Sarah Hatch Royal Liverpool Hospital

1. Sarah Hatch Royal Liverpool Hospital

2. Juvenile Polyposis Syndrome Polyposis defined by the presence of multiple polyps in the gut lumen. Juvenile polyps occur in 2% of children: 10 or more juvenile polyps for juvenile polyposis. 1/3 of cases are hereditary: juvenile polyposis syndrome, autosomal dominant (1:100,000). Predisposion to hamartomatous GI polyps and subsequent cancer: 17% risk of malignancy at 33 years, increasing to 68% by age 60. Polyposis is a condition defined by the presence of multiple polyps in the gut lumen. Juvenile polyps occur sporadically in around 2% of children, thus juvenile polyposis is defined as the presence of 10 or more juvenile polyps. Around 1/3 of cases of juvenile polyposis are familial, a rare condition known as juvenile polyposis syndrome. The primary feature of JPS is the development of multiple polyps in the gut lumen. The subsequent risk of malignancy is extremely high and becomes an overwhelming probability with increasing age, rising from 17% at the age of 33 to 68% by the age of 60. Polyposis is a condition defined by the presence of multiple polyps in the gut lumen. Juvenile polyps occur sporadically in around 2% of children, thus juvenile polyposis is defined as the presence of 10 or more juvenile polyps. Around 1/3 of cases of juvenile polyposis are familial, a rare condition known as juvenile polyposis syndrome. The primary feature of JPS is the development of multiple polyps in the gut lumen. The subsequent risk of malignancy is extremely high and becomes an overwhelming probability with increasing age, rising from 17% at the age of 33 to 68% by the age of 60.

3. Presentation Infancy: Acute/chronic gastrointestinal bleeding, intussusception, rectal prolapse, protein-losing enteropathy, failure to thrive. Adulthood: Gastrointestinal blood loss. Patients usually JPS may manifest at any age, though the age of onset is typically at around 18. Infants typically present with acute or chronic gastrointestinal bleeding, intussusception (which is telescoping of one portion of the bowel into another), rectal prolapse, protein-losing enteropathy and failure-to-thrive. The primary presenting symptom in adults is acute or chronic gastrointestinal bleeding, and examination typically reveals the presence of 50 to 200 polyps, mostly in the rectosigmoid region.JPS may manifest at any age, though the age of onset is typically at around 18. Infants typically present with acute or chronic gastrointestinal bleeding, intussusception (which is telescoping of one portion of the bowel into another), rectal prolapse, protein-losing enteropathy and failure-to-thrive. The primary presenting symptom in adults is acute or chronic gastrointestinal bleeding, and examination typically reveals the presence of 50 to 200 polyps, mostly in the rectosigmoid region.

4. Histology of Juvenile Polyps Hamartomatous polyps throughout the GI tract continue to be formed during individual�s lifetime. Gross infiltration of lamina propia by inflammatory cells causes attenuation of underlying smooth Juvenile polyps are formed throughout an individual�s lifetime and are hamartomatous in origin, which means that they are benign masses of abnormally developed native tissue. Histological examination reveals gross infiltration of the lamina propia by lymphocytes and plasma cells, leading to attenuation of the underlying smooth muscle and pathognomonic cystic dilation of glands. Histological confusion with other polyposis syndromes, such as Cowden, is common and may hinder optimal clinical management. JPS is diagnosed/defined histologically. Hamartoma: a benign mass of indigenous tissue resulting from faulty development in an organ and composed of an abnormal mixture of tissue elements that develop and grow at the same rate as normal elements but are not likely to compress adjacent tissue. Polyps are smooth and unilobular, ranging in size from mm to cm. Lamina propia is a lumenal muscle layer of the intestine (between the epithelium and smooth muscle?)Juvenile polyps are formed throughout an individual�s lifetime and are hamartomatous in origin, which means that they are benign masses of abnormally developed native tissue. Histological examination reveals gross infiltration of the lamina propia by lymphocytes and plasma cells, leading to attenuation of the underlying smooth muscle and pathognomonic cystic dilation of glands. Histological confusion with other polyposis syndromes, such as Cowden, is common and may hinder optimal clinical management. JPS is diagnosed/defined histologically. Hamartoma: a benign mass of indigenous tissue resulting from faulty development in an organ and composed of an abnormal mixture of tissue elements that develop and grow at the same rate as normal elements but are not likely to compress adjacent tissue. Polyps are smooth and unilobular, ranging in size from mm to cm. Lamina propia is a lumenal muscle layer of the intestine (between the epithelium and smooth muscle?)

