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Physiol. Genomics 28: 203-212, 2007. First published August 22, 2006; doi:10.1152/physiolgenomics.00133.2006
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Received 25 June 2006; accepted in final form 16 August 2006.
Physiological Genomics 28:203-212 (2007)
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Ultrafine mapping of Dyscalc1 to an 80-kb chromosomal segment on chromosome 7 in mice susceptible for dystrophic calcification

Zouhair Aherrahrou, Lars C. Doehring, Piotr M. Kaczmarek, Henrike Liptau, Eva-Maria Ehlers, Andrea Pomarino, Sandra Wrobel, Anika Götz, Bjoern Mayer, Jeanette Erdmann and Heribert Schunkert

Medizinische Klinik II, University of Luebeck, Luebeck, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In mice, dystrophic cardiovascular calcification (DCC) is controlled by a major locus on proximal mouse chromosome 7 named Dyscalc1. Here we present a strategy that combines in silico analysis, expression analysis, and extensive sequencing for ultrafine mapping of the Dyscalc1 locus. We subjected 15 laboratory mouse strains to freeze-thaw injury of the heart, and association with respective genotypes allowed condensation of the Dyscalc1 locus to 1 Mb. Within this region, 51 known and predicted genes were studied in DCC-susceptible C3H/He and DCC-resistant C57BL/6 mice with respect to mRNA expression in response to injury. Five genes displayed differential expression. Genotyping of seven novel single nucleotide polymorphisms (SNPs) within these genes revealed an 80-Kb region in NZB mice that were found positive for calcification though carrying otherwise alleles from DCC-resistant mice. This microheterogeneity in NZB mice was evolutionary conserved in all DCC-susceptible mouse strains and contains the genes EMP-3, BC013491, and Abcc6 (partially). The flanking SNPs are rs3703247 and NT_039420.5_2757991. mRNA levels of EMP-3 were found to be upregulated in response to injury in both C57BL/6 and C3H/He mice. Sequencing of EMP-3 revealed an SNP leading to an amino acid substitution (p.T153I) that was found in all mouse strains susceptible for DCC but not in resistant strains such as C57BL/6 mice. Thus, the p.T153I changes might affect the biological function of EMP-3 gene product after injury. Using this combined approach, we ultrafine-mapped the Dyscalc1 locus to an 80-Kb region and identified EMP-3 as a new candidate gene for DCC.

cardiovascular calcification; EMP-3; Abcc6


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
DYSTROPHIC CARDIOVASCULAR CALCIFICATIONS (DCC) occur at sites of inflammation and necrosis independently of plasma calcium phosphate homeostasis. In the cardiovascular system the process accompanies atherosclerosis, cardiomyopathy, and heart valve degeneration, thereby contributing tremendously to cardiovascular morbidity and mortality (12, 13).

Converging evidence suggests that such cardiovascular calcification as well as myocardial infarction are significantly influenced by genetic background (7, 15, 17). Specifically, the Framingham Heart Study revealed that coronary risk factors account for ~40% of the observed interindividual variability in coronary artery calcium deposition, while at least another 40–50% is determined by independent genetic factors (5). Moreover, recent family studies document that dystrophic coronary calcifications are at least in part genetically determined (7). Currently no specific genetic causes of cardiovascular calcifications have been identified. For this reason rodents were used to further examine the molecular genetic basis of this trait. C3H/He and DBA/2 inbred strains served as a model for DCC, while C57BL/6 mice are known to be resistant to DCC after tissue injury (4, 9).

In a quantitative trait locus (QTL) analysis between C57BL/6 and C3H/He mice, a major locus named Dyscalc1 was identified on mouse chromosome 7 that contributed both to myocardial and vascular calcification (4, 8, 9). We confirmed the pathogenetic relevance of this locus for cardiac calcification by breeding a congenic B6.C3HDyscalc1 mouse strain that contained the Dyscalc1 locus of C3H/He on a C57BL/6 genetic background (1). Congenic B6.C3HDyscalc1 animals regularly display dystrophic cardiac calcinosis subsequent to tissue trauma by ischemia or freeze-thaw injury (1). Very recently, Korff et al. (11) reported fine mapping of the Dyscalc1 region by analysis of novel congenic mouse strains to an interval <1 million base pairs (Mb) wide and suggested the ATP-binding cassette C6 (Abcc6) as a new candidate gene for DCC.

