BACKGROUND
peutz-jeghers syndrome  is a rare autosomal dominant disorder typified by hamartomatous polyposis of the gastrointestinal tract and melanin pigmentation of the oro-facial region  <cit> .
although germline mutations in the coding sequence of the serine-threonine kinase gene stk11/lkb <dig> have been found to cause pjs  <cit> , such mutations only account for up to 80% of cases  <cit> .
in addition to locus heterogeneity  <cit>  mutations in regulatory sequences of stk11/lkb <dig> may cause pjs.
identifying regulatory genomic sequences through functional assays is time consuming and problematic.
as natural selection is more likely to tolerate sequence changes in redundant, non-functional sequence than in functionally important sequences, regulatory elements in non-coding sequence will be highly conserved through evolution.
comparison of sequence between both closely related and highly divergent species therefore allows for the identification of non-coding sequence that has a high probability of being important to the regulation of gene transcription.
this alternative approach to the identification of promoter elements is termed "phylogenetic foot printing"  <cit> .
here we describe the phylogenetic foot printing of the 5' upstream region of stk11/lkb <dig> and the sequence analysis of this region in a series of pjs cases in whom exonic and splice site mutations in the gene had been excluded.
RESULTS
additional analysis using the tfbs prediction programs transplorer  and proscan v <dig>   <cit>  identified  <dig> binding sites between nucleotides - <dig> and - <dig>  and  <dig> binding sites between nucleotides - <dig> and - <dig> respectively, confirming the presence of regulatory elements within the consensus region.
ecr browser/ rvista and consite have previously been shown to correctly identify 88% and 66% of tfbs in functionally verified promoter elements respectively <cit>  and it is highly likely, therefore, that the consensus region encompassing nucleotides - <dig> to - <dig>  contains stk11/lkb <dig> promoter elements.
dna from a series of  <dig> pjs patients that did not harbour germline stk11/lkb <dig> mutations  was studied for mutations in the region between nucleotides - <dig> to - <dig>  encompassing all tfbss predicted by rvista and consite.
seven of the cases had a documented family history of pjs.
the diagnosis of pjs in all cases was based on established criteria  <cit> .
three sequence changes were identified.
the change g → c at position - <dig> was found in four cases  and represented a previously documented single nucleotide polymorphism .
an additional single nucleotide change at position - <dig>  was identified in eight cases .
a single sample of control dna used as a sequence reference was also found to be homozygous for the change.
finally, a two base pair deletion coupled with a single base pair insertion at position - <dig>  was identified in one case.
a series of healthy population controls  was screened by high performance liquid chromatography  and one individual  was found to harbour the sequence change.
none of these three sequence changes identified were therefore deemed to be potentially pathogenic.
there is a high degree of redundancy in promoter elements of genes, however point mutations in promoter regions of pten and mlh <dig> have been reported to be disease causing  <cit> .
to investigate the possibility of large-scale deletions or insertions undetectable by straightforward pcr primers p1fwd and p3rev were used to amplify an  <dig> bp fragment with products visualised on a 2% agarose gel.
no large-scale deletions or insertions were detected in any of the patients.
CONCLUSIONS
as understanding of the contribution of coding sequence changes to disease becomes clearer, attention will focus on regulatory elements of genes.
phylogenetic foot printing using programs such as ecr browser and consite present potentially powerful tools in identifying regulatory elements, enabling the analysis of these sequences without time consuming functional studies.
although the efficiency of in-silico delineation of promoter elements has not been rigorously evaluated, ecr browser/ rvista and consite have been shown to correctly identify 88% and 66% of tfbs respectively <cit> .
on the basis of our findings, however, it appears unlikely  that mutations in the promoter region of stk11/lkb <dig> are responsible for pjs cases not attributable to exonic sequence changes.

