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Human Molecular Genetics Advance Access originally published online on April 20, 2005
Human Molecular Genetics 2005 14(11):1549-1558; doi:10.1093/hmg/ddi163
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Genome-wide linkage identifies novel modifier loci of aganglionosis in the Sox10Dom model of Hirschsprung disease

Sarah E. Owens1, Karl W. Broman2, Tim Wiltshire3, J. Bradford Elmore1, Kevin M. Bradley1, Jeffrey R. Smith1 and E. Michelle Southard-Smith1,*

1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, 529 Light Hall, 2215 Garland Avenue, Nashville, TN 37232-0275, USA, 2Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, 615 N Wolfe Street, Baltimore, MD 21205-2179, USA and 3Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA

* To whom correspondence should be addressed. Tel: +1 6159362172; Fax: +1 6159362661; Email: michelle.southard-smith{at}vanderbilt.edu

Received February 17, 2005; Accepted April 12, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Hirschsprung disease (HSCR) is a complex disorder that exhibits incomplete penetrance and variable expressivity due to interactions among multiple susceptibility genes. Studies in HSCR families have identified RET-dependent modifiers for short-segment HSCR (S-HSCR), but epistatic effects in long-segment (L-HSCR) and syndromic cases have not been fully explained. SOX10 mutations contribute to syndromic HSCR cases and Sox10 alleles in mice exhibit aganglionosis and pigmentary anomalies typical of a subset of HSCR patients categorized as Waardenburg–Shah syndrome (WS4, OMIM 277580 [OMIM] ). Sox10 mutant alleles in mice exhibit strain-dependent variation in penetrance and expressivity of aganglionic megacolon analogous to the variation observed in patients with aganglionosis. In this study, we focused on enteric ganglia deficits in Sox10Dom mice and defined aganglionosis as a quantitative trait in Sox10Dom intercross progeny to investigate the contribution of strain background to variation in enteric nervous system deficits. We observe that the phenotype of Sox10Dom/+ mutants ranges over a continuum from severe aganglionosis to no detectable phenotype in the gut. To systematically identify genes that modulate Sox10-dependent aganglionosis, we performed a single nucleotide polymorphism-based genome scan in Sox10Dom/+ F1 intercross progeny. Our analysis reveals modifier loci on mouse chromosomes 3, 5, 8, 11 and 14 with distinct effects on penetrance and severity of aganglionosis. Three of these loci on chromosomes 3, 8 and 11 do not coincide with previously known aganglionosis susceptibility genes or modifier loci and offer new avenues for elucidating the genetic network that modulates this complex neurocristopathy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Phenotypes that display non-mendelian inheritance have challenged geneticists to define the genes and mechanisms that contribute to complex disease. Hirschsprung disease (HSCR) is a well-recognized complex disorder characterized by multigenic inheritance, with minimally nine genes (RET, GDNF, NTN, EDN3, EDNRB, ECE-1, SOX10, ZFHX1B and PHOX2B) contributing to aganglionosis susceptibility in patients (1Go,2Go). HSCR is the most common cause of neonatal intestinal obstruction, with a general population incidence of one in 5000 live births. The disorder is clinically identified by the absence of enteric ganglia in a variable portion of the distal gastrointestinal tract. To facilitate genetic analysis, aganglionosis phenotypes in HSCR patients have been subdivided based on the extent of the affected gut segment. Long-segment HSCR (L-HSCR) includes patients with aganglionosis of, and proximal to, the splenic flexure, whereas the remaining cases are grouped as short-segment (S-HSCR) (1Go,3Go). Segregation analysis among S-HSCR patients suggests distinct models of segregation with multifactorial or recessive inheritance in effect for S-HSCR, whereas rarer dominant allele models are more consistent with L-HSCR (3Go). In the cases of isolated HSCR, where aganglionosis is the only documented neural crest defect, RET mutations are the predominant genetic alteration. Syndromic HSCR, aganglionosis in the context of additional neural crest phenotypes, appears to be the consequence of genetic alteration in genes that are more broadly expressed across neural crest lineages. Regardless of the categorical classification of aganglionosis phenotype (S-HSCR versus L-HSCR; isolated HSCR versus syndromic HSCR), the variable penetrance and expressivity of this disease are attributed to complex genetic interactions between susceptibility loci and undiscovered modifiers in the genetic background that predispose to deficiencies of enteric neural crest (4Go–6Go).

