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Analysis of genetic and phenotypic heterogeneity in juvenile polyposis
  1. K Woodford-Richensa,
  2. S Bevanb,
  3. M Churchmanc,
  4. B Dowlingc,
  5. D Jonesd,
  6. C G Norburyd,
  7. S V Hodgsone,
  8. D Desaif,
  9. K Nealef,
  10. R K S Phillipsf,
  11. J Youngg,
  12. B Leggettg,
  13. M Dunloph,
  14. P Rozeni,
  15. C Engj,
  16. D Markiek,
  17. M A Rodriguez-Bigasl,
  18. E Sheridanm,
  19. T Iwaman,
  20. D Eccleso,
  21. G T Smithp,
  22. J C Kimq,
  23. K M Kimq,
  24. J R Sampsonr,
  25. G Evanss,
  26. S Tejpart,
  27. W F Bodmeru,
  28. I P M Tomlinsona,
  29. R S Houlstonb
  1. aMolecular and Population Genetics Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, UK, bCancer Genetics, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK, cTumour Genetics Group, Nuffield Department of Clinical Medicine, University of Oxford, Wellcome Trust Centre for Human Genetics, Windmill Road, Oxford OX3 7BN, UK, dMolecular Genetics Laboratory, Churchill Hospital, Oxford OX3 7LJ, UK, eDepartment of Clinical Genetics, Guy's Hospital, St Thomas's Street, London SE1 9RT, UK, fPolyposis Registry, St Mark's Hospital, Watford Road, Harrow HA1 3UJ, UK, gQueensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Australia, hMRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK, iDepartment of Gastroenterology, Tel Aviv Medical Center and Medical School, IL-64239 Tel Aviv, Israel, jHuman Cancer Genetics Program, Ohio State University Comprehensive Cancer Center, Columbus, OH 43210, USA, kMolecular Genetics Laboratory, Pathology Department, Dunedin School of Medicine, Dunedin, New Zealand, lRoswell Park Cancer Institute, Buffalo, New York 14263, USA, mDepartment of Clinical Genetics, Royal Hospital for Children, St Michael's Hill, Bristol, UK, nCentre for Polyposis and Intestinal Diseases, 1–5–45, Yushima, Bunkyo-Ku, Tokyo 113, Japan, oWessex Regional Genetics Service, Southampton SO16 5YA, UK, pDepartment of Histopathology, Worcester Royal Infirmary, WR1 3AS, UK, qDepartment of Paediatrics, University of Ulsan College of Medicine, and Asan Institute, Seoul, Korea, rInstitute of Medical Genetics, University of Wales College of Medicine, Cardiff CF4 4XN, UK, sRegional Genetic Service, St Mary's Hospital, Manchester M13 0JH, UK, tCentre for Human Genetics, Campus Gasthuisberg, O and N6, B-3000, Leuven, Belgium, uCancer Immuno-genetics Laboratory, ICRF, Institute of Molecular Medicine, Oxford OX3 9DS, UK
  1. R Houlston, Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK. Email:r.houlston{at}


BACKGROUND Juvenile polyposis syndrome (JPS) is characterised by gastrointestinal (GI) hamartomatous polyposis and an increased risk of GI malignancy. Juvenile polyps also occur in the Cowden (CS), Bannayan-Ruvalcaba-Riley (BRRS) and Gorlin (GS) syndromes. Diagnosing JPS can be problematic because it relies on exclusion of CS, BRRS, and GS. Germline mutations in the PTCH, PTENand DPC4 (SMAD4)genes can cause GS, CS/BRRS, and JPS, respectively.

AIMS To examine the contribution of mutations in PTCH,PTEN, and DPC4(SMAD4) to JPS.

METHODS Forty seven individuals from 15 families and nine apparently sporadic cases with JPS were screened for germline mutations inDPC4, PTEN, andPTCH.

RESULTS No patient had a mutation in PTEN orPTCH. Five different germline mutations were detected in DPC4; three of these were deletions, one a single base substitution creating a stop codon, and one a missense change. None of these patients had distinguishing clinical features.

CONCLUSIONS Mutations in PTEN and PTCHare unlikely to cause juvenile polyposis in the absence of clinical features indicative of CS, BRRS, or GS. A proportion of JPS patients harbour DPC4 mutations (21% in this study) but there remains uncharacterised genetic heterogeneity in JPS.

