Point mutations in SMAD4 and BMPR1A
Using direct sequencing of individual exons, we identified 17 germline mutations in SMAD4 and 13 mutations in BMPR1A in the 80 patients, resulting in an overall mutation detection rate of 38%, or of 46% when only the unequivocal clinical cases were included (table 1). To our knowledge, 12 of the mutations have not been described previously (table 2).
SMAD4 point mutations
Of the 17 SMAD4 point mutations, 11 were predicted to lead to truncated proteins and were thus considered definitely pathogenic (5 nonsense, 6 frameshift mutations). Moreover, four missense mutations were localised at highly conserved amino acid positions and were thus considered likely to be disease‐causing (table 2). Two of the four missense mutations (patients JUV‐14 and JUV‐78) were proven to have occurred de novo. In a third patient (JUV‐81), only a faint mutant signal (c.1082G→A;p.Arg361His) was found during sequencing of exon 8, suggesting that this mutation was present as a mosaic. The same sequencing pattern was obtained in a second blood sample from the patient and was confirmed in a PCR product generated with primers localised outside the first primer pair used in the regular diagnostic setting. Thus, the faint signal was not due to unequal allele amplification based on a variant in the primer sequence. Moreover, sequencing of a DNA sample isolated from a polyp confirmed the presence of the mutation at a slightly higher level (data not shown). Both parents of this patient were reported to have no polyposis.
The mutation c.1139G→A in exon 8 of the SMAD4 gene (JUV‐44) is predicted to result in a missense mutation (p.Arg380Lys). However, this substitution, localised to the last position of the exon, interfered with splicing: a loss of the normal splice site (decrease in the splicing efficiency from 0.45 to <0.01) was predicted by the BDGP splice prediction program. Using mRNA analysis, we could show that the substitution led to the formation of a cryptic splice site localised within exon 8, resulting in a deletion of nucleotides 1003–1139 and formation of a premature stop codon due to a frameshift (fig 1). Thus, the correct designation of the mutation is c.1139G→A;r.1003_1139del137;p.Gly336AlafsX11. The PCR product obtained on mRNA with a forward primer localised within the deletion contained only the wild‐type nucleotide (G at position 1139), showing that no full‐length mRNA fragment was obtained from the mutant allele (not shown).
Figure 1Characterisation of the mutation in patient JUV‐44 on DNA and mRNA level. (A) Sequence analysis of genomic DNA showing the heterozygous substitution c.1139G→A localised at the last position of exon 8 of the SMAD4 gene; (more ...)
The variant c.425–6A→G in intron 2 of the SMAD4 gene (patient JUV‐51) was predicted to create a new splice acceptor site and might thus be pathogenic. Unfortunately, no mRNA was available from this patient.
BMPR1A point mutations
Of the 13 point mutations identified in BMPR1A, five were nonsense, 2 frameshift, 4 missense and 2 splice site mutations (table 2). One of the splice site mutations (JUV‐48) encompassed a deletion of 65 nucleotides localised to intron 4 of BMPR1A and included the highly conserved position −2 of the splice acceptor site of exon 5 (c.432‐2_432‐66del). This mutation was observed in two affected patients (mother and child). The variant was found because exons 4 and 5 were examined in the same PCR fragment. To date, no mRNA has become available for examination of the real effect on splicing.
Large deletions in SMAD4 and BMPR1A
All patients without identified point mutation (50) and patients with missense mutations or as yet unspecified variants (10) were examined by MLPA for the presence of large deletions or duplications.
Large SMAD4 deletions
Large SMAD4 deletions were found in six patients. Four exhibited a heterozygous deletion of all SMAD4 probes encompassing the entire SMAD4 gene and the promoter region. One patient had a deletion of coding exons 5–11 and another had a deletion of coding exons 6–11 (fig 2). All deletions were confirmed in a second independent MLPA test. In one of the families (JUV‐54), the deletion of the entire SMAD4 gene found in the index patient was confirmed in three other affected family members. The MLPA test kit readily found the large SMAD4 deletions in the 6 patients, whereas the remaining 54 patients and 5 normal controls revealed reproducible normal SMAD4 patterns with calculated relative values between 0.8 and 1.2.
