Article Text

This article has a correction. Please see:

Germline APC mutation spectrum derived from 863 genomic variations identified through a 15-year medical genetics service to French patients with FAP
  1. Arnaud Lagarde1,
  2. Etienne Rouleau2,
  3. Anthony Ferrari1,3,
  4. Tetsuro Noguchi3,
  5. Jinghua Qiu3,
  6. Adrien Briaux2,
  7. Violaine Bourdon3,
  8. Virginie Rémy3,
  9. Pascaline Gaildrat4,
  10. José Adélaïde1,3,
  11. Daniel Birnbaum1,3,
  12. Rosette Lidereau2,
  13. Hagay Sobol3,5,
  14. Sylviane Olschwang1,3
  1. 1Centre de Recherche en Cancérologie de Marseille INSERM UMR891, Marseille, France
  2. 2Centre René-Huguenin, Saint-Cloud, France
  3. 3Institut Paoli-Calmettes, Marseille, France
  4. 4INSERM U614, France
  5. 5Université Aix-Marseille II, Marseille, France
  1. Correspondence to Dr Sylviane Olschwang, Institut Paoli-Calmettes, Centre de Recherche en Cancérologie de Marseille, 232 boulevard Sainte-Marguerite, Marseille 13009, France; Sylviane.olschwang{at}


Heterozygous APC germline alteration is responsible for familial adenomatous polyposis, a colon cancer predisposition with almost complete penetrance. Point mutations generally lead to truncated proteins or no protein at all. They mainly involve exon 3 to codon 1700 (exon 15). The work presented here delineates precisely the APC mutation spectrum from 15 years of systematic molecular screening which identified 863 independent alterations in the French population.

  • APC gene
  • germline mutation
  • familial adenomatous polyposis
  • mutation spectrum
  • genomic rearrangements
  • genetic screening/counselling
  • molecular genetics
  • cancer: colon
View Full Text

Statistics from

Familial adenomatous polyposis (FAP) is a well-described, autosomal dominant inherited syndrome with near-complete penetrance associated with APC mutation.1 The phenotypic hallmark of FAP is the presence of more than 100 colorectal adenomas in combination with extracolonic manifestations; there is a subset of APC mutation carriers with fewer colorectal adenomas, and, except for desmoid tumours, lacking extradigestive manifestations. This variant disease was initially referred to as ‘attenuated adenomatous polyposis coli’, but more recently included in a clinical entity called ‘attenuated familial adenomatous polyposis’.2 3 We present here the genotypic description of 863 patients with FAP with a germline APC genomic alteration.

Patients referred from French outpatient genetic clinics for molecular screening to document colonic adenomatous polyposis from 1993 and 2008 were systematically screened for point mutations of the APC gene from exon 3 to codon 1700 of exon 15. Negative cases exhibiting at least one extraintestinal manifestation of FAP, as they were also negative for MYH mutations (data not shown), were selected for additional screening, including a search for point mutations upstream of exon 3 and downstream of codon 1700 and large genomic rearrangements. All patients or their legal representatives signed an informed consent form for genetic analyses related to their disease. DNA was extracted from peripheral blood cells using a standard procedure after cell lysis and proteinase K digestion. Several genomic analysis techniques were successively applied and compared. Exons of the APC gene (NM_000038, NT033772.6) and splicing junctions were analysed by sequencing after PCR amplification using primers reported by Groden et al.4 A search for large genomic rearrangements was performed first applying the QMPSF (quantitative multiplex PCR of short fragments) technique. Results were validated and more precisely characterised by comparative genomic hybridisation (CGH) on 244K oligoarrays (Agilent Technologies, Massy, France) and a specially designed 11 000 oligoarray, encompassing nucleotides 111 999 686 to 112 306 766 from the chromosome 5 hg17 build. Exons were fully covered by overlapping and duplicated probes, with an average of one oligonucleotide every 12 bases. Introns and adjacent regions were covered with one oligonucleotide every 350 to 420 bases. Sequences analysis was performed using the PhredPhrapConsed package v11, and CGH data were analysed with CGH-analytics from Agilent Technologies.