  • juvenile polyposis syndrome
  • germline mutations

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Juvenile polyposis (JPS; MIM 174 900) is a rare autosomal dominant condition characterised by hamartomatous polyps, usually within the colon but occasionally arising in the stomach and small bowel.1 These polyps are typified by a predominant stroma, cystic spaces, and an abundant lamina propria lacking smooth muscle, so distinguishing them from Peutz-Jeghers polyps. Unlike solitary juvenile polyps which may affect up to 2% of children and adolescents and have little or no malignant potential, JPS patients have an increased risk of gastrointestinal malignancy.2 3 JPS may occur in association with arteriovenous malformations (MIM 175 050) but it is not clear if this represents a distinct syndrome.

Juvenile polyps also occur as a manifestation of the dominantly transmitted familial cancer syndromes: Cowden syndrome (CS; MIM 158 350) characterised by multiple hamartomas, macrocephaly, trichilemmomas, and a high risk of benign and malignant neoplasms of the thyroid, breast, uterus and skin; Bannayan-Ruvalcaba-Riley syndrome (Bannayan-Zonana syndrome, BRRS, BZS; MIM 153 480) characterised by mental retardation, macrocephaly, lipomatosis, haemangiomas and genital pigmentation; and Gorlin syndrome (GS; MIM 109 400) characterised by multiple naevoid basal carcinomas, skeletal abnormalities, and odontogenic keratinocytes, macrocephaly, intracranial calcification, and craniofacial abnormalities. Compared with JPS the risk of gastrointestinal malignancy in CS, BRRS, and GS appears to be low.4

GS results from germline mutations in thePTCH gene (homologue ofDrosophila patched) on chromosome 9q22.1.5 Juvenile polyps appear to comprise a relatively minor and infrequent component of this disease although few GS patients undergo gastrointestinal screening. Nevertheless,PTCH remains a good candidate for JPS given the possibility that a different spectrum of mutations might cause juvenile polyps without the other features of GS. No study to date has tested PTCH for germline mutations in JPS patients.

The CS gene is PTEN (phosphatase and tensin homologue deleted on chromosome 10 (10q22-q23)),6 a ubiquitously expressed dual specificity phosphatase that acts as a tumour suppressor and is mutated in several sporadic tumour types.7-12 The inference from patients' clinical features that CS and BRRS might represent allelic forms of the same disease is supported by demonstration of germline mutations inPTEN in some13-15 but not all patients with BRRS.16 The shared clinical features of CS/BRRS and JPS, coupled with coincident somatic mutation data in juvenile polyps,17 raised the possibility thatPTEN could cause all of these syndromes. This hypothesis has been pursued by a number of workers with differing findings.18-22 Some studies found germlinePTEN mutations in individuals who had been diagnosed as having JPS but there is doubt that a diagnosis of CS/BRRS had been formally excluded in these patients. We have previously found no PTEN mutations in a relatively large set of JPS patients (including a subset of the cases reported below).

Recently it has been shown that constitutional mutations inDPC4 (deleted in pancreatic carcinoma locus 4; also referred to as SMAD4, small mothers against decapentaplegic deleted in pancreatic carcinoma, locus 4) can cause JPS.23 24 This gene, which codes for a protein involved in transforming growth factor β signal transduction, is mutated in a number of gastrointestinal cancers.25 26Previous studies have reported DPC4mutations in 5/9 (56%) and 1/21 (5%) JPS patients (the latter study including a subset of the cases reported below). Other members of the SMAD family are candidates for JPS but no mutations inSMAD1/2/3/5/6/7 have been found in JPS patients.27

Hereditary mixed polyposis syndrome (HMPS, MIM 601 228) is also characterised by atypical juvenile polyps with mixed features of hamartomas and adenomas.28 No gene for this syndrome has been identified but linkage to chromosome 6q has been reported in one large family.29

Hence there is evidence of considerable genetic heterogeneity in JPS. We have studied 56 individuals with a presumptive diagnosis of JPS, comprising 47 cases from 15 JPS families and nine apparently sporadic cases. These patients comprise an exceptionally large set of JPS cases. Our aims were: (i) to describe the clinical features of JPS patients (including features overlapping with CS, BRRS, and GS); (ii) to resolve the debate as to whether or not PTENmutations account for any cases of JPS; (iii) to testPTCH as a candidate gene for JPS; (iv) to determine the contribution of DPC4 mutations to JPS; (v) to assess the existence of any associations between germline mutations and clinical features; and (vi) to determine the proportion of JPS cases caused by as yet unidentified genes.