Efforts to identify the gene interactions that contribute to phenotypic variation of aganglionic megacolon have focused on the RET locus and identified modifiers at 3p21, 9q31, 16q23 and 19q12 (4Go,7Go). These loci are hypothesized to result in misregulation of RET. Studies in HSCR families and mouse models demonstrate that interactions between RET and EDNRB also impact disease susceptibility (5Go,6Go). Recent molecular and genetic analyses in mice have expanded the gene network to include Ednrb–Sox10 among the interactions that modulate neural crest cells during enteric nervous system (ENS) ontogeny (8Go,9Go). Although in vitro studies have suggested that Sox10 is capable of mediating transcriptional regulation of Ret (10Go), Sox10 may also impact Ret via its interaction with Ednrb. Strategies to elucidate the gene interactions that contribute to variation in penetrance and severity of aganglionosis continue to be of great interest because mutations are seldom documented in >30% of total HSCR cases (1Go).

SOX10 is an essential neural crest transcription factor that is altered in some syndromic HSCR patients (11Go–13Go) and in Dominant megacolon (Sox10Dom) mice (14Go,15Go). Patients with SOX10 alterations typically present with syndromic HSCR defined as aganglionosis associated with other deficits: hypopigmentation, deafness, iridia, nystagmus or ataxia. Sox10Dom mice exhibit strain-dependent variation in aganglionosis penetrance and severity analogous to the heterogeneous enteric phenotypes of HSCR patients. This study focuses on the variation in aganglionosis between individual Sox10Dom mice and uses this variation to identify modifiers that are influencing the aganglionosis aspect of the phenotype. These studies have been facilitated by the ability to control genetic background in inbred lines of Sox10Dom mice that are not possible in patient studies. Previous candidate gene analysis has identified contributions of alleles at Ednrb to the variation among individual Sox10Dom animals. Yet, only a minor proportion of aganglionosis variance in Sox10Dom mice is attributable to the Ednrb locus, and the phenotype of Sox10/Ednrb double mutants is significantly different on distinct genetic backgrounds (8Go), implying the existence of additional modifier loci.

The variation in HSCR phenotype and potential number of genes that influence development of the ENS are not surprising when considered in the context of the complex biological processes that neural crest cells endure to populate the gastrointestinal tract. These cells must emigrate from the neural tube, traffic to the foregut, and migrate throughout the intestine, all the while proliferating, and differentiating to give rise to the glia and neurons of the enteric ganglia. Interestingly, assays of gene expression in enteric neural crest stem cells (NCSC) have established that genes that play a role in aganglionosis susceptibility are highly expressed in these cells and suggest that HSCR is a consequence of NCSC dysfunction (16Go). Sox10 is among the genes upregulated in NSCS and maintains both glial and neuronal stem cell potential in the neural crest (17Go). Consequently, modifiers of Sox10 are likely to be relevant to neurobiology of enteric stem cells and should further elucidate interacting pathways that are essential for normal development of enteric ganglia.

Candidate gene approaches can be informative, but they are typically based on the biological processes under investigation and are often limited by complete knowledge of all components in a pathway. In contrast, genome-wide linkage studies have the potential to identify essential genes in complex diseases that otherwise would be missed by candidate gene studies (18Go,19Go). In this report, we describe a genome-wide survey performed to comprehensively identify Sox10 modifier loci that affect the penetrance and extent of intestinal aganglionosis. Our analysis reveals modifier loci on mouse chromosomes 3, 5, 8, 11 and 14 that have distinct effects on the penetrance and severity of aganglionosis. Fine-mapping in the modifier interval on chromosome 5, coupled with bioinformatics analysis of genes in the interval, identifies several candidates that are highly relevant based on their expression in enteric neural crest. Our analysis is consistent with the hypothesis that the Sox10Dom phenotype is modulated by loci in addition to Ednrb. This study contributes significantly to the epistatic network that modulates ENS deficits and will expand our understanding of neural development in the gut.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Evaluation of aganglionosis as a quantitative trait in Sox10Dom/+ F2 mice
To establish the range of phenotypes among Sox10Dom/+ F1 intercross progeny, we evaluated the extent of aganglionosis in postnatal day 7–10 (P7–P10) F2 pups by whole-mount acetylcholinesterase (AChE) histochemistry. AChE staining detects cholinergic neurons in the ENS and is a rapid, high definition method for visualizing ganglia architecture. This technique is advantageous over sectioning because it preserves the three-dimensional organization of the intestinal tissue. All F2 animals (n=2210) were phenotyped and then genotyped for the Sox10Dom mutation, with subsequent analyses focusing on Sox10Dom/+ mutants. Genotyping results were consistent with the anticipated 50% transmission ratio, identified 1092 Sox10Dom/+ mice, and confirmed the absence of enteric deficits in all pups that had wild-type genotype. Microscopic examination of Sox10Dom/+ intestines stained by AChE allowed us to score the extent of gut length affected by hypo- and/or aganglionosis in individual animals as a quantitative trait. Consequently, the ready identification of individual mice at the extremes of the phenotype distribution allowed selective genotyping of Sox10Dom/+ animals based either on penetrance (no hypo- or aganglionosis present) (Fig. 1B) or on severity (extent of gut length affected) in the F2 phenotype distribution (Fig. 1A and B).