Here we report our progress towards unveiling the Dyscalc1 gene using in silico mapping by dense single nucleotide polymorphism (SNP) genotyping in a large number of DCC-positive and -resistant mouse strains. Combining ultrafine mapping with gene expression analysis, we ultrafine mapped the Dyscalc1 locus to an 80-Kb region and suggest the epithelial membrane protein-3 (EMP-3) gene as a new candidate for DCC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Housing
Female mice from C57BL/6J (C57), C3H/HeJ (C3H), DBA/2J (DBA), 129S1/SvJ (129S1), BALB/cJ (BALB/c), CBA/J (CBA), AKR/J (AKR), FVB/NJ (FVB), MRL/MpJ (MRL), NZB/BINJ (NZB), and NZW/LacJ (NZW) inbred strains were purchased from Charles River (Sultzbach-Rosenberg, Germany). All animals had free access to water and food and were maintained in a pathogen-free environment with a 12-h day/night cycle.

Freeze-Thaw Injury
Animal studies were performed in accordance with the German animal studies committee of Schleswig-Holstein. Animals were subjected to freeze-thaw injury as reported previously (1, 3, 16). In brief, female animals (n = 5 mice per strain) were operated under deep anesthesia after an intraperitoneal injection of 250 mg/kg body wt of a 12.5-mg/ml 2,2,2-tri-bromo-ethanol solution, prepared freshly in 2-methyl-2-butanol (1). The abdominal cavity was opened via median abdominal incision, and the inferior myocardial wall was accessed transdiaphragmatically and frozen for 10 s by a steel rod (1/5 inch in diameter) that had been precooled in liquid nitrogen. The abdominal incision was closed, and the animals were allowed to recover and returned back to their cage. After 3 and 7 days, mice were killed under anesthesia by cervical dislocation, respectively. Hearts were excised, rinsed with phosphate-buffered saline (PBS; GIBCO, Invitrogen), embedded in tissue freeze medium (Leica Instruments, Leimen, Germany), frozen in liquid nitrogen, and stored at –20°C until histological examination. For total RNA extraction, the necrotic and healthy myocardium specimens were separated and stored in liquid nitrogen.

Quantitative Analysis of mRNA Induction
Primer design.
Genes and expressed sequence tags (ESTs) between SNP markers rs3689409 (39.08 Mb) and rs6379675 (40.55 Mb) were selected to span the entire Dyscalc1 region. Transcript sequences were obtained from the public Ensembl Mouse Genome Server http://www.ensembl.org/Mus_musculus/ (July 2004). Specific primer pair sets for each gene were designed online using the http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi database. PCR product lengths were <250 bp, an optimal length for relative real-time PCR (the list of primer pair sets used in this study might be obtained by directly writing to the corresponding author). Whenever possible, amplicons spanned small introns to allow distinction between cDNA (intended, short amplicon) and genomic DNA (contamination, long amplicon).

Reverse Transcriptase PCR
Relative real time reverse transcriptase (RT)-PCR was performed as previously described (1). In brief, total RNA was extracted from healthy and injured myocardium using RNAclean (Hybaid-AGS, Heidelberg, Germany). Total RNA was reverse transcribed using a mixture containing hexamer pd (N6) primers, dNTPs (Amersham Biosciences), RNAguard RNase inhibitor, and M-MLV reverse transcriptase (Invitrogen) according to standard protocols. Relative real-time RT-PCR was performed using the qPCR Corekit for SYBR Green I (Eurogentec EGT Group). A microtiter plate containing 5 µM primer pair sets was employed. Mixtures (10 µl) containing the template cDNA (20–80 ng), MgCl2, dNTPs, SybrGreen I, and Hot GoldStar enzyme were prepared according to the manufacturer and added to each well.

mRNA Quantification
Changes in mRNA levels were determined using the {Delta}{Delta}Ct method (1, 19). Briefly, we compared the threshold cycle numbers (Ct) determined by RT-PCR of the genes of interest in healthy myocardium as well as in necrotic myocardium (of treated animals) to sham-operated animals employing the {Delta}{Delta}Ct equation. We tested ß-actin, 18S, and GAPDH as internal standards. Both 18S and GAPDH were not significantly altered (data not shown).