Patients and methods

Patients with JPS were identified from sources in the UK, Australia, Israel, USA, Japan, and Korea. The diagnostic criteria for JPS used in this study were in accordance with the proposal of Ko and colleagues30 that affected individuals have either five or more juvenile polyps throughout the gastrointestinal tract or any number of juvenile polyps and a family history of JPS. All affected individuals had more than one typical juvenile polyp as confirmed by histology. The following clinical data were also obtained using a standardised proforma: family history; occurrence of benign and malignant tumours; dysmorphic features; arteriovenous malformations and other cardiovascular anomalies; pigmentation; and other notable clinical features including those of CS, BRRS, and GS. Where possible, material from JPS polyps and other tumours was obtained for histological review and confirmation of diagnosis.

Constitutive DNA from individuals was extracted from EDTA blood samples using a standard sucrose lysis method. Published oligonucleotide and reaction conditions were used to amplify specifically each exon of theDPC4, PTEN, andPTCH genes.20 24 31 The search for germline mutations in DPC4 was performed using conformation specific gel electrophoresis (CSGE). Two independent workers using either CSGE or single strand conformational gel polymorphism (SSCP) undertook screening for mutations inPTEN. A combination of CSGE and SSCP was used to screen the PTCH gene. All samples with band shifts were sequenced in duplicate and in forward and reverse orientations, after reamplification of the appropriate exon from genomic DNA in the polymerase chain reaction, using the ABI Ready Reaction Dye Terminator Cycle Sequencing kit and the 377 Prism sequencer.


Fifty six patients with JPS were studied; 47 were ascertained from 15 families and nine apparently sporadic patients had no known relatives affected with JPS, although in three cases there was a strong family history of colorectal cancer. Table 1 details the clinical characteristics of all patients. Mean age at presentation of juvenile polyposis was 23 (SD 16) years (range 1–65). All affected individuals from whom histological material was available had juvenile polyps of a typical type.

Table 1

Clinical features of the patients studied

The extent of polyposis within the gastrointestinal tract varied considerably between families in terms of both the numbers and sites of polyp formation. Some of the families showed marked evidence of intrafamilial differences in disease expression (table 1) even allowing for factors such as patient age and screening by colonoscopy. Furthermore, in a number of patients there was documented evidence of adenomatous intestinal polyposis although none had the atypical juvenile polyps found in HMPS.

The increased risk of gastrointestinal cancer reported in association with JPS was clearly present in our patients. Fifteen (27%) had developed some type of gastrointestinal cancer by the age of 65. Of these, six were small bowel carcinomas, two gastric cancers, and seven colorectal cancers. Based on cancer incidence rates in the UK,32 this equates to about a 16-fold increased risk of gastrointestinal malignancy in JPS.

None of the patients had prototypical dermatological (e.g. tricholemmomas) or skeletal phenotypic features indicative of CS, BRRS, or GS. There was some overlap between the clinical features of JPS patients and these other syndromes—three patients had macrocephaly for example—but no specific features to suggest misdiagnosis. Furthermore, none of the patients had a past history of thyroid cancer and only one had developed breast cancer. Three patients had hyper- or hypothyroidism but no tissue diagnosis suggestive of CS. The increased risk of cancer in these JPS patients appeared to be confined to the gastrointestinal tract in contradistinction to CS and GS. Arteriovenous and other cardiovascular anomalies were present in three patients (5%). The reported association between JPS and these anomalies may therefore be genuine.

No JPS patient had a germline PTEN mutation. Control samples with known PTEN mutations showed aberrant SSCP bands and previously reported polymorphisms were detected. PTEN mutations are typically found in 80% of CS and 50% of BRRS cases which adhere to operational diagnostic criteria established by the International Cowden Consortium.33 Also, germlinePTCH mutations were not detected in our JPS cases. PTCH mutations are typically detectable in 39% of GS patients.31

Five germline DPC4 mutations were identified (table 2). Three of these were deletions ranging in size from two to 11 base pairs in exons 1, 4, and 11. One of the mutations was a single base substitution creating a stop codon in exon 10. The fifth mutation was a missense mutation in exon 8. This variant has been reported previously.24 Our series therefore confirms that mutations in DPC4 are associated with JPS but the mutations we detected did not involve the trimerisation domain of theDPC4 protein. There were no distinguishing clinicopathological features in those patients harbouringDPC4 mutations.

Table 2

Description of DPC4 mutations identified in JPS patients


Several syndromes—JPS, CS, BRRS, GS, and HMPS—include juvenile polyps in their phenotype. These diseases can have subtle manifestations and some of their clinical features are not unique. Hence diagnosis is not straightforward. However, it is important to distinguish between these syndromes as the types of cancer associated with each appear to be quite different. A more reliable and objective means of differentiating syndromes than reliance solely on clinical features is clearly desirable, especially in the case of JPS the diagnosis of which is made in part by exclusion. Determining the molecular basis of each of these syndromes clearly offers the best method of establishing a diagnosis.