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  		Figure 1. Evaluation of aganglionosis as a quantitative trait in Sox10Dom/+ F2 mice. Phenotype distribution of aganglionosis (A) and total length of the gut affected (B) by ENS deficits in Sox10Dom/+ F2 mice. Numbers of mice (y-axis) in each phenotype class defined in 2.5% gut length increments are plotted along the x-axis. Bracket in (A) identifies the most severely aganglionic 10% of the phenotype distribution (n=109) selected for genotyping. Plot of total affected phenotype in (B) includes both hypoganglionosis and aganglionosis. Animals representing the mildest 10% affected mice (M, n=107) and the non-penetrant animals (asterisk; phenotype of 0% gut length affected, n=107) were selected for genotyping.

 
Genome-wide linkage analysis identifies modifiers of Sox10Dom aganglionosis penetrance and severity
Selective genotyping of individual animals at the extremes of the phenotype distribution increases the power to detect quantitative trait loci (20Go,21Go). We used this approach in our genome survey and determined genotypes of individual F2 mice with the most severe, mildest or non-penetrant phenotypes (n=323) using single nucleotide polymorphism (SNP) markers. The average initial map resolution of SNP markers spanning all 19 autosomes and the X chromosome was ~18 cM. Because >10% of individual F2 mice were unaffected, linkage analysis to identify modifier loci was performed using a two-part model (22Go) that allows for separate effects on penetrance and severity. Significant LOD scores (P≤0.05) found on chromosomes 3, 5, 8, 11, and 14 indicated the presence of five modifier loci (Fig. 2).



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  		Figure 2. Genome-wide interval mapping of total affected gut length in the phenotypic extremes of the Sox10Dom F2 population. LOD scores from a two-part model indicating the combined effects on penetrance and severity are plotted for all mouse chromosomes revealing five significant modifier loci on chromosomes 3, 5, 8, 11 and 14. Individual chromosomes are listed across the x-axis, with tick marks representing the location of SNP markers used in genotyping. Dashed and dotted horizontal lines represent the significant (P≤0.05) or suggestive (P≤0.25) thresholds, respectively.

 
Regions of significant linkage detected in the initial genome scan were investigated further by genotyping all Sox10Dom/+ F2 mice (n=1068) with the marker closest to each significant modifier. In this analysis, the ability to accurately quantify hypoganglionosis, aganglionosis and the total length of gut affected in Sox10Dom/+ mice allowed us to assign effects of individual modifiers either on penetrance or severity or on both (Fig. 3). The most significant modifier, on chromosome 5 (chr5) affects the penetrance and severity of both aganglionosis and total affected gut length phenotypes. The other modifier loci on chromosomes 8, 11 and 14 greatly affect penetrance of aganglionosis. Although multipoint LOD plots for these chromosomes demonstrate a notable effect on the severity of the total affected phenotype, this is largely due to individual mice that exhibit hypoganglionosis but no aganglionosis. When the 245 animals with hypoganglionosis but no aganglionosis are excluded (green curves in Fig. 3B), the loci on chromosomes 3, 8, 11 and 14 are seen to have little impact on the severity of the total affected phenotype.