GAPDH was chosen as internal standard in this investigation, and baseline was defined as fold induction = 1.

DNA Sequencing
Direct sequencing was performed as described previously (1) on PCR fragments amplified from cDNA obtained from C57BL/6 and C3H/He mice in the 5'-untranslated region (UTR), coding region, and 3'-UTR of five candidate genes: Kcnj14, EMP-3, Rcn3, Bcl2-associated X protein (Bax), and CD37. Sequencing of PCR products was performed on both strands by a commercial sequencing service (Seqlab, Göttingen, Germany). Primer sequences can be obtained from the authors by request.

PCR-Single-Strand Conformation Polymorphism Analysis
DNA was isolated from tail biopsies by a standard protocol. Information on SNPs was retrieved using the "compare transcript SNPs" option at http://www.ensembl.org/Mus_musculus/ for every gene containing polymorphic SNPs between C57BL/6 and DBA/2J mice: Kcnj14, Kdelr1, EMP-3, BC013491, Abcc6, and Nomo1 (Table 1).


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  		Table 1. PCR fragment and corresponding SNPs used for PCR-SSCP

 
Primer pairs were designed flanking SNPs, and PCR products were amplified using an Eppendorf MasterMix (2.5x) (Eppendorf) according to the manufacturer's specifications. Amplicons were analyzed by single-strand conformation polymorphism (SSCP) on DNA from C57, C3H, DBA, 129S1, MRL, NZB, AKR, CBA, and FVB mice (6).

Statistical Analysis
Using RT-PCR we measured the fold induction of the genes EMP-3 and BC013491 in healthy and injured myocardium of C57 and C3H mice, respectively. We used the Mann-Whitney U-test to test for differences between necrotic and healthy myocardium of C3H and C57 mice. Differences were defined as significant for P values < 0.05 after adjustment for multiple testing by Bonferroni.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Genetic and Physical Mapping of the Dyscalc1 Locus
Using BXH-RI strains and congenic intercross analysis, the Dyscalc1 locus had been mapped to a 6.5-cM chromosomal segment on mouse chromosome 7 between microsatellite markers D7Mit270 (18 cM, MGI) and D7Mit230 (24.5 cM, MGI) (1, 4, 9). In the physical map by [http://www.ensembl.org/Mus_musculus/ (February 2006)], this region corresponds to a 9.4-Mb stretch between 36.30 Mb (D7Mit270) and 45.73 Mb (D7Mit230) (Fig. 1A). We retrieved SNP genotype information on this region from Mouse Resources at the Wellcome Trust Centre for Human Genetics (http://zeon.well.ox.ac.uk/rmott-bin/strains.cgi) (April 2006), and fine mapped the Dyscalc1 locus. In the first step, we used phenotype information from a set of four BXH recombinant inbred strains (BXH-7, BXH-8, BXH-9 and BXH-10 RI) and their parental C57BL/6 and C3H/He inbred strains.


Figure 1
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  		Fig. 1. Ultrafine mapping of Dyscalc1. A: localization of the quantitative trait locus, Dyscalc1 (black box), on mouse chromosome 7 between the simple sequence length polymorphion markers D7Mit270 and D7Mit230 using BXH-RI and congenic intercross analysis as published previously (4, 9). B: fine mapping of Dyscalc1 to 2 Mb using in silico mapping in BXH RI strains (BXH-7, BXH-8, BXH-9, and BXH-10). C: additional laboratory mouse strains allocate Dyscalc1 to ~1 Mb between the UT_7_38.436442 (39.5 Mb) and rs6379675 (40.5 Mb). D: genotyping of novel single nucleotide polymorphisms (SNPs) in NZB mice further decreased the region to 80 kb using PCR-single-strand conformation polymorphism (SSCP) analysis of genes flanking EMP-3 (highlighted in black).

