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Human Molecular Genetics, 2003, Vol. 12, No. 24 3207-3214
DOI: 10.1093/hmg/ddg354
© 2003 Oxford University Press

Functional haplotypes of the RET proto-oncogene promoter are associated with Hirschsprung disease (HSCR)

Guido Fitze1,*, Hella Appelt2, Inke R. König3, Heike Görgens2, Ulrike Stein4, Wolfgang Walther4, Manfred Gossen4, Matthias Schreiber5, Andreas Ziegler3, Dietmar Roesner1 and Hans K. Schackert2

1Department of Pediatric Surgery, University of Technology Dresden, Fetscherstr. 74, D-01307 Dresden, Germany, 2Department of Surgical Research, University of Technology Dresden, D-01307 Dresden, Germany, 3Institute of Medical Biometry and Statistics, University at Lübeck, D-23538 Lübeck, Germany, 4Max-Delbrück-Center for Molecular Medicine, D-13092 Berlin, Germany and 5Department of Pediatric Surgery, University of Erlangen, D-91054 Erlangen, Germany

Received June 18, 2003; Revised October 7, 2003; Accepted October 15, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The activation of the RET signaling pathway during embryogenesis is a crucial prerequisite for a directional migration of enteric nervous system progenitor cells. Loss-of-function germline mutations of the RET proto-oncogene are reported in familial and sporadic cases of Hirschsprung disease (HSCR) with a variable frequency. Furthermore, variants of several RET polymorphisms are over- or under-represented in HSCR populations. Specifically, the c.135A RET variant has been previously shown to be strongly associated with the HSCR phenotype. We have reported an HSCR-phenotype modifying effect of the RET c.135G>A polymorphism due to a within-gene interaction in patients harboring RET germline mutations, yet the function of the c.135G>A variant is unknown. The basic RET promoter region was investigated by DNA sequencing approach in 80 HSCR patients. Identified polymorphisms were genotyped in the HSCR and in a control population and haplotypes were reconstructed. The dual-luciferase assay was used to evaluate the activity of different RET promoter haplotypes. We demonstrate that variants of two RET promoter polymorphisms -5G>A and -1C>A from the transcription start site are associated with HSCR. Furthermore, the -5G>A polymorphism is in strong linkage disequilibrium with the c.135G>A polymorphism. The promoter haplotype -5/-1AC associated with HSCR has a significantly lower activity in an in vitro dual-luciferase expression assay compared with those haplotypes identified in the majority of normal controls. These data suggest a role for RET haplotypes containing the -5A promoter variant in the etiology of HSCR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The RET proto-oncogene is expressed in human tissues of neural crest origin and has been recognized as a susceptibility gene for several autosomal inherited diseases such as the multiple endocrine neoplasia type 2 (MEN 2) syndromes and Hirschsprung disease (HSCR) (1). In particular, HSCR is considered a dysgenetic neurocristopathy characterized by a congenital absence of the intramural ganglia in the hindgut causing intestinal obstruction in newborns. The incidence of 1 in 5000 live births worldwide, the familial occurrence and various syndrome presentations of Hirschsprung disease suggest a genetic background implicated in this disease (2,3). Complex segregation analyses suggest a non-Mendelian mode of inheritance with incomplete penetrance, a variable phenotype in HSCR families and a higher incidence in males than females (4). Linkage analyses of multiplex HSCR families revealed that the RET gene locus at 10q11.2 is the major gene locus for Hirschsprung disease (5), although RET germline mutations (which are primarily point mutations scattered throughout the extracellular domain and within the intracellular tyrosine kinase domain) were only detected in up to 20% of sporadic and in 50% of familial cases (6,7). In addition, RET modifying gene loci at 3p21 and 19q12 were described in association with a HSCR phenotype that involved exclusively the rectosigmoid colon (8). Another RET independent HSCR susceptibility gene has been mapped to 9q31 by linkage analysis of six families in which an exclusive link to this locus was shown in one family (5).