5. Clinical Management Regular invasive monitoring by endoscopy for JPS patient and first degree relatives: surgical resection of occasional polyps, prophylactic colectomy or gastrectomy if polyposis is diffuse. Compliance is typically poor. (1) The clinical management of JPS patients and their first-degree relatives is an invasive regime of regular endoscopic monitoring, commencing at age 15 and repeated every three years, for which compliance is poor. (2) Occasional polyps are surgically resected, (3) but radical prophylactic gastrectomy or colectomy is recommended if the polyposis is diffuse. (4) Follow-up screening is then annual.(1) The clinical management of JPS patients and their first-degree relatives is an invasive regime of regular endoscopic monitoring, commencing at age 15 and repeated every three years, for which compliance is poor. (2) Occasional polyps are surgically resected, (3) but radical prophylactic gastrectomy or colectomy is recommended if the polyposis is diffuse. (4) Follow-up screening is then annual.

6. There is a need to develop an effective JPS screening tool for elimination of non-sufferers from endoscopic surveillance and to focus appropriate clinical management on those at risk. Thus, the development of a genetic screening test to eliminate non-carriers would minimise unnecessary invasive testing and enable more focussed clinical surveillance for those known to be at risk. Will allow a definitive diagnosis of JPS (i.e. no more confusion with other polyposes), ensuring that appropriate care is given.Thus, the development of a genetic screening test to eliminate non-carriers would minimise unnecessary invasive testing and enable more focussed clinical surveillance for those known to be at risk. Will allow a definitive diagnosis of JPS (i.e. no more confusion with other polyposes), ensuring that appropriate care is given.

7. A variety of causative mutations for JPS have been identified in genes encoding two different tumour suppressors, (1) SMAD4 and (2) BMPR1A. A variety of causative mutations for JPS have been identified in genes encoding two different tumour suppressors, (1) SMAD4 and (2) BMPR1A.

8. Both SMAD4 and BMPR1A are involved in TGF-� signal transduction. (1) Phosphorylation of cytoplasmic SMADs occurs following receptor dimerisation by TGF-�. (2) The common mediator SMAD4 binds to other phosphorylated SMADs and causes them to be translocated to the nucleus, where they act to inhibit growth by affecting transcription. (3) The effect of a fault in this pathway is therefore to allow escape from growth control and apoptosis.Both SMAD4 and BMPR1A are involved in TGF-� signal transduction. (1) Phosphorylation of cytoplasmic SMADs occurs following receptor dimerisation by TGF-�. (2) The common mediator SMAD4 binds to other phosphorylated SMADs and causes them to be translocated to the nucleus, where they act to inhibit growth by affecting transcription. (3) The effect of a fault in this pathway is therefore to allow escape from growth control and apoptosis.

9. PCR Basics Specific amplification of oligonucleotides

10. DHPLC is a sensitive and specific tool for mutation detection. The first step in DHPLC is PCR amplification of genomic DNA. The amplified DNA fragments are then rapidly denatured by heating, followed by slow cooling to allow re-annealing. Some heteroduplexes will form whenever a mutant allele is present: these contain a region in which the DNA is looped-out around the mismatched region.DHPLC is a sensitive and specific tool for mutation detection. The first step in DHPLC is PCR amplification of genomic DNA. The amplified DNA fragments are then rapidly denatured by heating, followed by slow cooling to allow re-annealing. Some heteroduplexes will form whenever a mutant allele is present: these contain a region in which the DNA is looped-out around the mismatched region.