 
Analyzing the distribution of SNP markers among DCC-susceptible C3H, BXH-10, and DCC-resistant C57, BXH-7, BXH-8, BXH-9 mice allowed allocation of the Dyscalc1 segment to a 2-Mb region between the SNPs rs4226572 (38.38 Mb) and rs6379675 (40.55 Mb) (Table 2, Fig. 1B). Based on chromosomal segregation in these strains, we determined two polymorphic chromosomal segments between [rs4226572 and UT_7_37.999111] and [UT_7_38.436442 and rs6379675], respectively, that might contain the Dyscalc1 locus. The two regions are separated by a 500-Kb nonpolymorphic region located between the SNPs UT_7_37.999111 and UT_7_38.436442.


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  		Table 2. SNP genotypes and DCC phenotype information in four BXH R1 (BXH-7, BXH-8, BXH-9, and BXH-10)

 
We next tested further SNPs that are polymorphic between the DCC-susceptible and DCC-resistant mice within the 2-Mb chromosomal segment in additional inbred mouse strains. C3H, DBA, 129S1, BALB/c, BXH-10, NZW, and NZB tested positive for dystrophic myocardial calcification upon freeze-thaw injury whereas C57, CBA, AKR, FVB, BXH-7, BXH-8, BXH-9, and MRL mice were resistant for this trait (Table 3). The noncalcifying phenotype observed in CBA mice excluded the region containing the haplotype block [rs4226572 to UT_7_37.999111], thus further narrowing the Dyscalc1 locus to the [UT_7_38.436442 - rs6379675] region (1 Mb) (Fig. 1C).


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  		Table 3. Detailed SNP genotypes within the condensed 2-Mb region and DCC phenotype information in additional laboratory inbred strains

 
Unexpectedly, the NZB mice were found positive for DCC after freeze-thaw injury even though they carried only alleles found in DCC-resistant strains in this initial analysis (i.e., of a genotype similar to C57 mice).

Gene Expression Profile Analysis
Within the 1-Mb region linked to DCC in this two-step in silico mapping approach, we studied the 51 known and predicted genes (ESTs) spanning our region of interest between SNP markers rs3689409 (39.08 Mb) and rs6379675 (40.55 Mb) (Table 3). Of these, 38 genes displayed detectable mRNA expression in the heart. Gene expression levels in healthy myocardium and regulation following freeze-thaw injury were tested in both DCC-susceptible C3H and DCC-resistant C57 mice by quantitative real-time RT-PCR as previously described (1). Four mice in each group (healthy and injured C3H and C57 mice, respectively) were examined.

Four genes were found to be highly upregulated in response to freeze-thaw injury in both DCC-susceptible and DCC-resistant mice: EMP-3; Bax; CD37 antigen; and reticulocalbin 3, EF-hand calcium binding domain (Rcn3) (Fig. 2).


Figure 2
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  		Fig. 2. mRNA gene induction of genes and expressed sequence tags (ESTs) spanning the region linked to dystrophic cardiovascular calcification (DCC) between Ush1c (40.32 Mb) and Rcn3 (39.17) genes, respectively. Gene induction in necrotic lesion (injury) was compared with healthy myocardium (healthy) 3 days after freeze-thaw injury in C57BL/6 (C57) and C3H mice (n = 4 in each group). Results were obtained by relative real-time RT-PCR after normalization to GAPDH (see MATERIALS AND METHODS). Fold of induction is expressed as means ± 1 SD compared with base line (1x).

 
Independently from freeze-thaw injury, the mRNA levels of Kcnj14 (potassium inwardly rectifying channel subfamily J member 14) were found to be higher in C57 mice than in C3H mice (Fig. 2).

Sequencing of Selected Potential Candidate Genes
We sequenced the 5'-UTR, coding region, and 3'-UTR of the four upregulated and one strain-specific genes in DCC-resistant C57 and DCC-susceptible C3H mice to identify differences between noncalcifying and calcifying mice. No SNPs were detected in Bax gene and CD37 gene, respectively. As shown in Table 4, sequence differences between C57 and C3H mice were found in EMP-3 gene and Rcn3 gene. Four SNPs were detected in EMP-3: two SNPs in the 5'-UTR and two SNPs in the coding region. One silent mutation bearing no effect on the amino acid sequence was found as well as a C-to-T base-pair mutation [Sanger SNP ID: NT_039420.5_2758108 (Table 5)] leading to an amino acid exchange from threonine (ACT) to isoleucine (ATT) at position c.662C/T (p.T153I) in DCC-susceptible C3H mice.