Mutations in several genes such as EDNRB, EDN3, GDNF, NTN, SOX10, ECE1 and SIP1 were identified in up to 5% of cases, supporting a genetic heterogeneity for this disorder (2,3,8–10). Yet, rare heterozygous germline mutations of GDNF, NTN and EDNRB have been detected in patients showing the HSCR phenotype in combination with a RET germline mutation (11–13). This observation supports the concept of synergistic heterozygosity for HSCR, which considers the disease phenotype as a result of a cumulative effect of at least two mutations in different genes (14–16).

Variants of several RET polymorphisms are more frequently associated with the HSCR phenotype than in normal controls. Specifically, the c.135A RET variant has been previously shown to be strongly associated with the HSCR phenotype, compared with the other characterized RET variants (17,18). In addition to the mutation-independent effect of the c.135A variant in the etiology of HSCR per se or the genetic variants in linkage disequilibrium with this c.135A variant, we have reported a HSCR phenotype modifying effect of the RET c135G>A polymorphism due to a within-gene interaction in patients harboring RET germline mutations (16). A functional mechanism of action for RET haplotypes in the etiology and modification of HSCR phenotypes in any of the reported RET polymorphisms is unknown, despite the evidence that these variations are associated with HSCR. Notably, it has been suggested that the silent c.135G>A polymorphism is in linkage disequilibrium with a functional genetic variant possibly located in the RET promoter region (16,19).

In this study we investigate the RET promoter region using a direct DNA sequencing approach. Polymorphisms at positions -5 and -1 from the transcription start site were characterized. Haplotypes comprising the -5G>A and -1C>A and c.135G>A polymorphisms were reconstructed to evaluate the association of specific RET haplotypes with Hirschsprung disease. Furthermore, we have cloned the four different promoter haplotypes (three observed haplotypes and one haplotype generated by site-directed mutagenesis) and used the dual-luciferase assay to estimate the activity of these promoter haplotypes. Finally, an electrophoretic mobility shift assay was established to test a potentially protein binding site changed by the RET promoter polymorphisms.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequence analysis of the putative promoter region of the RET proto-oncogene (20) between -169 and +98 from the transcription start site at +1 (as per GenBank NT_033985) did not reveal any germline mutation in the 80 HSCR patients studied. We have further analyzed two known polymorphic nucleotide substitutions at base pairs -5G>A and -1C>A from the transcription start site (21) and the c.135G>A polymorphisms in exon 2. No significant deviation from the Hardy-Weinberg equilibrium was detected at the -5G>A, -1C>A and the c.135G>A polymorphisms in either patients or controls (all nominal P>0.05). In comparison to the control population, genotype frequencies were different at all three polymorphisms in the affected population (Table 1). In particular, the -5G>A variant was characterized by an almost inverse genotype distribution, with an over-representation of the homozygous -5A genotype in the HSCR population (41 homozygous -5A compared with 11 homozygous -5G genotypes out of 80 HSCR patients versus seven -5A compared with 67 homozygous -5G genotypes out of 120 controls; OR 29.35; 95% CI 10.90–87.48; adjusted exact two-sided P<0.0001, Cochrane–Armitage trend test). These findings are almost identical to those of the c.135G>A polymorphism (40 homozygous c.135A compared with 10 homozygous c.135G genotypes out of 80 HSCR patients versus eight compared with 65 out of 120 controls; OR 31.07; 95% CI 11.25–95.65; adjusted exact two-sided P<0.0001, Cochrane–Armitage trend test).