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  		Figure 3. Modifier loci defined by genotyping the total Sox10Dom/+ F2 population exhibit distinct effects on penetrance and severity. Multipoint LOD plots for the five modifier loci on chromosomes 3, 5, 8, 11 and 14 are shown. For each modifier locus, LOD scores indicating the effect on penetrance (the chance of being affected, blue curves), severity (the average amount affected given that there is an effect, red curves) or both (black curves) was evaluated for two phenotypic subsets: aganglionosis (A) and total length of affected gut (B). Tick marks on the x-axis representing the location of markers used to genotype each chromosome. The horizontal dashed lines indicating the 95% LOD thresholds for LOD (p, µ) in black and the 95% LOD thresholds for LOD (p) and LOD (µ) in blue. Inset tables list the position (in centiMorgans) of each modifier locus, the closest marker used for genotyping in the total F2 distribution, and LOD scores, P-values and 95% confidence intervals (CI). LOD curves for severity in (A) (in red) derive from considering animals with aganglionosis more than zero (n=700). LOD curves for severity in (B) (in red) derive from considering animals with total affected gut length more than zero (n=945). In (B) several modifiers appear to affect the severity of total affected gut length, but this is largely attributable to individuals with hypoganglionosis greater than zero and with aganglionosis equal to zero. When the severity of total affected phenotype is considered for only those animals with aganglionosis greater than zero (green curves, B), it becomes clear that the apparent contribution of loci on chromosomes 3, 8, 11 and 14 to severity of total affected phenotype is attributable to animals with hypoganglionosis but not aganglionosis.

 
Allele effects of Sox10Dom modifier loci
Modifier loci either can increase susceptibility and severity of phenotype or can act protectively to confer resistance to disease in the face of a predisposing mutation (23Go). To assess the effects of individual Sox10Dom modifiers (one at a time) on the penetrance and severity of aganglionosis, we evaluated complete genotype information in the total F2 distribution. B6 alleles at modifier loci on chromosomes 5, 8, 11 and 14 increased both the proportion of individuals affected by aganglionosis and the extent of gut length affected by aganglionosis (Fig. 4). This is consistent with prior reports of more severe phenotype in Sox10Dom/+ mutants on the B6 background (8Go,24Go,25Go). In contrast, the chromosome 3 locus exerted an opposite effect with B6 alleles protecting mutants from the presence of aganglionosis and decreasing the severity of aganglionosis. All allele effects observed were approximately additive, with heterozygotes exhibiting phenotypes mid-way between the phenotype ranges of the homozygous animals, consistent with semi-dominant effects of each locus. Complete dominance was not detected for any of the modifier loci.



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  		Figure 4. Effect of alleles at Sox10Dom modifier loci on the penetrance and severity of aganglionosis. Genotypes from the total F2 population obtained from the marker closest to each significant modifier were used to assess the effects of individual loci (one at a time) on penetrance (A) and severity (B) of phenotype. In (A) the estimate (midline) and 95% confidence interval (flanking brackets) of the probability that an individual with a certain genotype at the individual modifier loci will exhibit aganglionosis is plotted for each genotype class to illustrate the relationship between the number of B6 or C3Fe alleles at each modifier locus and the presence of aganglionosis. (B) The mean of the log2 transformed percentage aganglionosis is plotted for each genotype class to show the relation of the number of B6 or C3Fe alleles and the extent of aganglionosis. Markers used to generate genotype information are listed beneath the plot for each locus. Genotype groups are defined as B6/B6 (BB), B6/C3Fe (BC) and C3Fe/C3Fe (CC).

 
1.5-LOD support intervals define genomic intervals of Sox10Dom modifier loci
To identify an approximate chromosomal location for each locus, 1.5-LOD support intervals were calculated for each modifier (26Go) (Fig. 5). Additional genotyping was performed for chromosomes 5, 11 and 14 using 23 additional markers to refine the positions and determine if these peaks were due to more than one modifier on each chromosome. Information for all markers used for genotyping is provided in Supplementary Material Table S1. Although the shape of the LOD curve for chromosome 11 suggested the possibility of multiple modifiers, after controlling for the locus at 42 cM, no further evidence for additional chromosome 11 loci was detected. Analysis of chromosomes 5 and 14 also supported the presence of only a single modifier. The refined mapping narrowed the 1.5-LOD support interval for chr5 to a location at 15–23 cM, whereas the chromosome 11 modifier (chr11) resides in the 1.5-LOD interval at 28–50 cM. The chromosome 14 modifier (chr14) is consistent with the position of Ednrb that has been previously reported as a modifier of Sox10Dom (8Go). The chr14 locus was initially detected by our genome-wide linkage analysis using SNP markers on chromosome 14, which were evenly distributed along the chromosome and independent of the markers previously used in candidate gene studies (8Go). Subsequently, we incorporated genotype data from additional markers flanking the Ednrb locus into the analysis to obtain the narrowed linkage interval centered at 54 cM.