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  		Table 4. Sequence analysis of candidate genes

 

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  		Table 5. Genotyping of SNPs in genes identified by mRNA induction analysis in multiple mouse strains

 
Likewise, an amino acid substitution was found for Rcn3: at c.844G/A (Sanger SNP ID: NT_039420.5_1923171), where a charged amino acid, glutamine, is substituted by a noncharged amino acid, arginine (p.R260Q).

Kcnj14 showed a highly polymorphic sequence. A total of 17 SNPs, two insertions, and two deletions were found (Table 4). In the 5'-UTR of C3H/He mice, an insertion of a 2-nucleotide GT-motif and of a 6-nucleotide (ACCCCC)-motif were observed, as well as two deletions of a C, and six SNPs. Six SNPs were found in the 3'-UTR. Six SNPs were observed in the coding region, of which five were silent. One amino acid substitution was identified: at position c.205G/A, alanine is exchanged for threonine (NT_039420.5_2660081).

Ultrafine In Silico Mapping
To further examine the association between genotype and phenotype, we extended genotyping of these partially novel SNPs in EMP-3 (NT_039420.5_2757991 and NT_039420.5_2758108), Kcnj14 (NT_039420.5_2660081, NT_039420.5_2660061, NT_039420.5_2660046 and NT_039420.5_2659974), and Rcn3 (NT_039420.5_1923171) to the following laboratory mouse strains: DCC-susceptible NZW, 129S1, C3H, DBA, BXH-10, BALB/c, and NZB and DCC-resistant C57, CBA, FVB, MRL, AKR, BXH-7, BXH-8, and BXH-9.

When genotype and phenotype data were taken together, of all examined SNPs, only the genotype tagged by the SNP leading to an amino acid exchange (p.T153I) in Emp-3, NT_039420.5_2758108, was consistently found in all DCC-positive mouse strains but not in DCC-resistant mice. The smallest segment with DCC-positive SNPs on a DCC-negative background was obtained from NZB mice (Table 5).

Ultrafine Mapping of the Dyscalc1 Locus by PCR-SSCP
The predisposition of NZB mice to dystrophic cardiac calcification led us to hypothesize that some evolutionarily conserved genetic microheterogeneity might control the susceptibility to DCC. For this reason we tested additional closely spaced SNPs flanking EMP-3 using PCR-SSCP analysis to determine the genetic variation among DCC-positive C3H, DBA, 129S1, and NZB mice and DCC-resistant C57, CBA, FVB, MRL, and AKR mice on a single nucleotide scale (Table 6). This analysis revealed a small genetic microheterogeneity within an 80-Kb region, between genes Abcc6 (rs3703247, 40.08 Mb) and EMP-3 (NT_039420.5_2757991, 40.00 Mb), flanking BC013491 (Table 6, Fig. 1D).


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  		Table 6. Genotyping of SNPs in genes flanking EMP-3 in multiple laboratory inbred strains

 
RT-PCR Analysis of mRNA Expression in Response to Injury of the Genes Within the 80-Kb Region in Myocardial Tissue
Since Abcc6 displayed mRNA expression in neither necrotic nor healthy myocardium (as analyzed in Gene Expression Profile Analysis), we focused our analysis on mRNA changes in EMP-3 and BC013491 using quantitative real-time RT-PCR. Four mice in each group were killed 3 days after freeze-thaw injury. Compared with healthy myocardium, upregulation of EMP-3 was demonstrated in necrotic tissue of both DCC-resistant C57 and DCC-susceptible C3H mice (Fig. 3). mRNA levels of EMP-3 were similar in healthy myocardium of both C57 (DCC-resistant) and C3H (DCC-susceptible) mice [1 ± 0.3 and 1.3 ± 0.3; not significant (NS), respectively]. Compared with healthy myocardium, induction in necrotic lesions was 5.9 ± 2.0 (P < 0.01) and 5.4 ± 0.7 (P < 0.01) in C57 and C3H mice, respectively, with no significant difference between the two strains.