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  		Table 1. Genotype frequencies of polymorphic variants of RET in 80 HSCR patients and 120 control individuals
 
In 11 of 80 HSCR patients (13.8%) and in 21 of 120 control individuals (17.5%) we found a compound heterozygosity of promoter variants, which did not allow the reconstruction of haplotypes from genotypes. Therefore, PCR products of the basic RET promoter were subcloned and sequenced. None of the 80 HSCR patients and 120 controls harbored the -5/-1AA haplotype. Within the HSCR population, we observed in 68.8% (110 of 160 alleles) the -5/-1AC haplotype. In contrast, the same haplotype was found in the control population only in 25% (60 of 240 alleles), which reveals a significant difference in haplotype frequencies in the HSCR and control populations (exact two-sided P<0.0001; Table 2).


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  		Table 2. Exact haplotype frequencies at promoter -5 and promoter -1 polymorphic positions of RET in 80 HSCR patients and 120 control individuals
 
Since the promoter and the c.135G>A polymorphisms are separated by about 23 kb of genomic sequence, haplotypes comprising all three variants were estimated. The haplotype reconstruction revealed only three haplotypes (-5/-1/c.135ACA; -5/-1/c.135GAG; -5/-1/c.135GCG) with a frequency greater than 2%. Besides the observation that the -5/-1AA haplotype was not found in any tested individual, two further haplotypes (-5/-1/c.135ACG; -5/-1/c.135GCA) could only be reconstructed in the HSCR but not in the control population (Table 3). We found a strong linkage disequilibrium between the -5G>A and c.135G>A variants and consequently an association of the -5/-1/c.135ACA haplotype with the HSCR phenotype (66.9%), compared with the normal phenotype (25%), which ascertains a significant difference in haplotype frequencies in the HSCR and control populations (adjusted Monte-Carlo P<0.001, Table 3).


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  		Table 3. Haplotype frequencies at promoter -5, promoter -1 and c.135G/A polymorphic positions of RET in 80 HSCR patients and 120 control individuals
 
To determine the effect of promoter haplotypes on the promoter activity, we subcloned the three identified haplotypes -5/-1GC, -5/-1GA and -5/-1AC and generated the fourth haplotype -5/-1AA using site-directed mutagenesis.

Neuroblastoma cell lines NMB and Vi-856 expressing RET (Fig. 1) were transiently transfected with dual-luciferase reporter constructs. The expression assays revealed a significantly lower promoter activity in basic RET promoter constructs containing the -5A variant, as compared with the -5G variant. Notably, the fourth haplotype -5/-1AA, which could not be found in 400 chromosomes, showed the lowest promoter activity. These differences in the activity of the four RET promoter haplotypes were unveiled in both cell lines (Fig. 2, global P<0.0001). Post-hoc pair-wise comparisons using the model including main effects showed that expression levels of all RET haplotypes were significantly different for both the NMB and Vi-856 cell lines (all adjusted P<0.05), except for the -5/-1GA and the -5/-1GC haplotypes and the -5/-1A/A and the -5/-1AC haplotypes.



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  		Figure 1. RT–PCR determination of RET mRNA in NMB and Vi-856 neuroblastoma cells. After total RNA isolation and reverse transcription according to standard protocols, a 445 bp cDNA fragment (spanning from exon 6 to exon 8; sense-primer, 5'-GCGGGCGTCCTCTTGCTCCACTT-3'; antisense-primer, 5'-GGCCGCCACACTCCTCACACT-3') was amplified in a Perkin-Elmer 9600 thermocycler and resolved on an agarose gel: lane 1, 1 kb DNA-ladder; lane 2, negative control; lane 3, NMB; lane 4, Vi-856.

 