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  		Figure 5. Refined intervals of modifier loci. Plots of 1.5-LOD support intervals show the position and size of significant intervals for the modifier loci on chromosomes 3, 5, 8, 11 and 14. Chromosomes containing significant modifiers are plotted against map position. Vertical lines represent the entire length of each chromosome with tick marks representing the position of the markers used to genotype the chromosome. The vertical brackets in bold to the left of each chromosome indicate the 1.5-LOD support interval for aganglionosis and the tick mark in the middle of the bracket shows the estimated position of the modifier. The total span (in centiMorgans) of the support interval is reported at the top of each bracket. The name of the markers flanking the 1.5-LOD support interval are shown.

 
Identification of candidate genes in chromosome 5 modifier interval
Bioinformatics approaches combining genome annotation with literature searches have been used successfully to identify biologically relevant genes within modifier intervals (27Go). We used the positions of the closest markers flanking the 1.5-LOD interval on chr5 to define the boundaries of this interval on the mouse genome assembly and search for genes that might be involved in development of enteric neural crest based on their expression profiles in the literature and public databases. A total of 59 genes within this interval were identified by Ensembl Martview (28Go). This listing of candidates was narrowed to include only those genes with reported expression in the embryo and in the gastrointestinal tract based on information in the PubMed and in the Gene Expression Database (29Go) (Table 1). Within this focused list, several highly relevant candidates were identified based on their documented expression in enteric neural crest cells in the developing gut. These include Uchl1 (PGP9.5), Atp8a1 and Phox2b. Uchl1, a ubiquitin ligase, is expressed early in the development of enteric neurons and maintained in mature neurons. Expression of Atp8a1, an aminophospholipid translocase, has been reported in embryonic enteric precursors but the developmental timing or cell-type specificity has not been determined (30Go). Phox2b is expressed in early enteric neural progenitors, maintained in differentiating neurons (31Go–33Go) and ablation of the gene leads to complete loss of enteric neurons in mice (34Go). It is notable that PHOX2B mutations have been reported in Haddad syndrome patients that exhibit features of central congenital hypoventilation defects syndrome in association with aganglionosis (2Go,35Go). Expansions of the second polyalanine tract in PHOX2B have been associated with severity of the neural crest defects in this syndrome (36Go) and specific haplotypes at PHOX2B are over-represented in HSCR patients in case–control studies (37Go). Phox2b is a logical and exciting candidate modifier for future study.


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  		Table 1. Candidate genes identified within the chromosome 5 modifier interval
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Variation in expressivity and penetrance of HSCR is the consequence of multiple gene interactions that modulate the ability of enteric neural crest cells to populate the developing gut. Sox10 is a key transcriptional regulator, which is integral to the processes of ENS development. To identify the genome locations of new genes that impact ENS development and thus aganglionosis, we have undertaken a genome-wide linkage analysis in Sox10Dom/+ mice. Our study was designed to detect genetic modifiers, regions of the genome that do not exhibit an obvious phenotype, but in the context of the Sox10Dom mutation, exert an effect on the penetrance and severity of aganglionosis. Using this comprehensive approach, we have successfully identified five modifier loci of Sox10Dom on mouse chromosomes 3, 5, 8, 11 and 14.

We have demonstrated the value of defining aganglionosis as a quantitative trait in mouse models to map modifiers of aganglionic megacolon. Our approach allows us to estimate the genetic contribution to this phenotype and establishes that there is a significant genetic contribution to Sox10Dom hypoganglionosis (h2=0.52), aganglionosis (h2=0.60) and total affected phenotypes (h2=0.66). This is consistent with previous reports of genetic background effects on Sox10 phenotypes (8Go,12Go,24Go,38Go and 39Go). Importantly, phenotype evaluation in mouse mutants like Sox10Dom is not limited by practical challenges associated with obtaining biopsy materials in human HSCR patients. Our characterization of Sox10Dom phenotype illustrates that enteric deficits are present as a continuum ranging from extremely severe aganglionosis to unaffected animals. In human HSCR, SOX10 mutations have been more frequently documented in syndromic HSCR cases (11Go,13Go), but reports of non-penetrant individuals with SOX10 alterations have been documented (12Go). Previous genetic studies of HSCR have attempted to simplify phenotypes by classifying patients into L-HSCR and S-HSCR categories based on the extent of gastrointestinal tract affected (3Go). Our characterization of Sox10Dom phenotype suggests that categorical classification of patients may be an over-simplification that could dilute the power to detect all modifier loci involved in a complex phenotype like aganglionosis. Although comparable studies have not been performed in Ret mouse models to investigate the effects of genetic background and range of phenotype, studies in HSCR patients indicate that aganglionosis also varies widely in RET patients (1Go,7Go,40Go).