11. Chromatographic separation is achieved using a reverse-phase ion-pair system and a gradient of an organic mobile phase, with UV detection of eluted fragments. The column oven temperature is selected to cause partial denaturation of the marginally less stable heteroduplexes, leading to earlier elution. Thus, up to three additional early-eluting peaks may be observed in the presence of an affected allele. 4 theoretical peaks. TEAA ion-pairing agent. Partially denatured DNA has exposed hydrophobic bases, increasing the partitioning into the organic mobile phase and thereby causing earlier elution.Chromatographic separation is achieved using a reverse-phase ion-pair system and a gradient of an organic mobile phase, with UV detection of eluted fragments. The column oven temperature is selected to cause partial denaturation of the marginally less stable heteroduplexes, leading to earlier elution. Thus, up to three additional early-eluting peaks may be observed in the presence of an affected allele. 4 theoretical peaks. TEAA ion-pairing agent. Partially denatured DNA has exposed hydrophobic bases, increasing the partitioning into the organic mobile phase and thereby causing earlier elution.

12. Previously-published PCR primers were employed for the amplification of each of the eleven exons of SMAD4, with optimal conditions determined on a gradient of increasing annealing temperature. Choose the lowest temperature that gives a bright band.Previously-published PCR primers were employed for the amplification of each of the eleven exons of SMAD4, with optimal conditions determined on a gradient of increasing annealing temperature. Choose the lowest temperature that gives a bright band.

13. WAVEMAKER� software was used to analyse the theoretical melting of each sequence, yielding profiles of helical fraction versus nucleotide base position across a range of temperatures. Here you can see the progression from partially unwound fragment ends to full denaturation. A range of partially denaturing column oven temperatures was selected for the analysis of each exon. WAVEMAKER� software was used to analyse the theoretical melting of each sequence, yielding profiles of helical fraction versus nucleotide base position across a range of temperatures. Here you can see the progression from partially unwound fragment ends to full denaturation. A range of partially denaturing column oven temperatures was selected for the analysis of each exon.

14. Wild type samples were analysed across the range of partially-denaturing column oven temperatures to characterise the DHPLC profiles of unaffected individuals. Samples containing known mutations were analysed whenever possible. For this purpose, DNA from a number of different colorectal cancer cell lines containing SMAD4 mutations was obtained by kind donation from Professor Ian Tomlinson of Cancer Research UK. Wild type samples were analysed across the range of partially-denaturing column oven temperatures to characterise the DHPLC profiles of unaffected individuals. Samples containing known mutations were analysed whenever possible. For this purpose, DNA from a number of different colorectal cancer cell lines containing SMAD4 mutations was obtained by kind donation from Professor Ian Tomlinson of Cancer Research UK.

15. Following screening of around 15 JPS patient samples a single family affected with a SMAD4 exon 1 mutation were identified by the presence of three discrete peaks in two siblings at 53 oC. A third sibling was unaffected. This mutation was subsequently sequenced and identified as a large insertion.Following screening of around 15 JPS patient samples a single family affected with a SMAD4 exon 1 mutation were identified by the presence of three discrete peaks in two siblings at 53 oC. A third sibling was unaffected. This mutation was subsequently sequenced and identified as a large insertion.

16. In total, five different known mutations were successfully characterised. The first, shown here, was a nonsense mutation in exon 2 from a JPS patient. The presence of the mutation is distinguished by two additional early-eluting peaks at 57 oC, which indicate the presence of heteroduplexes. In total, five different known mutations were successfully characterised. The first, shown here, was a nonsense mutation in exon 2 from a JPS patient. The presence of the mutation is distinguished by two additional early-eluting peaks at 57 oC, which indicate the presence of heteroduplexes.

17. Nonsense Mutation in Exon 5 A nonsense mutation in exon 5 was identified by an additional peak at 59 oC.A nonsense mutation in exon 5 was identified by an additional peak at 59 oC.

18. Missense Mutation in Exon 8 Here, a missense mutation in exon 8 has produced a couple of earlier peaks at 56 oC.Here, a missense mutation in exon 8 has produced a couple of earlier peaks at 56 oC.

19. Missense Mutation in Exon 11 Finally, a missense mutation in exon 11 was identified by a small additional peak at 60 oC.Finally, a missense mutation in exon 11 was identified by a small additional peak at 60 oC.