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  		Figure 2. Effects of basic promoter variants of the RET proto-oncogene on promoter activity in neuroblastoma cell lines NMB and Vi-856. Dual-Luciferase® Reporter Assay was used to study activity of the four different basic promoter haplotypes -5/-1GC, -5/-1AC, -5/-1GA and -5/-1AA relative to the transcription start site of the RET proto-oncogene in neuroblastoma cell lines NMB and Vi-856 after transient transfection of constructs. pGL3-basic-RET-promoter driven luciferase-levels were normalized to pRL-SV40 early enhancer/promoter-driven Renilla luciferase-levels as transfection control and expressed as relative luciferase activity. The pGL3-control plasmid containing the SV40 early enhancer/promoter region upstream of the firefly luciferase (control) and the promoter- and enhancerless plasmid pGL3-Basic containing firefly luciferase (basic) served as positive and negative controls, respectively. In addition, four deletion variants of the basic RET promoter were constructed and used for analysis of promoter activities as described above: variant {Delta}192 (deletion of -142 to +50 from the transcription start site), variant {Delta}243 (deletion of bases -145 to +98), variant {Delta}134 (deletion of -43 to +91), variant {Delta}129 (deletion of -41 to +88). Forty-eight hours after transient transfection, cells were washed in PBS and harvested by addition of 100 µl of passive lysis buffer. The activity of both luciferases was measured sequentially in a 1450 Micro Beta Trilux Luminometer using Dual-Luciferase® Reporter Assay reagents. The analysis of each RET promoter construct was performed in three independent experiments, which were repeated four times. (A) In neuroblastoma cell line NMB basic promoter activities containing the -5/-1AC or -5/-1AA haplotypes were significantly lower compared with the -5/-1GC or -5/-1GA haplotypes (adjusted P=0.022 and P=0.008, respectively). Moreover, basic promoter activities containing -5/-1AA or -5/-1AC haplotypes were significantly lower compared with -5/-1GC or -5/-1GA haplotypes (adjusted P=0.025 and P=0.0069, respectively). In contrast, promoter activities containing the -5/-1AC versus -5/-1AA haplotype and -5/-1GC versus -5/-1GA haplotype were not significantly different. Promoter activities of deletion variants {Delta}192 and {Delta}243 were similar to pGL3-Basic construct, while markedly reduced in {Delta}134 and {Delta}129 deletion variants as compared to the four RET promoter haplotypes tested. (B) In neuroblastoma cell line Vi-856 basic promoter activities containing the -5/-1AC or -5/-1AA haplotypes were significantly lower compared to the -5/-1GC or -5/-1GA haplotypes (adjusted P=0.0023 and P=0.0003, respectively). Moreover, basic promoter activities containing -5/-1AA or -5/-1AC haplotypes were significantly lower compared to -5/-1GC or -5/-1GA haplotypes (adjusted P=0.0004 and P=0.0015, respectively). In contrast, promoter activities containing the -5/-1AC versus -5/-1AA haplotype and -5/-1GC versus -5/-1GA haplotype were not significantly different. Promoter activities of deletion variants {Delta}192 and {Delta}243 were similar to pGL3-Basic construct, while markedly reduced in the {Delta}134 and {Delta}129 deletion variants as compared with the four RET promoter haplotypes tested.

 
The RET promoter contains neither a TATA-box (20) nor a canonical initiator sequence (22) around the transcriptional start site, in the immediate vicinity of the -5/-1 haplotype positions. Furthermore, inspection of the sequence in the immediate vicinity of the -5/-1 position using the TRANSFAC software (23) did not reveal any consensus binding sites for known transcription factors. Despite this, we tested the possibility of discrete binding of trans-acting protein factors to this region in an electrophoretic mobility shift assay. Such binding in dependence of the haplotype sequence could account for the differences in activity observed among the promoter variants. Using nuclear extracts of NMB cells, we could not detect any discrete or differential DNA binding activity recognizing the DNA sequence around the position -5/-1 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The activation of the RET signaling pathway during embryogenesis is a crucial prerequisite for a directional migration of enteric nervous system progenitor cells (24). Loss-of-function germline mutations of the RET proto-oncogene are reported in familial and sporadic cases of HSCR with a variable frequency (3,5–7,16). Independent from these RET germline mutations, variants of several RET polymorphisms are over- or under-represented in HSCR populations. Specifically, the c.135A RET variant has been previously shown as strongly associated with the HSCR phenotype (17,18). We have reported an HSCR-phenotype modifying effect of RET haplotypes characterized by the c.135G>A polymorphism due to a within-gene interaction in patients harboring RET germline mutations (16). These findings could be considered as a synergistic heterozygosity, in which the phenotype is determined by the cumulative effect of at least two genetic heterozygous mutations (15,16).