Figure 3
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  		Fig. 3. mRNA gene induction of Emp-3 and BC013491. Gene induction in necrotic lesion (injury) was compared with healthy myocardium (healthy) 3 days after freeze-thaw injury in C57BL/6 (C57) and C3H mice (n = 4 in each group). Results were obtained using relative real-time RT-PCR after normalization to GAPDH (see MATERIALS AND METHODS). Fold induction is expressed as means ± 1 SD compared with baseline (fold induction = 1).

 
mRNA levels of BC013491 remained unchanged in necrotic lesions (0.7 ± 0.2 in C57 and 0.7 ± 0.5 in C3H mice; NS of C57 vs. C3H mice) as well as in healthy myocardium (1.0 ± 0.3 and 0.9 ± 0.2; NS in C57 and C3H mice, respectively).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The in-depth investigation of QTLs is one of the most pertinent challenges in molecular genetics. Traditional segregation analysis often leaves the investigator with regions harboring hundreds of genes without the option of further discrimination. In the present study we took advantage of a phenotype (DCC) with differential occurrence in multiple inbred laboratory mouse strains. Linking this phenotype with genotype information available on these strains allowed an in silico segregation analysis of giant dimensions. Further studies on tissue expression of genes located in the remaining chromosomal region revealed that mRNA levels of only a few were altered in DCC. By sequencing these differentially expressed genes, we discovered novel SNPs and exploited this information for narrowing down the region of interest to microheterogeneity of only 80 kb that transferred the phenotype.

The present study exploited the fact that dystrophic calcification occurs in multiple mouse strains. Interestingly, a single major locus appears to determine the phenotype, as a highly conserved region on chromosome 7 is found in practically all mouse strains that develop DCC (4, 9, 11). The genotypes of these DCC-positive strains can be compared with those that do not display calcifications upon tissue injury. Within such an in silico mapping approach, the number of meioses contributing to the ultrafine mapping of the trait is way beyond that of congenic strains bred under experimental conditions. In the context of the present study, this evolutionary experiment led firstly to substantial condensation of the region of interest (1 Mb) and secondly to the identification of a mouse strain that developed DCC yet carried almost exclusively markers characteristic for DCC-resistant strains.

To date, no gene has been singled out to be responsible for DCC. Systematic testing of potential candidate genes had been inefficient due to the high number of genes left in the 6.5-cM interval (~200 genes and ESTs). The condensation of the region after the in silico strategy allowed testing of a small number of genes by 1) sequencing and 2) gene expression profiles in healthy vs. injured myocardium following freeze-thaw injury in DCC-resistant and DCC-susceptible mice. This approach detected no variants in the genes Bax and CD-37, excluding them for DCC. On the other hand, polymorphisms were found in EMP-3, Rcn-3, and Kcnj14, suggesting them as potential candidate genes. Genotyping of these partially novel genetic variants in multiple laboratory mouse strains revealed that within all DCC-susceptible mouse strains, only one novel mutation in the EMP-3 gene is conserved.

To link this search with the phenotype, we closely examined each gene in the region with respect to expression and regulation in myocardial tissue. In doing so, we detected Abcc6, a gene that was previously suggested to cause DCC (11), at mRNA level in neither necrotic nor healthy myocardial tissue. Moreover, genotyping of three SNPs (two SNPs in exon 1, one SNP in exon 2) by direct sequencing, as well as the SNP in intron 15–16 (rs3703247) from the Wellcome Trust Centre for Human Genetics in Abcc6 revealed DCC-resistant alleles for Abcc6 in DCC-susceptible mice (Table 6). Thus, in addition to not being found at the mRNA level, large parts of the Abcc6 gene are excluded for the Dyscalc1 locus. In conjunction it is unlikely that Abcc6 is a candidate gene for DCC. Nevertheless, further sequencing of the whole Abcc6 locus in DCC-susceptible and DCC-resistant mice is necessary to investigate the Abcc6 3'-region (in this case, downstream) of rs3703247.