 peutz-jeghers syndrome is a rare autosomal dominant disorder typified by hamartomatous polyposis of the gastrointestinal tract and melanin pigmentation of the oro-facial region. although germline mutations in the coding sequence of the serine-threonine kinase gene stk11/lkb have been found to cause pjs, such mutations only account for up to 80% of cases. in addition to locus heterogeneity mutations in regulatory sequences of stk11/lkb may cause pjs. identifying regulatory genomic sequences through functional assays is time consuming and problematic. as natural selection is more likely to tolerate sequence changes in redundant, non-functional sequence than in functionally important sequences, regulatory elements in non-coding sequence will be highly conserved through evolution. comparison of sequence between both closely related and highly divergent species therefore allows for the identification of non-coding sequence that has a high probability of being important to the regulation of gene transcription. this alternative approach to the identification of promoter elements is termed "phylogenetic foot printing". here we describe the phylogenetic foot printing of the 5' upstream region of stk11/lkb and the sequence analysis of this region in a series of pjs cases in whom exonic and splice site mutations in the gene had been excluded.  additional analysis using the tfbs prediction programs transplorer and proscan v identified binding sites between nucleotides - and - and binding sites between nucleotides - and - respectively, confirming the presence of regulatory elements within the consensus region. ecr browser/ rvista and consite have previously been shown to correctly identify 88% and 66% of tfbs in functionally verified promoter elements respectively and it is highly likely, therefore, that the consensus region encompassing nucleotides - to - contains stk11/lkb promoter elements. dna from a series of pjs patients that did not harbour germline stk11/lkb mutations was studied for mutations in the region between nucleotides - to - encompassing all tfbss predicted by rvista and consite. seven of the cases had a documented family history of pjs. the diagnosis of pjs in all cases was based on established criteria. three sequence changes were identified. the change g → c at position - was found in four cases and represented a previously documented single nucleotide polymorphism. an additional single nucleotide change at position - was identified in eight cases. a single sample of control dna used as a sequence reference was also found to be homozygous for the change. finally, a two base pair deletion coupled with a single base pair insertion at position - was identified in one case. a series of healthy population controls was screened by high performance liquid chromatography and one individual was found to harbour the sequence change. none of these three sequence changes identified were therefore deemed to be potentially pathogenic. there is a high degree of redundancy in promoter elements of genes, however point mutations in promoter regions of pten and mlh have been reported to be disease causing. to investigate the possibility of large-scale deletions or insertions undetectable by straightforward pcr primers p1fwd and p3rev were used to amplify an bp fragment with products visualised on a 2% agarose gel. no large-scale deletions or insertions were detected in any of the patients.  as understanding of the contribution of coding sequence changes to disease becomes clearer, attention will focus on regulatory elements of genes. phylogenetic foot printing using programs such as ecr browser and consite present potentially powerful tools in identifying regulatory elements, enabling the analysis of these sequences without time consuming functional studies. although the efficiency of in-silico delineation of promoter elements has not been rigorously evaluated, ecr browser/ rvista and consite have been shown to correctly identify 88% and 66% of tfbs respectively. on the basis of our findings, however, it appears unlikely that mutations in the promoter region of stk11/lkb are responsible for pjs cases not attributable to exonic sequence changes. 
germline mutations or large-scale deletions in the coding region and splice sites of stk11/lkb do not account for all cases of peutz-jeghers syndrome. phylogenetic foot printing and transcription factor binding site prediction of sequence 5' to the coding sequence of stk11/lkb was performed to identify non-coding sequences of dna indicative of regulatory elements. a series of pjs cases in whom no mutation in stk11/lkb could be identified were screened for sequence changes in the putative promoter defined by nucleotides - to - two novel sequence changes were identified, but were found to be present in healthy individuals. these findings indicate that promoter sequence changes are unlikely to contribute to pjs. it is conceivable that, on the basis of data from other diseases, inherited variation in promoter elements of stk11/lkb may cause pjs.
 germline mutations or large-scale deletions in the coding region and splice sites of stk11/lkb do not account for all cases of peutz-jeghers syndrome. it is conceivable that, on the basis of data from other diseases, inherited variation in promoter elements of stk11/lkb may cause pjs.  phylogenetic foot printing and transcription factor binding site prediction of sequence 5' to the coding sequence of stk11/lkb was performed to identify non-coding sequences of dna indicative of regulatory elements. a series of pjs cases in whom no mutation in stk11/lkb could be identified were screened for sequence changes in the putative promoter defined by nucleotides - to - two novel sequence changes were identified, but were found to be present in healthy individuals.  these findings indicate that promoter sequence changes are unlikely to contribute to pjs. 