Although it has been speculated that the c.135A variant may result in alternative splicing (25) neither for the c.135G>A polymorphism nor for any other RET variants associated with HSCR, a functional mechanism of action has been reported. Therefore, we have hypothesized that the c.135G>A polymorphism is a marker rather than a functional variant which is linked with an unknown variant that contributes to the HSCR phenotype.

In the present study we have tested the hypothesis that functional variants are located in the RET promoter region. Although no mutation was identified in the basic RET promoter in 80 HSCR patients, we have revealed an association of the RET promoter -5A and the -1C variants with the HSCR phenotype. By reconstructing haplotypes, a strong linkage disequilibrium between the -5G>A and the c.135G>A polymorphisms and a strong association of the -5/-1/c.135ACA haplotype with the HSCR phenotype could be demonstrated. Our findings indicate stability of the haplotype comprising -5G>A and c.135G>A variants, challenging the notion that there is a broken allelic association of exon 2 with the RET promoter (21).

To investigate the functional relevance of RET promoter variants, we tested the activity of all four promoter haplotypes in a dual-luciferase assay, showing that the occurrence of the -5A variant caused a significantly lower promoter activity. These findings are supported by the generation of RET promoter variants in which the sequence containing the -5G>A and the -1C>A polymorphisms were deleted, resulting in a marked reduction or almost complete disruption of the promoter activity. Loss of AP2, ETF and also of Sp1 binding sites (26,27) in the deletion variants {Delta}192 or {Delta}243 dramatically reduced basal activity of the promoter. However, if at least one Sp1 site and both AP2 and ETF sites are still present, a reduced (but measurable) RET promoter activity remains. This has been shown by the reporter constructs {Delta}134 and {Delta}129, where sequences from -43 to +91 or -41 to +88 were deleted.

Even though direct protein binding assays to a relatively small DNA sequence around promoter haplotype positions were not conclusive, by no means was a function of these nucleotides in transcription control excluded, since transcription initiation is a complex, multi-step process in which several components of the initiation machinery contact up to 100 nucleotides of the core promoter region, including sequences around the transcription initiation site (28). For example, the transcription factor Sp1 can bind in vitro two consensus sites at around position -40 of the RET promoter. This binding activity does not, however, spread downstream towards the transcription start site in the DNAseI footprint analysis (26).

Here, we provide evidence that the c.135G>A polymorphism is a marker for a functional variant in the RET promoter. Our findings substantiate the assumption of a putative functional RET variant linked with the c.135G>A polymorphism located about 25–30 kb upstream the c.135G>A polymorphism (19).

Notably, 36 of 58 HSCR non-mutation carriers (62.1%) reported in our previous study (16) harbor the homozygous -5A/-1C haplotype, while only three of 18 patients with a mutation (16.7%) harbor this homozygous haplotype. Interestingly, in 13 of these mutation carriers, the non-mutated RET allele retains the c.135A variant (72.2%), which is in strong linkage disequilibrium with the -5A/-1C variant, as shown in this study. Hence 49 of 76 HSCR patients studied (64.5%) carried two alleles which were mutated and/or predicted to be expressed at lower levels as compared to the wild type allele.

Based on data generated by us (16,17) and others (18,19), we come to the following conclusions. Several lines of evidence support the idea that expression of both RET alleles contributes to the pathogenesis of Hirschsprung disease in a dose-dependant fashion. In addition, we show that there is a mutation-independent etiological impact of certain haplotypes comprising the -5A RET promoter variant, which is in strong linkage disequilibrium with the c.135A variant. Likewise, we demonstrate that the activity of the basic RET promoter containing the -5A variant is significantly reduced compared with those containing the -5G variant.