Although chr14 position and allele effects are consistent with prior reports of Ednrb affecting aganglionosis in Sox10 mutants, three of the five modifier loci we identify map to novel regions on chromosomes 3, 8, and 11 that have not been previously associated with HSCR. These novel modifier intervals do not coincide with syntenic regions of known aganglionosis susceptibility loci (RET, GDNF, NTN, EDN3, EDNRB, ECE-1, SOX10 and ZFHX1B) or previously mapped modifiers in HSCR patients (3p21, 19q12, 9q31 or 16q23) (4Go,5Go,7Go). Moreover, we determined that essential genes for neural crest development including Grb10, Pax3, Phox2a, dHand, Hox11L1, Krox20, Mash1, Pou3f1, Kit, Ednrbm1, Hoxa4, Dlx2 and Ikbkap do not map to these regions. Sox8 has been recently reported as a modifier of aganglionosis in mice haploinsufficient for Sox10 based on the increased extent of aganglionosis in double mutant Sox10LacZ/+, Sox8LacZ/LacZ mice (41Go). However, none of the modifier loci identified in our study map to the same location as Sox8, and no previous evidence suggests that naturally occurring variants at SOX8 influence HSCR.

It is possible that the chr5 modifier locus we identified is equivalent to Phox2b and if substantiated would offer significant support for the mechanistic view of HSCR as a stem cell disorder. Sox10–Phox2b interaction would be consistent with prior descriptions of HSCR as a stem cell defect (16Go) because the most likely temporal window for interaction between these two genes would occur in the NCSC just before or during early migration into the gut. In support of this hypothesis, Kim et al. (17Go) have reported co-expression of Phox2b in Sox10+ neural crest cells and loss of Phox2b expression in homozygous Sox10Dom/Dom embryos.

HSCR inheritance is complicated by epistasis, gender and parent-of-origin effects (1Go,7Go). The modifier loci identified in this study are, by definition, in epistasis with Sox10. However, when we tested pairwise interactions between the modifiers identified in our analysis, we saw no evidence for epistasis between these loci (data not shown). Gender effects in isolated human HSCR where RET alterations are the predominating alteration show a marked 4:1 predominance of males/females (3Go). Gender effects on penetrance of aganglionosis have also been reported for patients with alterations in ENDRB (42Go). There is not a greater incidence or severity of aganglionosis in Sox10Dom/+ males than in females. We previously reported a small gender specific effect for the direction of dominance at the Ednrb locus on the severity of aganglionosis in Sox10Dom/+ mutants (8Go). Our current findings are consistent with that result. In both cases, the analysis derived from comparison of C57BL/6J with C3 substrains, C3FeLe.B6-a and C3HeB/FeJ, respectively, and may reflect the common origin of these substrains. In our genome survey, we anticipated the potential to identify significant gender or parent-of-origin effects and established our crosses to facilitate detection; however, we observed little evidence for notable gender or parent-of-origin effects when we tested for these effects at the newly identified modifier loci. This is consistent with prior observations that notable gender effects have only been documented for RET and EDNRB in human HSCR cases (1Go,42Go). It is possible that genome regions capable of exerting gender and parent-of-origin effects on Sox10Dom phenotypes are equivalent between the B6 and the C3Fe strains that we have investigated. Future studies of Sox10Dom/+ mutants in additional strain backgrounds will determine whether Sox10 phenotypes are subject to the same gender and parent-of-origin effects observed in human HSCR or if these characteristics are unique to the RET and EDNRB loci and their modifiers.

We have summarized the gene interactions detected in this study and tried to define them in the context of what is known to date about epistatic effects in HSCR (Fig. 6). Others have established epistasis between Ret and Ednrb (5Go,6Go), while prior studies from our group identified effects of Sox10–Ednrb on aganglionosis phenotypes in Sox10Dom/+ mutants. We did not detect any genetic interaction between Ret and Sox10 in our analysis despite prior in vitro studies suggesting Sox10 directly regulates Ret transcription (10Go). However, we strongly suspect that the B6 and C3Fe strains carry equivalent haplotypes through the Ret interval because screens to identify polymorphic markers in the immediate proximity of the Ret locus were not successful (V.A. Cantrell and E.M. Southard-Smith, unpublished data). Ret and Sox10 may exhibit epistasis in other strains that are divergent through the Ret interval. Given that Sox10 directly modulates Ednrb transcription (9Go), it is also possible that Sox10 may influence Ret activity via its interaction with Ednrb.