A total of 784 patients had variants that were predicted to shorten the length of the APC protein. The functional effect of variants observed in 48 additional patients was more ambiguous. In addition to in silico analysis, nucleotide substitutions were tested after PCR amplification from patient genomic DNA together with ∼150 bp of flanking sequences inserted into a splicing reporter minigene as described by Tournier et al.5 Fifteen variants found in 22 patients had an effect on the splicing process and were subsequently classified as deleterious. The g.20 377 206A>T substitution located in exon 0.1, identified in eight patients, cosegregated in two families with a cumulate LOD score of 5.6. A 40 bp deletion was also found in exon 0.1 that removed one splicing junction. Sixteen unique variants from 17 patients remained unclassified, none of which occurred in highly conserved domains involved in β-catenin binding or degradation.6

Finally a total of 815 point mutations were retained to delineate the APC mutation spectrum, corresponding to 390 different mutations (online supplementary table 1, figure 1). All will be referred to the APC mutation database developed with LOVD v2.0 Build 17, available at, where 91 are already described. All but nine mutations were located upstream of codon 1700 and thus detected through the standard screening. No mutation was found in exons 1 and 2. Focusing on the 286 nonsense mutations, half (140) involved one of the 15 CGA codons leading within this part of the gene (except for codon 348 located in the alternatively transcribed part of exon 9), up to 25 times for codon 213. Most insertions were duplications of one nucleotide. Most of the deletions involved short tandem repeats of one to five nucleotides. The two hotspots described at codons 1061 and 1309 were involved 56 and 92 times, respectively—that is, one-third of the ins/del mutations. One larger deletion was found twice, also flanked by a 5 bp repeat.

Figure 1

APC germline mutation spectrum. The APC gene is represented from the 5' genomic part on the left to the Stop codon on the right. Coding exons are shown in boxes. Introns are not drawn to scale. Vertical bars indicate mutations according to the number of events found independently; frequent events (more than 20) are indicated next to the corresponding position. Nucleotide substitutions within the coding sequence are in black, those involving consensus splicing regions are in grey; they are positioned above the exons. Short deletions/insertions are positioned under the exons. Large genomic deletions are shown horizontally under the genomic region encompassed.

Large genomic deletions were detected by QMPSF in 48 patients—that is, 5.5% of all germline alterations (figure 1). No duplications were observed. All were also detected with the P043 MLPA kit (MRC-Holland, Amersterdam, The Netherlands). Thirty-five deletions were studied on CGH arrays. All were confirmed on the dedicated array. Five deletions spanning exon 9 (two cases) and 11–13 (three cases) were missed on the 244K array, no corresponding oligonucleotide being spotted on this array. The dedicated array allowed the characterisation of the deletion boundaries, ranging from 2.7 kb to over 17 Mb without any recurrent breakpoint as previously reported.7 In two patients, the deletion of exons 11–13 was replaced with a 351 bp Alu Ya5 sequence.

In a subgroup of 341 patients with sporadic FAP, parental DNA was available, and correct assignment of parentage was confirmed using the Amp FlSTR SGM Plus kit (Applied Biosystems, Courtabeouf, France), so that it was clear that the mutation identified in the index case arose de novo. This means that at least 40% of the detected pathogenic mutations arose as de novo mutations in the index case, a proportion slightly lower than that observed in other rare cancer predispositions, but much higher than that usually noted in FAP.8

A total of 863 genomic variants were identified through this systematic approach applied to patients exhibiting at least one extracolonic common manifestation of FAP. No APC genomic alteration was found in 71 cases, leading to a 93% mutation detection rate in FAP. One allelic imbalance was noted after reverse transcription-PCR (RT-PCR), but the causative genomic event could not be documented. As RNA was not available for most patients, this phenomenon is probably more common and could be investigated in relation to promoter hypermethylation or somatic mosaicism as possible causes of FAP.9 10 The presence of desmoid tumour was recorded for 110 patients—that is, 13% as commonly reported.

In summary, this experience highlights the strong yield of APC germline mutations in FAP. The spectrum of mutations is wide, from point mutations to large rearrangements, which can be complex. Finally, because of the high frequency of de novo mutations, we recommend APC screening in MutYH-negative cases of sporadic FAP even in the absence of extradigestive manifestations.


This work was supported by the French National Cancer Institute (INCa) through a grant to the PHRATries network (Prédispositions Héréditaires Rares Aux Tumeurs, Réseau d'Identification et de Soins). We are grateful to the French clinicians who faithfully entrust our institution with molecular genetics tests of the APC gene.


View Abstract

Supplementary materials

  • Web Only Data

    Files in this Data Supplement:


  • Funding INCa, INSERM.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval This study was conducted with the approval of the St-Antoine Hospital, Paris France.

  • Provenance and peer review Not commissioned; externally peer reviewed.

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

Linked Articles

  • Correction
    BMJ Publishing Group Ltd