These findings are substantiated by our findings that haplotypes comprising the promoter variant at -5 and a RET mutation seem to modulate the mutation-derived HSCR phenotype through a within-gene interaction between mutation and polymorphism. RET haplotypes containing the -5A promoter variant and a HSCR-associated mutation in the coding region on the same chromosome may result in lower expression levels of this allele, conceivably compensated by the second allele (16,25). Based on our data on the transcriptional activity of different RET promoter haplotypes, we speculate that the low -5/-1AA haplotype frequency of <0.0025, which is due to a low promoter activity, is possibly below a threshold level necessary for migration and differentiation of neural crest derived progenitor cells, thus resulting in severe impairment of embryogenesis (29).

In conclusion, the present findings support the concept that the involvement of both RET gene copies occurs in a dose-dependent fashion in RET-associated diseases and confirm the notion of a within-gene interaction between mutation and polymorphisms, with the consecutive modulation of RET-associated phenotypes (16).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Populations
We studied a population of 80 consecutive German Hirschsprung disease patients who fulfilled the histological and immunohistochemical criteria for HSCR: absence of neuronal ganglia on histological evaluation of the aganglionic segment and increased acetylcholinesterase immunoreactivity in nerve fibers on suction-biopsies of the rectal submucosa. Males were 3.7 times more frequently affected than females, and 24 out of 80 (30%) patients had long segment HSCR phenotype in which the aganglionosis reached beyond the rectosigmoid colon. Seventy-six of the 80 patients analyzed in this study have been reported previously. Eighteen of these 76 patients harbour a RET germline mutation (16). A total of 120 anonymous healthy blood donors from Germany served as controls. This population was matched for gender and race but not for age since Hirschsprung disease is a congenital disorder.

All patients and parents (if the patient was a minor) gave written informed consent to participate in the study. The ethics committee of the University of Technology of Dresden approved the study protocol.

RET sequence analysis
Genomic DNA of all tested individuals was obtained from leukocytes in peripheral venous blood samples isolated by standard protocols. The sequence analysis of RET exon 2 was performed applying the primers and methodology reported previously (16). Moreover, we analyzed the basic RET promoter region (20,27) comprising bases -169 to +98 from the transcription start site (as per GenBank NT_033985) after PCR amplification of a 603 bp DNA fragment. Sequence analysis was carried out applying the Thermo SequenaseTM Fluorescent Cycle Sequencing kit (Amersham Pharmacia Biotech, Freiburg, Germany). The sequencing primers were identical to the PCR primers with an additional Cy5 labeling, allowing sequence analysis on A.L.F.express devices (Amersham Pharmacia Biotech, Freiburg, Germany). PCR conditions and primer sequences used are available on request.

Experimental haplotype analysis
Unambiguous haplotype reconstruction from the two genotypes at -5 and -1 from the transcription start site of the RET promoter was possible if one or both genotypes were homozygous. Otherwise, PCR products of the basic RET promoter were subcloned into the pCR®2.1-TOPO® vector (Invitrogen, Karlsruhe, Germany) and replicated after being transformed into competent E. coli TOP10 (Invitrogen, Karlsruhe, Germany) bacteria. Vectors of up to five bacterial clones were purified and sequenced to identify both haplotypes of each PCR product using dideoxynucleotide method of DNA sequencing.

Cell lines and RET expression
The neuroblastoma cell lines NMB (30) and Vi-856 (31) were kindly provided by M. Schwab (DKFZ Heidelberg). Both cell lines were cultered in RPMI 1640 supplemented with heat inactivated 15% FCS, 2 mM;L-glutamine and 5% HEPES, 100 U/ml penicillin and 0.1 mg/ml streptomycin in 5% CO2 at 37°C. All materials were obtained from PAA Laboratories GmbH, Cölbe, Germany.