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  		Figure 6. Summary of gene interactions that contribute to heterogeneity of aganglionosis in HSCR. Heavy black arrows represent genetic interactions validated by analysis of double mutants in vivo. Lighter gray arrows indicate RET-dependent modifiers detected in human genetic studies. Effects of the 16q23 locus, although apparently confined to inbred Mennonite populations (5Go) may further inform the process of ENS development. Dashed arrow emphasizes potential for gene interaction between Sox10 and Ret based on in vitro studies. Modifiers of Sox10 identified in this study (Sox10m1-5) are shown in black text accompanied by gray shadowed arrows.

 
Our study does not exclude the potential for other HSCR modifiers and interactions that might be detected in additional strains, but instead emphasizes the value of investigating heterogeneous disorders like HSCR in mouse models where genetic background and input alleles can be controlled to provide additional power for genetic analysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mice
All animal protocols were approved by the Institutional Animal Care and Use Committee at Vanderbilt University. Lines of Sox10Dom/+ mice congenic on the C57BL/6J background (B6N14.Sox10Dom/+) were crossed with C3HeB/FeJ (C3Fe) mice to generate B6N15C3Fe.Sox10Dom/+ (F1) progeny. Intercrosses were performed by crossing male B6N15C3Fe.Sox10Dom/+ (F1) mice to wild-type B6C3Fe or C3FeB6 females as well as reciprocal intercrosses between female B6N15C3Fe.Sox10Dom/+ (F1) mice and wild-type B6C3Fe or C3FeB6 males in order to explore the effects of imprinting (Table 2). From these crosses, 2210 F2 mice were generated. Of the total cohort, tissues were collected from 2184 animals for evaluation of ENS deficits. Gut tissues from P7–P10 pups were collected and processed for acetycholinesterase whole-mount staining (AChE) using routine protocols (8Go) to visualize enteric ganglia. The extent of gut regions affected by either hypoganglionosis or aganglionosis was determined by microscopic examination. The entire length of the gut, as well as any hypo- or aganglionic regions, was measured. Lengths of the hypo- or aganglionic segment were divided by the total length of the gut to yield percentage of hypoganglionosis or percentage of aganglionosis. Total affected percentage was defined as the sum of percentage of hypoganglionosis and percentage of aganglionosis. Subsequent to AChE staining and quantitation of ENS deficits, animals were genotyped for gender and the presence of the Sox10Dom allele as described previously (15Go,43Go). Selective genotyping of individual animals at the extremes of the phenotype distribution was performed as described by Silver (44Go). Briefly, from the total distribution of 1068 Sox10Dom/+ animals, 109 F2 mice that represented the most severely aganglionic 10% from the tail of the phenotype distribution were selected and 107 F2 mice that represented the 10% most mildly affected were selected from the opposite tail of the distribution. To evaluate modifiers of penetrance, 107 (out of 126 total) non-penetrant F2 animals (phenotype of 0% gut length affected) were selected for genotyping.


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  		Table 2. Source crosses for generation of F1 intercross progeny
 
Marker selection and detection
SNP markers selected on the basis of polymorphism between C57BL/6J and C3H/HeJ strains of mice were identified either from publicly available databases at the Whitehead Institute (www.genome.wi.mit.edu/SNP/mouse) and the Roche database (http://mouseSNP.roche.com) or from SNP screens performed at Novartis Research Foundation. A total of 98 SNP markers spanning all 19 autosomes and the X chromosome were chosen for the initial genome scan with an average intermarker distance of ~18 cM. Twelve additional markers were applied in genotyping the tails of F2 phenotype distribution to refine the modifier interval on chromosome 5. Eight additional markers on chromosome 11 were likewise selected for genotyping to refine the broad interval and allow for further analysis of epistatic effects. Simple sequence length polymorphism markers neighboring Ednrb and Sox10 were also genotyped and included as components of the genome scan. In total, 121 markers (Supplementary Material, Table S1) were used for genotyping.