Figure 2Examples of normalised peak areas showing deletions in the SMAD4 and BMPR1A gene. Deletion of (A) the entire SMAD4 gene including the promoter region in patient JUV‐88; (B) exon 5–11 of the SMAD4 gene in patient JUV‐58; (more ...) Large BMPR1A deletions
Deletions in the BMPR1A gene were found in three patients. One patient (JUV‐38) had a deletion of four BMPR1A probes (the two first noncoding exons and the two probes designed for the first coding exon of the gene (table 1, fig 2). A heterozygous deletion of the two probes for coding exon 1 was found in patient JUV‐22 and his affected father. Owing to the large introns localised to both sides of exon 1 (37 kb and 14.2 kb, respectively), we were unable to verify this deletion by long‐range PCR on genomic DNA. In one patient (JUV‐26), a deletion of the entire BMPR1A and PTEN genes was observed. The clinical phenotype of this patient and details of the deletion will be reported elsewhere.
Given the high homology between the BMPR1A gene and a pseudogene, reliability for BMPR1A is not as good as for SMAD4. In particular, wide variability was found with the MLPA probes designed for BMPR1A coding exon 4 and exon 10 (MLPA fragments of 154 and 382 bp, respectively), often showing nonreproducible relative peak heights of between 0.5 and 3.
Mutations in PTEN
In patient JUV‐16, the MLPA test revealed an isolated “deletion” of PTEN exon 7. Sequencing this exon, we found the heterozygous nonsense mutation c.697C→T;p.Arg233X localised close to the hybridisation site of the MLPA probe for PTEN exon 7. The mutation was also found in the affected father of the index patient.
Subsequently, all the remaining mutation‐negative patients were screened for PTEN germline mutations by direct sequencing. Of the 40 patients, 1 (JUV‐18) was found to have a pathogenic splice site mutation in intron 4 (c.253+1G→T).
The colorectal phenotype of SMAD4
mutation carriers was indistinguishable: There was no significant difference in the median age at diagnosis of JPS between carriers of the SMAD4
(12 years) and the BMPR1A
(14 years) mutations (p
0.48; table 3). Both groups had a comparable number and histological spectrum of colorectal polyps.
Table 3Genotype–phenotype correlation (age at diagnosis, gastric polyposis, hereditary haemorrhagic telangiectasia phenotype) in carriers of SMAD4 and BMPR1A mutations Gastric polyposis
In a previous study on 29 unrelated patients with JPS with 12 identified mutations, we found an over‐representation of gastric polyposis among carriers of SMAD4
mutations compared with carriers of BMPR1A
A similar trend was observed when only the 27 patients (22 families) with known status of gastric polyposis who had not been analysed in our previous study were considered: 11 of 17 patients with SMAD4
mutations, but none of 11 patients with BMPR1A
mutations, had gastric polyposis (p<0.01). In the combined sample (previously and newly analysed cases) information on results of gastroscopy was available for 30 patients with SMAD4
mutations (20 unrelated index patients and 10 affected relatives) and for 13 patients with BMPR1A
mutations (nine index patients and four affected relatives) (table 3). Of the 30 patients with a SMAD4
mutation, 22 (73%) were found to have gastric polyposis. In contrast, only 1 of the 13 patients (8%) with BMPR1A
mutations had gastric polyps (p<0.001). The over‐representation of gastric polyposis in SMAD4
mutation carriers remained true even when age at gastroscopy was considered. Although the median age at gastroscopy was 35 (range 11–60) years for patients with SMAD4
mutations and 26 (range 4–73) years for patients with BMPR1A
mutations, the difference was not significant (p
0.71) (table 3).