After further reducing the number of candidate genes by studying mRNA expression levels, we returned our attention to the NZB mouse that displayed DCC despite carrying DCC-resistant genotypes. The occurrence of DCC on the chromosomal background of DCC-resistant mice allowed two possible explanations. First, phenocopy, i.e., a genetic variation located at some other chromosomal locus, might cause a DCC-like disorder in these NZB mice. Alternatively, such mice may carry a small stretch of chromosomal DNA from DCC-susceptible mice that was not detected by the dense SNP set offered by the databanks [Mouse Resources at Wellcome Trust Centre for Human Genetics (http://zeon.well.ox.ac.uk/rmott-bin/strains.cgi)]. We therefore aimed at the identification of additional SNPs in the region. Here it was helpful that we had identified several genes with a marked upregulation of their mRNA levels in response to tissue injury. Sequence information from novel SNPs within these genes was used for ultrafine mapping of the Dyscalc1 locus in NZB mice. Thereby, we discovered a small, 80-kb region that displayed a uniform pattern of SNP distribution in all strains tested for DCC including NZB mice that (in testing the other SNP markers) had been the exception to the rule.

We focused our research on the two genes left in the region of interest, EMP-3 and BC013491. At present, the function of the predicted BC013491 gene is unknown. This gene is not regulated in DCC tissue as its mRNA was not altered 3 days after tissue trauma by myocardial freeze-thaw injury. EMP-3 mRNA, however, is expressed in the heart and is upregulated in response to freeze-thaw injury in both C3H and C57 mice. This information is important since we found in a previous study employing bone marrow transplantation from DCC-resistant C57BL/6 mice to DCC-susceptible congenic B6.C3HDyscalc1 mice, that DCC occurs in the injured heart only when cardiac cells (rather than circulating cells) harbor the Dyscalc1 locus (10). This implicates that the gene underlying the Dyscalc1 locus must be expressed by resident cells (e.g., cardiomyocytes, smooth muscle cells, or fibroblasts).

The EMP-3 gene encodes a 163-amino acid protein of 18 kDa that consists of four transmembranous domains and two N-linked glycosylation sites in the first extracellular loop (18). Sequencing cDNA from DCC-susceptible C3H and DCC-resistant C57 mice revealed four SNPs in EMP-3. One of these mutations results in an amino acid substitution from threonine to isoleucine at position c.662C/T in C3H mice. Interestingly, a functional role of this allelic variation was previously reported in an immunological context by Yadav et al. (20). While conducting research on graft-vs.-host disease and chronic allograft rejection, the authors found minor histocompatibility antigen disparities. The authors reported posttranslational phosphorylation of the H4a minor histocompatibility antigen (containing isoleucine) but not the H4b epitope (containing threonine) (20). Using an ex vivo system, they demonstrated that CD8+ T lymphocytes bind more efficiently to phosphorylated antigen tetramers than nonphosphorylated ones, facilitating T cell response.

Moreover, EMP-3 has been reported to play a role as a tumor suppressor gene in glioma and neuroblastoma (2). The overexpression of members of the EMP family (EMPs) leads to cell blebbing, binding of annexin V, and cell death by a caspase-dependent pathway (18). Not only involved in cell apoptosis, EMPs have also been shown to interact with neuronal voltage-dependent calcium channels (14).

The pathophysiological mechanisms leading to DCC are not fully understood but might involve cell-cell interaction, apoptosis, immunological reactions, and/or calcium phosphate homeostasis. Thus, given its assumed role in immune response, apoptosis and Ca2+ homeostasis, EMP-3 offers an excellent candidate for DCC.

Further research is necessary to examine the pathogenetic mechanisms of DCC and to confirm the role of each gene within the 80-kb region. Currently, transgenic mice are generated to further pursue their functional roles.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The study was supported by Forschungsförderung der Medizinischen Fakultät Lübeck 2003 (J14-2003; Z. Aherrahrou), Bundesministerium für Bildung und Forschung [National Genome Network 2 (NGFN2) (01GS0418; H. Schunkert, J. Erdmann)], the Deutsche Forschungsgemeinschaft (Schu 672/14-1), and the European Union-sponsored project Cardiogenics.


   ACKNOWLEDGMENTS
 
We thank Dr. B. Ivandic for greatly contributing to our work by initiating research on DCC. We are indebted to Dr. R. Noel for advice in matters of animal maintenance and welfare.


   FOOTNOTES
 
Address for reprint requests and other correspondence: Z. Aherrahrou, Medizinische Klinik II, Universitätsklinikum Schleswig-Holstein, Campus Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany (e-mail: aherrazou{at}o2online.de).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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