SNP genotypes were generated by single nucleotide primer extension with detection by fluorescence polarization (FP) (45Go). Reaction processing entailed three steps: a 4.2 µl PCR reaction, addition of 4 µl of an ExoI and CIP reagent mix to degrade unincorporated primer and dephosphorylate dNTPs and a final addition of 4 µl of an Acyclopol and Acycloterminator reagent mix for the primer extension reaction (AcycloPrimeTM-FP SNP Detection System, Perkin–Elmer, Boston, MA, USA). Each 4.2 µl of PCR mixture included 0.1 unit AmpliTaq Gold DNA polymerase, 1x Buffer II (Applied Biosystems, Foster City, CA, USA), 2.5 mM MgCl2, 0.25 mM dNTPs, 335 nM of each primer, and 2 ng DNA. Integrated automation was employed for genotyping in 384-well format (Tecan Genesis Workstation 200, Tecan GenMate, Velocity11 VSpin and MJ Tetrads). Incorporation of R110- and TAMRA-labeled Acycloterminators was detected by FP on a Molecular Devices/LJL Analyst HT. Samples were genotyped in duplicate and in a few cases in triplicate to obtain the least number of uncalled genotypes possible for each marker in the total 326 samples assayed in the initial genome scan and for the closest marker to each modifier locus in the total F2 progeny of 1068 samples.

Statistical analysis
The heritability (h2) of ENS deficits in Sox10Dom mice was established by calculating the proportion of genetic variance to total variance [(Vf2–Vf1)/(Vf2)], where Vf2 is the phenotypic variance observed within the Sox10Dom F2 intercross progeny (n=1068) and Vf1 is the phenotypic variance within the Sox10Dom F1 parents (n=39) used to perform the intercross. The heritability factor was calculated for three phenotypes: aganglionosis (h2=0.60), hypoganglionosis (h2=0.52), and total affected (h2=0.66). The significant genetic contribution to these phenotypes is consistent with previous reports of genetic background effects in congenic lines (8Go,24Go).

The total distribution of Sox10Dom F2 animals comprised the following subsets in Table 3.


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  		Table 3. Phenotype classes of Sox10Dom F2 mice
 
Statistical analyses to identify modifiers of Sox10Dom were performed via a two-part model (22Go), an extension of standard interval mapping appropriate for the case in which many individuals exhibit a non-penetrant phenotype of zero. We considered a single-modifier model and assumed that an individual with modifier genotype g has probability pg of having a non-zero phenotype; and that, given its phenotype is greater than zero, it follows, approximately, a normal distribution with mean µg and standard deviation {sigma}. We calculated three sets of LOD scores, to indicate the evidence for the presence of modifiers: LOD(p) concerns the test of the hypothesis that pg=p for all g, the penetrance; LOD(µ) concerns the test of the hypothesis that µg=µ for all g, the severity. LOD(p, µ) combines the two. A log base 2 transformation was performed on all three phenotypes to attenuate the great skew in their distributions. Statistical analyses were performed with R/qtl version 0.99–9 (46Go).

Statistical significance was assessed via permutation tests (47Go), with 1000 permutation replicates. Interval estimates of modifier location were calculated via 1.5-LOD support intervals, which correspond to ~95% confidence intervals (48Go).

Gender and parent-of-origin effects were assessed by splitting the data according to such covariates and by the inclusion of sex and/or parent-of-origin as additive covariates in the modifier analyses.

The possibility of epistasis (interactions between modifiers) was assessed by testing for pairwise interactions between the loci identified in the single-modifier analyses. The possibility of multiple modifiers on chromosome 11, suggested by the multiple peaks in the LOD curve, was assessed by a two-dimensional, two-QTL scan of chromosome 11 only, and with the loci on chromosomes 3, 5, 8 and 14, included as additive covariates.

Nomenclature
The chromosome intervals that exert the specific effects on Sox10Dom penetrance and aganglionosis described in this manuscript have been approved by the Mouse Genome Informatics Nomenclature committee for designation as modifier loci: Sox10m1 (chr14), Sox10m2 (chr3), Sox10m3 (chr5), Sox10m4 (chr8) and Sox10m5 (chr11).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Drs Al George, David Airey and David Threadgill for critical reading and thoughtful discussion of the manuscript. This work was supported by a Foundation for Digestive Health and Nutrition Research Scholar Award and US National Institutes of Health grants from NINDS (NS43556) and NIDDK (DK60047) to E.M.S2.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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