Generally, gastric polyposis in SMAD4
mutation carriers is diagnosed later in life (median age at diagnosis 41 years) compared with diagnosis of colorectal polyps (12 years) (p<0.001). The difference in age at gastroscopy between SMAD4
mutation carriers with and without gastric polyps was highly significant: gastric polyps were diagnosed at a median age of 41 years, whereas patients without gastric polyps had gastroscopy at a median age of 16 years (p<0.001). No significant difference in the age at gastroscopy was observed between SMAD4
mutation carriers without gastric polyposis (p
Consistent with this over‐representation of gastric polyposis, all seven cases of gastric cancer were reported in families with SMAD4 mutations (index patient JUV‐55; one affected relative in families JUV‐55 and JUV‐37; four affected relatives in family JUV‐4). The brother of index patient JUV‐4 had an early tubular adenocarcinoma diagnosed at 38 years of age; the histology results of the other three affected family members (two uncles and an aunt) were not available. In the relative of JUV‐37, an early gastric cancer of the diffuse–infiltrating type surrounded by hyperplastic tissue was found at 42 years of age. This woman died from an adenocarcinoma of the small bowel. Index patient JUV‐55 had an adenocarcinoma, and his brother had a well‐differentiated adenocarcinoma diagnosed within a juvenile polyp.
To exclude a germline E‐cadherin mutation as underlying cause of gastric cancer, mutation analysis of the CDH1 gene was performed in the affected brother of index patient JUV‐4. No mutation was identified. DNA was not available from the other six patients.
Hereditary haemorrhagic telangiectasia
In addition to gastrointestinal polyposis, 5 of the 39 index patients with identified germline mutations (JUV 14, 44, 51, 58, 78) had a clinical diagnosis of hereditary haemorrhagic telangiectasia (HHT, Osler–Weber–Rendu disease). All five patients belong to the 23 index patients harbouring a SMAD4 mutation, thus the frequency of HHT among SMAD4 mutation carriers is 22% (5/23) in our sample.
Large deletions versus point mutations
No significant difference with respect in age at diagnosis between carriers of point mutations and large deletions of each gene (SMAD4
0.12) was found; however, the statistical analysis in BMPR1A
mutation carriers was limited because of the small number of patients with BMPR1A
3). In addition, the difference in presence of gastric polyposis (p
0.3) and HHT (p
1.0) between carriers of SMAD4
deletions and point mutations was not significant.
Polyp histology and differential diagnoses
The documented histological results of removed colorectal polyps varied considerably among patients as well as between different examinations in the same patient (table 2). Adenomatous components including dysplasia (intraepithelial neoplasia according to the revised World Health Organization classification) were described in many juvenile polyps. In addition, in the majority of patients with proven germline mutations in the SMAD4 or BMPR1A genes, presence of juvenile polyps (with or without intraepithelial neoplasia), hyperplastic polyps, pseudopolyps and adenomas were reported to different extents. In many cases, the initial histological results delayed the diagnosis of JPS; in some cases, juvenile polyps were only diagnosed when the tissue blocks were re‐evaluated by an experienced pathologist. Similar diagnostic difficulties were evident for gastric polyps.
The accompanying infiltrate often leads to the assumption of inflammatory pseudopolyps, thus ulcerative colitis was a common initial diagnosis in our patients with JPS. Rare differential diagnoses include Morbus Ménétrier (giant hypertrophic gastritis) (patient JUV‐36) and Cronkhite–Canada syndrome (CCS) (patient JUV‐88). The latter patient, with a deletion of the entire SMAD4 gene, had been diagnosed at age of 12 years due to numerous polyps throughout the entire colon (diagnosed histologically as inflammatory pseudopolyps, granulation tissue polyps or juvenile polyps), severe anaemia and protein‐losing enteropathy. Gastroduodenoscopy showed normal findings.
In both patients harbouring a germline PTEN mutation (JUV‐16, JUV‐18) a variety of different polyp types was reported, encompassing juvenile, hyperplastic, adenomatous and inflammatory polyps, although JPS was diagnosed in JUV‐16 after histological re‐evaluation by an experienced pathologist (table 2). Patient JUV‐18 presented with additional extraintestinal tumours; he had a renal cell carcinoma and an intramuscular mixed benign tumour in the gluteal region composed of a lipoma and a haemangioma component.