The RET expression was evaluated by amplification of a cDNA fragment. After whole RNA isolation and reverse transcription according to standard protocols, a 445 bp cDNA fragment (spanning from exon 6 to exon 8; sense-primer, 5'-GCGGGCGTCCTCTTGCTCCACTT-3'; antisense-primer, 5'-GGCCGCCACACTCCTCACACT-3') was amplified in a Perkin-Elmer 9600 thermocycler (Perkin Elmer Applied Biosystems, Weiterstadt, Germany) using 1 µl cDNA reagent in a volume of 25 µl containing 0.1 µM of each oligonucleotide primer, 1.5 mM MgCl2, and 0.75 U Taq polymerase (Invitek, Berlin, Germany). Thermocycling conditions consisted of an initial denaturation at 94°C for 5 min, followed by 35 cycles of 30 s denaturation at 94°C, 30 s annealing at 60°C, and 30 s extension at 72°C, completed by a final extension step for 7 min at 72°C. The amplified cDNA fragment was resolved on 1% agarose gel and verified by DNA sequencing.

Cloning of RET-promoter and construction of luciferase-reporter plasmids
The promoter of RET was PCR-amplified from genomic DNA harboring the three different haplotypes comprising variants at -5 and -1, using primers flanking the basic promoter sequence (20,21). The fourth haplotype -5/-1AA was generated by site-directed-mutagenesis with the megaprimer-method (32). The PCR products were cloned into the pCR®2.1-TOPO® vector (Invitrogen, Karlsruhe, Germany) and sequence analysis was performed with Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit with 7-deaza-dGTP (Amersham Biosciences, Freiburg, Germany) on an automated laser fluorescence sequencer (A.L.F.express, Amersham, Biosciences, Freiburg, Germany). Four 418 bp ApaI–BtgI restriction fragments (reaching from position -246 to +172, respectively) containing the basic RET promoter (20) sequence from -169 to +98 relative to the transcription start site at +1 (as per GenBank clone NT_033985) with the four haplotypes comprising variants at -5 and -1, respectively, were generated. Overhangs were filled using Klenow enzyme. Fragments were cloned into the SmaI site upstream of the firefly luciferase gene of the pGL3-Basic plasmid (Promega, Mannheim, Germany). Constructs were verified by the dideoxynucleotide method of DNA sequencing. PCR conditions, primer sequences and restriction enzyme conditions used are available on request.

Transient transfection and luciferase-reporter assay of various RET promoter haplotypes
To analyze the activity of the four basic RET promoter variants, we used the Dual-Luciferase® Reporter Assay System (Promega, Mannheim, Germany), which allows the normalization of the experimental reporter to the activity of internal controls. Neuroblastoma cell lines NMB and Vi-856 were used for determination of promoter activity after transient transfection. Cells were cotransfected with 5 µg of each plasmid, i.e. pRL-SV40 containing the SV40 early enhancer/promoter region upstream the Renilla luciferase cDNA as internal control and pGL3 containing the basic RET promoter upstream the firefly luciferase cDNA, using Lipofectin®Reagent (Life Technologies, Karlsruhe, Germany) according to the manufacturer's protocol. In addition, we used the pGL3-Control plasmid containing the SV40 early enhancer/promoter region upstream the firefly luciferase and the promoter and enhancerless plasmid pGL3-Basic containing firefly luciferase, which served as positive and negative control, respectively. All plasmids were obtained from Promega, Mannheim, Germany. After transient transfection for 48 h, cells were washed in PBS and harvested by addition of 100 µl of passive lysis buffer. The activity of both luciferases was measured sequentially on a 1450 Micro Beta Trilux Luminometer (Wallac OY, Perkin Elmer Life Sciences, Gaithersburg, MD, USA) using Dual-Luciferase® Reporter Assay reagents according to the manufacturer's protocol. The ratio of firefly luciferase levels to Renilla luciferase levels was used to determine RET promoter activity and expressed as relative luciferase activity. The analysis of each RET promoter construct was performed in three independent experiments, which were repeated four times.

