No TGFBRII germline mutations in juvenile polyposis patients without SMAD4 or BMPR1A mutation

Juvenile polyposis (JPS) is an autosomal dominant disorder characterised by the presence of multiple gastro-intestinal juvenile polyps and an increased risk of colorectal cancer (CRC).¹ JPS is caused by germline mutation of SMAD4 or BMPR1A, both involved in the transforming growth factor β/bone morphogenic protein (TGFβ/BMP) signalling pathway. A recent study by van Hattem et al, published in this journal (Gut 2008;57:623–7), showed that a germline defect in one of these genes is found in approximately 50% of JPS patients, with 30–40% being a point mutation or small deletion and 10–15% a large genomic deletion. Since no germline defect is found in ~50% of JPS patients, it is likely that other genes exist which cause JPS.²

Several candidate genes, mostly involved in TGFβ/BMP signalling, have been investigated for a role in JPS pathogenesis. No mutations have been found in these genes.^3–6 (table 1) Recently, the TGFβ co-receptor endoglin was proposed as a JPS susceptibility gene, but other studies could not confirm this.² Also, PTEN, the gene originally linked to Cowden syndrome (CS) and Bannayan–Riley–Ruvalcaba syndrome (BRRS), has been suggested as a JPS gene. The current consensus, however, is that PTEN mutations in patients with juvenile polyps likely represent CS or BRRS patients that have not (yet) developed extra-intestinal clinical features specific to these conditions.⁷ Lastly, the CDX2 gene was investigated in juvenile polyposis, since mice with a heterozygous mutation of CDX2 develop intestinal hamartomatous polyps, but no pathogenic mutations were found in 37 JPS families.⁸

Table 1.

Candidate genes investigated in the pathogenesis of juvenile polyposis

Gene	Patients studied/mutations found	Reference (first author and year)
BMPR1B (ALK6)	32/0	Howe 20043
BMPR2	59/0*	Howe 20043, van Hattem 20082
ACVR1 (ALK1)	66/0†	Howe 20043, Gallione 20046, van Hattem 20082
SMAD1	30/0	Bevan 19994
SMAD2	34/0	Bevan 19994, Roth 19995
SMAD3	34/0	Bevan 19994, Roth 19995
SMAD5	30/0	Bevan 19994
SMAD7	34/0	Bevan 19994, Roth 19995
CDX2	37/0	Woodford-Richens 20018

^*32 patients investigated by sequencing (Howe ³) and 27 by multiplex ligation-dependent probe amplification (MLPA) (van Hattem²).

^†39 patients investigated by sequencing (Howe³ and Gallione⁶) and 27 by MLPA (van Hattem²).

The TGFβ receptor type II (TGFBRII) is a component of the TGFβ pathway and is mutated within a polyadenine tract in exon 3 in up to 90% of CRCs with microsatellite instability and in 15% of microsatellite stable malignancies.⁹ In addition, germline mutation of TGFBRII has been reported in a patient with hereditary CRC (944C>T, reference sequence NM_003242).¹⁰ Also, mice with conditionally knocked out TGFBRII in fibroblasts develop intra-epithelial neoplasia of the prostate and invasive squamous cell carcinoma of the forestomach and loss of TGFBRII in intestinal epithelium promotes invasion and malignant transformation of tumors in Apc^1638N/wt mice.^{11 12} Because of its role in TGFβ signalling and in (colorectal) carcinogenesis, we investigated whether germline mutation or deletion of the TGFBRII gene is involved in JPS pathogenesis.

Nineteen JPS patients from 18 families, in whom germline mutation or deletion of SMAD4, BMPR1A, PTEN or ENG was previously excluded,² were investigated for germline defects in the TGFBRII gene. JPS was defined according to accepted clinical criteria.¹ All exons and intron–exon boundaries of the TGFBRII gene were analysed by direct sequencing and the possibility of germline deletion of (parts of) the TGFBRII gene was investigated by multiplex ligation-dependent probe amplification (MLPA) (P065 MLPA kit, MRC-Holland BV, Amsterdam, The Netherlands). No pathogenenic germline mutations or deletions in TGFBRII were found in this cohort. Known polymorphic variations were found in intron 3, intron 4, exon 4, and intron 7 (table 2).

Table 2.

Polymorphisms found in TGFBRII

Location	Nucleotide	Amino acid change	Number of JPS patients	refSNP ID
Intron 3	c.338+7 A>G	Intronic	9/18	rs1155705
Intron 4	c.530−4 T>A	Intronic	7/18	rs11466512
Exon 4	c.1242 C>T	p.N414N	6/18	rs2228048
Intron 7	c.1600−8 C>T	Intronic	1/18	rs11466530

Reference sequence: NM_001024847.

JPS, juvenile polyposis; TGFBRII, transforming growth factor receptor type II.

TGFBRII germline mutation is linked to Marfan syndrome type 2.¹³ Surprisingly, these patients do not have an increased risk of cancer.¹⁴ Possibly, diverging phenotypic effects of the different TGFBRII mutations are responsible for the absence of malignancies in Marfan patients carrying a TGFBRII mutation.¹³ Alternatively, the germline variation (944C>T) found in the patient with hereditary CRC could be a rare polymorphism without significance for CRC development. Although this alteration was not found in 119 control subjects,¹⁰ others found it at a similar frequency in normal controls (7 of 492) and individuals with sporadic CRC (6 of 228).¹³ Moreover, no additional germline mutations in TGFBRII have been found in patients with hereditary non-polyposis colorectal cancer (HNPCC) or in patients with familial or early onset CRC.^{15 16}

Because of its role in TGFβ signalling and CRC pathogenesis we hypothesised that TGFBRII may be a JPS susceptibility gene. Linkage analysis could not be performed due to the lack of large JPS kindreds in our cohort. It is nevertheless felt that TGFBRII is unlikely to be involved in JPS pathogenesis since no germline mutations or deletions in TGFBRII were found in the current study. Still, about half of JPS patients remain without molecular diagnosis and the search for other JPS causing genes should continue apace. Candidate genes could include other, perhaps less obvious, components of the TGFβ/BMP pathway.