Until recently the molecular basis of the hamartomatosis polyposis syndromes was unknown. Early somatic data suggested the existence of a putative locus for JPS at 10q22–24, encompassingPTEN.17 This observation led several researchers to examine PTEN as a candidate for JPS. No linkage to 10q22 or mutation inPTEN was detected in either the JPS families or isolated cases studied by Marsh and colleagues.20However, both Olschwang and colleagues21 and Lynch and colleagues18 reported germlinePTEN mutations in patients with multiple juvenile polyps, suggesting that PTEN was the cause of some JPS cases. Eng has recently questioned their conclusion.33 In the study by Olschwang and colleagues,21 the adult male patient (G116) also had a laryngeal carcinoma and a heterogeneous thyroid nodule, which is highly suggestive of CS. Furthermore, although both children in the report (G796 and G710) did not have features indicative of CS or BRRS, they were less than 15 years of age. As the penetrance of CS is only 10% by this age34 it is possible that both children will develop features of CS later in life. Similarly, the affected members of the family reported by Lynch and colleagues18 had major clinical features consistent with a diagnosis of CS.

Our mutational analysis of JPS patients is the most comprehensive to date. The findings concur with previous work suggesting that the probability of detecting a constitutive mutation inPTEN is not high if a patient's clinical phenotype does not adhere to the International Cowden Consortium operational diagnostic criteria for CS33 or to the specific clinical features of BRRS.

PTCH is a good candidate for JPS but has not previously been tested for germline mutations in JPS patients. We have excluded this gene as a cause of juvenile polyps outside the setting of GS. Although PTCH mutations may directly lead to gastrointestinal hamartoma formation it remains possible that reports of juvenile polyps in GS result from a chance association or from contiguous deletion of PTCH and at least one other gene.

Recent work has identified germline mutations in theDPC4 (SMAD4) gene on 18q21.1 as a cause of some cases of JPS.23 In our study 21% of JPS could be ascribed to germline mutations inDPC4 (five of 24; comprising 15 families and nine sporadic cases tested). This provides further support for the role of mutations in this gene as a cause of JPS. Failure to detect mutations in DPC4 in the other patients is unlikely to reflect problems in the screening method alone: it is possible that some mutations may be in the UTRs, introns, or promoter region of the gene but we have screened all exons and splice sites and have found that CSGE can detect all small insertions, deletions, and 90% of single base substitutions under such conditions.

DPC4/SMAD4 is a pivitol component of the transforming growth factor β signal transduction pathway.35 Through hetero-oligomer formation by interaction with SMAD1 (and possibly 5) andSMAD2 (and possibly 3),SMAD4 mediates apoptotic and growth inhibition responses. Hamartoma formation in JPS presumably results from disruption of the transforming growth factor β signal transduction pathway.35 In our study mutations inSMAD4 were detected in exons 1, 4, 8, 10, and 11. Pathogenic mutations previously reported were in exons 5, 8, and 9.23 A wide range of mutations can clearly affect cellular responses to transforming growth factor β leading to hamartoma formation. Whether specific genotype-phenotype relationships exist await further studies.

We conclude that the clinical features of Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome, and Gorlin syndrome can be used to distinguish patients who are likely to carry germline mutations inPTEN and PTCH and who do not, therefore, have a diagnosis of JPS. The rare syndrome HMPS can probably be distinguished from JPS on histological grounds, and there is no evidence for linkage of JPS to the HMPS locus (although this cannot be confirmed until the HMPS gene is identified). Combining our findings and those reported by Howe and colleagues23suggests that up to 40% of JPS cases might be caused by germline mutations in DPC4 although these patients cannot be distinguished from the majority of JPS patients who harbour germline mutations in uncharacterised genes. Current evidence does not suggest any common mode of action of PTEN, PTCH, and DPC4. Therefore, there are few clues as to the nature of the uncharacterised genes for JPS as it appears that several different cellular defects are associated with hamartomatous juvenile polyps of identical appearance.


We thank the patients that took part in this study. Part of this work was supported by BREAKTHROUGH Breast Cancer. KW-R is supported by the EEC, SB is in receipt of a Fellowship from the Coeliac Society, MC is supported by the CRC, and BD by the MRC.



  • Abbreviations used in this paper:
    juvenile polyposis syndrome
    Cowden syndrome
    Bannayan-Ruvalcaba-Riley syndrome
    Gorlin syndrome
    conformation specific gel electrophoresis
    single strand conformational gel polymorphism