20. Final screening protocol restricted to minimum number of temperatures required to cover melting across entirety of each fragment with minimal overlap. The number of column oven temperatures included in the final screening protocol was minimised in order to optimise the practicality. An unknown mutation could occur anywhere within the gene, and mutation detection is reported to be optimal at a helical fraction of 70-85%, therefore temperatures that give rise to melting domains spanning the entire length of each exon with minimal overlap were chosen. The clarity of elution profiles was also considered, as clearly defined homoduplexes are essential for reliable mutation detection. Generally, the lowest possible temperatures were chosen in order to minimise peak broadening. The number of column oven temperatures included in the final screening protocol was minimised in order to optimise the practicality. An unknown mutation could occur anywhere within the gene, and mutation detection is reported to be optimal at a helical fraction of 70-85%, therefore temperatures that give rise to melting domains spanning the entire length of each exon with minimal overlap were chosen. The clarity of elution profiles was also considered, as clearly defined homoduplexes are essential for reliable mutation detection. Generally, the lowest possible temperatures were chosen in order to minimise peak broadening.

21. Practicality of Screening SMAD4 The final protocol devised for the screening of all exons of SMAD4 involves 10 PCRs and 35 DHPLC analyses, which represents a significant workload for the investigation of even a single index case. It is therefore considered practical to refine the protocol further by assigning exon priorities according to the frequency with which mutations have previously been observed. As can be seen, mutation in exons 8, 9, 10 and 11 are particularly frequent. Estimated time for a single index case: one day to prepare all fragments by PCR (depends upon availability of machines), DHPLC for single index plus control estimated at 12 hours (performed overnight). Batch-wise run unlikely. Spectrum of germline mutations in SMAD4. Mutational �hotspot� in exon 9 of SMAD4.The final protocol devised for the screening of all exons of SMAD4 involves 10 PCRs and 35 DHPLC analyses, which represents a significant workload for the investigation of even a single index case. It is therefore considered practical to refine the protocol further by assigning exon priorities according to the frequency with which mutations have previously been observed. As can be seen, mutation in exons 8, 9, 10 and 11 are particularly frequent. Estimated time for a single index case: one day to prepare all fragments by PCR (depends upon availability of machines), DHPLC for single index plus control estimated at 12 hours (performed overnight). Batch-wise run unlikely. Spectrum of germline mutations in SMAD4. Mutational �hotspot� in exon 9 of SMAD4.

22. Method Limitations Inclusion of introns? Likelihood of mutation - how much to include? High sequence variation gives rise to multiple peaks for wild type samples. The biggest limitation of the current method relates to the decision to save time by using published PCR primers. The relatively large intronic sequences present at the ends of the amplicons were originally considered a bonus as they would enable identification of splicing mutations. However, it is now apparent that the inclusion of poorly conserved introns can lead to multiple anomalous peaks for unaffected individuals, as exemplified by the exon 5/6 fragment. Sequencing revealed a long polymorphic sequence in intron 7.The biggest limitation of the current method relates to the decision to save time by using published PCR primers. The relatively large intronic sequences present at the ends of the amplicons were originally considered a bonus as they would enable identification of splicing mutations. However, it is now apparent that the inclusion of poorly conserved introns can lead to multiple anomalous peaks for unaffected individuals, as exemplified by the exon 5/6 fragment. Sequencing revealed a long polymorphic sequence in intron 7.

23. Method Limitations (cont.) Cannot detect mutations that do not amplify. Gross deletion of gene not detected. Allelic drop-out could occur (would only see homoduplexes�). SMAD4 mutations only account for 20% of JPS DHPLC will fail to detect mutations that do not amplifyDHPLC will fail to detect mutations that do not amplify

24. Established routine method at London North West Thames Regional Genetics Service (Kennedy-Galton Centre) UKGTN gene dossier for JPS Planned development of DHPLC for BMPR1A (20% of JPS cases) Approved for publication as a short report in Annals of Clinical Biochemistry Somatic mutational inactivation of SMAD4 reported in pancreatic, prostatic and colorectal carcinomas. Mutations in SMAD4 account for around 18.2% of index cases reported to date, with 20.8% for BMPR1ASomatic mutational inactivation of SMAD4 reported in pancreatic, prostatic and colorectal carcinomas. Mutations in SMAD4 account for around 18.2% of index cases reported to date, with 20.8% for BMPR1A

25. Acknowledgements Ms. Anne Trewick, Clinical Biochemistry Department, Northwick Park Hospital. Mr. Stewart Payne, Kennedy-Galton Genetics Centre. Professor Ian Tomlinson, Cancer Research UK.

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27. Any Questions?