Generation of deletion variants of the pGL-3 RET promoter reporter constructs
The pGL3 vector containing the basic RET promoter upstream the firefly luciferase cDNA was linearized with BssHII restriction endonuclease. The linearized plasmid DNA was treated with BAL-31 exonuclease for 1, 2, 2.5, 3, 3.5 and 4 min at 37°C to generate deletions of varying lengths within the RET promoter. The reactions were stopped by addition of phenol–chloroform. The ethanol-precipitated DNA was religated with T4 DNA ligase (Invitrogen, Karlsruhe, Germany) and the extent of deletions was analyzed by the dideoxynucleotide method of DNA sequencing. Four variants were selected for analysis of the promoter activity: variant {Delta}192 (deletion of -142 to +50 from the transcription start site), variant {Delta}243 (deletion of bases -145 to +98), variant {Delta}134 (deletion of -43 to +91), variant {Delta}129 (deletion of -41 to +88).

Electrophoretic mobility shift assay
Nuclear protein extracts of logarithmically grown NMB cells were prepared according to Andrews and Faller (33). Individual DNA oligonucleotides covering the promoter position -20 to +15 with all four -5/-1 haplotype combinations were synthesized, 32P end-labeled with polynucleotide kinase according to standard protocols and pairwise annealed to 35 bp duplex-DNA probes. Binding reactions (10 µl total, in 10 mM Tris–HCl pH 7.5, 5 mM MgCl2, 0.1% BSA, 5% glycerin, 2 µl nuclear extract, 5 fmol DNA probes and varying amounts of poly dIdC competitor DNA) were incubated for 20 min at room temperature. Samples were loaded on a 5% polyacrylamid gel (AA/BAA 60 : 1, 1xTBE) and electrophoresed for 3 h in 1xTBE buffer at 10 V/cm. Dried gels were subjected to autoradiographic analysis using X-ray films.

Statistical analysis of molecular genetics
To evaluate deviation from the Hardy-Weinberg equilibrium, observed and expected genotype frequencies were compared by an exact goodness of fit test separately in cases and controls. Genotype frequencies between the groups were compared performing Cochrane–Armitage trend tests. Odds ratios, exact 95% confidence intervals, and exact P-values are presented.

Frequencies of haplotypes constructed from the -5G>A, -1C>A and the c.135G>A polymorphisms were estimated with the expectation–maximization algorithm (34). Haplotype frequencies were compared between cases and controls using an exact version of Pearson's {chi}2 test for the experimentally resolved haplotypes. Estimated haplotype frequencies were compared using a score test with simulated P-values from 10 000 replications (35). To account for multiple testing, test-wise P-values were adjusted according to a step-down procedure in conjunction with Sidak. The global significance level was set to 0.05.

Statistical analysis of RET promoter haplotype activity
To investigate functional differences in the four haplotypes -5/-1AA, -5/-1AC, 5/-1GC, -5/-1GA, we have applied a repeated measurement one-way ANOVA separately for NMB and Vi-856. The dependent variable was the log ratio of firefly luciferase and Renilla luciferase after adjusting each expression for blank expression. We estimated the model using the four haplotypes. We set the global significance level to 5% and adjusted the P-value for multiple testing according to a step-down procedure in conjunction with Sidak.

We conducted post-hoc pair-wise comparisons of haplotype functions using Scheffé's test and used graphical methods to investigate deviations from normality and goodness-of-fit.


   ACKNOWLEDGEMENTS
 
This study was supported in part by the Deutsche Forschungsgemeinschaft-grant FI 809/1-1. We thank the patients involved in this study and their parents, and the clinicians for providing samples from their patients. We are grateful to M. Schwab who has kindly provided the neuroblastoma cell lines. The authors also thank Yvonne Kemnitz, Felicitas Zachow and Dr Cordula Büttner for their excellent technical assistance.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +49 3514583598; Fax: +49 3514585343; Email: guido.fitze{at}mailbox.tu-dresden.de Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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