mead1／Mead1)-Eya1~(bor／bor)小鼠耳蜗周数不同。这说明CAST／EiJ和C57BL／6这两个鼠种在Mead1位点的发挥抑制性的作用不同。Mead1和Mead2位点对突变小鼠耳蜗发育不全的抑制作用与Mead3位点有很大不同。当三种保护性等位基因(CAST／EiJ，C57BL／6J，BALA／cJ)在Mead1和Mead2位点纯合时，对耳蜗发育不全的抑制作用表现出完全外显性；而在Mead3位置，C3Fe．CAST~(Mead3／Mead3)-Eya1~(bor／bor)小鼠存在不完全外显性(50％)，以及广泛的耳蜗周数变化。
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 crosses
Objective:
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/bor)F2 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 turns
Objective:
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/bor)exhibited 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 N2
Objective:
To map genetic modifier loci of Eya1~(bor/bor) in C3HeB/FeJ×CAST/EiJ N2
Methods:
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/bor)hypomorphs (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 strains
Objective:
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 strains
Objective:
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 part
4.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/bor)congenic 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/bor)mice 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/bor)mice 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).
引文
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2.Melnick M, Bixler D, Nance WE, Silk K, et al. Autosomal dominant branchiootorenal dysplasia: Anew addition to the branchial arch syndromes. Clin Genet 1975(7)25-34.
3.Fraser F.C, Ling D, Clogg D, et al. Genetic aspects of the BOR symdrome-branchial fistulas, ear pits, hearing loss, and renal anomalies. Am J Med Genet. 1978(2)241-252.
4.Chen A, Francis M, Ni L, et al. Phenotypic manifestations of branchiootorenal syndrome. Am J Med Genet. 1995(58)365-370.
5.Amit Kochhar, Stephanie M. Fischer, William J. Kimberling, et al. Branchio-Oto-Renal Symdrome. Am J Med Genet. 2007(143A)1671-1678.
6.Brown S.D, Steel K.P. Genetic deafness-progress with mouse models. Hum Mol Genet. 1994(3)1453-1456.
7.Steel K.P. Inherited hearing defects in mice. Annu Rev Genet. 1995(29)675-701.
8.Kenneth R.Johnson, Susan A. Cook, Lawrence C. Erway, et al. Inner ear and kidney anomalies caused by LAP insertion in an intron of the Eyal gene in a mouse model of BOR sundrome. Hum Mol Genet. 1999(8)645-653.
9.Xu P X, Woo I, Her H, et al. Mouse Eya homologues of the Drosophila eyes absent gene require Pax6 for expression in lens and nasal placode Development. 1997(124)219-231.
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