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Molecular Cancer

, 17:23 | Cite as

Germline mutations in the spindle assembly checkpoint genes BUB1 and BUB3 are infrequent in familial colorectal cancer and polyposis

  • Pilar Mur
  • Richarda M. De Voer
  • Rubén Olivera-Salguero
  • Sandra Rodríguez-Perales
  • Tirso Pons
  • Fernando Setién
  • Gemma Aiza
  • Rafael Valdés-Mas
  • Angelo Bertini
  • Marta Pineda
  • Lilian Vreede
  • Matilde Navarro
  • Silvia Iglesias
  • Sara González
  • Joan Brunet
  • Alfonso Valencia
  • Manel Esteller
  • Conxi Lázaro
  • Geert J. P. L. Kops
  • Miguel Urioste
  • Xose S. Puente
  • Gabriel Capellá
  • Laura Valle
Open Access
Letter to the Editor

Abstract

Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC.

Keywords

Colorectal cancer predisposition Hereditary colorectal cancer High-penetrance genes Variegated aneuploidy Mechanism 

Abbreviations

a.a.

amino acid

CRC

colorectal cancer

EBV

Epstein Barr virus

ExAC

Exome Aggregation Consortium (http://exac.broadinstitute.org/)

GnomAD

Genome Aggregation Database (http://gnomad.broadinstitute.org/)

LOH

loss of heterozygosity

MAF

minor allele frequency

SAC

spindle assembly checkpoint

SEM

standard error mean

Letter to the editor

Much of the heritability associated with colorectal cancer (CRC) cannot be explained by the currently known genetic risk factors for CRC. Genome-wide genetic and genomic screenings have tried to identify novel high penetrance genes for CRC. Despite the identification of novel candidate genes for CRC predisposition, overall, they account for a very low number of familial cases, and for many of the suggested genes, identification of additional mutated families is essential to conclusively define their actual implication in CRC predisposition [1].

Based on genome-wide copy number profiling and exome sequencing in early-onset and familial CRC, De Voer et al. identified germline mutations in BUB1 and BUB3, components of the spindle assembly checkpoint (SAC) and thus controllers of correct chromosome segregation, as cause for CRC predisposition [2, 3]. Six germline mutations affecting BUB1 and BUB3 were identified in 6 independent families. Recently, Broderick et al. assessed the presence of germline mutations in proposed genes for CRC predisposition, including BUB1 and BUB3, finding no increased frequency of mutations in cases compared to controls in either gene [4]. In view of the controversial results, the purpose of the present study is to evaluate the impact, supported by functional studies, of BUB1 and BUB3 germline variants in the predisposition to CRC and/or polyposis.

Using a strategy that combines pooled DNA amplification and massively parallel sequencing, we sequenced BUB1 and BUB3 in 456 familial colorectal cancer cases (60 Amsterdam-positive families), and in 88 polyposis cases, without identified mutations in known high-penetrance genes. Considering novel and rare (population minor allele frequency (MAF) < 1%) non-synonymous genetic changes, a total of 4 variants, BUB1 c.1965-1G>A, BUB1 c.2296G>A (p.E766K), BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I), were detected in 4 families (Table 1, Fig. 1). Only BUB1 c.2473C>T was reported in public databases. Loss of heterozygosity (LOH) causing the elimination of the wild-type allele or promoter methylation was not detected in any of the tumors studied (Table 1, Additional file 1: Table S1), as previously observed [2].
Table 1

Germline genetic variants in BUB1 and BUB3 identified in 544 CRC and/or polyposis families

aMutation [protein domain]

BUB1 c.1965-1G>A

(r.1965_1975del; p.S655Rfs*32)

BUB1 c.2296G>A

(p.E766K)

BUB1 c.2473C>T (p.P825S)

[Kinase domain]

BUB3 c.77C>T (p.T26I)

[WD40 repeat 1]

Population MAF (%) (1000G / ESP

/ ExAC/ gnomAD)

n.r.

n.r.

rs748392521; n.r. / n.r. /

0.004 / 0.005

n.r.

Protein function prediction

(score)

p.S655Rfs*32

bPPH2: PsD (0.934) / N

(0.349)

bPPH2: PrD (1 / 0.997)

bPPH2: PsD (0.770 /

0.548)

SIFT: N (0.2)

SIFT: D (< 0.0001)

SIFT: N (0.06)

Condel: N (0.463)

Condel: D (0.542)

Condel: D (0.524)

Mut. Taster: D

Mut. Taster: D

Mut. Taster: D

Align GVGD: N (C0)

Align GVGD: D (C65)

Align GVGD: N (C0)

Protein stability prediction

(ddG, Kcal/mol)

p.S655Rfs*32

CUPSAT: destabilizing

(−0.69)

CUPSAT: destabilizing

(−1.16)

CUPSAT: destabilizing

(− 0.29)

ERIS: destabilizing (1.57)

ERIS: stabilizing (−0.71)

ERIS: destabilizing

(1.10)

I-Mutant: destabilizing

(−0.53)

I-Mutant: destabilizing

(−0.64)

I-Mutant: destabilizing

(− 0.68)

PoPMuSIC: destabilizing

(0.04)

PoPMuSIC: stabilizing

(0.17)

PoPMuSIC:

destabilizing (0.33)

Splice site predictions

(Alamut v2.9)

Disrupts acceptor site

No change

No change

No change

RNA study: 11 bp deletion

RNA study: no change

RNA study: no change

cEvolutionary conservation

(PhyloP / PhastCons)

1.239 / 0.95

4.358 / 1

5.572 / 1

Variegated aneuploidy

No

No

n.a.

No

Somatic LOH / promoter

methylation

n.a. / n.a.

No LOH; No methylation

(II.6 and III.2)

No LOH; No methylation

No LOH; n.a.

Functional studies in LCL

Damaging effects

No effect (incomplete

functional assessment)

n.a.

n.a.

d,eVariant classification

ePathogenic

Likely benign / Uncertain

significance

Uncertain significance

Uncertain significance

a. RefSeq GRCh37: BUB1 NM_004336, BUB3 NM_004725

b. Polyphen 2: HumDiv / HumVar scores.

c. PhyloP score (values between -14 and +6): Sites predicted to be conserved are assigned positive scores. PhastCons score (values between 0 and 1): the closer the value is to 1, the more probable the nucleotide is conserved.

d. According to the standard guidelines for the interpretation of sequence variants (recommendations of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology) [7].

e. Interpret with caution. BUB1 and BUB3 have not yet been defined as genes with clinical value in hereditary cancer.

Abbreviations: 1000G, 1000 Genomes; bp, base pairs; ca., cancer; D, damaging or deleterious; ESP, NHLBI GO Exome Sequencing Project; ExAC, Exome Aggregation Consortium; GnomAD, Genome Aggregation Database (http://gnomad.broadinstitute.org); LCL, EBV-transformed lymphoblastoid cell line; N, neutral; n.a., not available; n.r., not reported; PPH2, polyphen 2; PrD, probably damaging; PsD, possibly damaging

Fig. 1

Pedigrees of the families with BUB1 and BUB3 mutations. Filled symbol, cancer. Ages at information gathering or at death (†), when available, are indicated on the top-right corner, and ages at cancer diagnosis, between brackets after tumor type. Abbreviations: AP, adenomatous polyp; HP, hyperplastic polyp; JP, juvenile polyp; ca, cancer; CRC, colorectal cancer; PTC, papillary thyroid cancer; N.C., normal colonoscopy; MUT, mutation carrier; WT (wildtype); non-carrier of the mutation identified in the family

BUB1 c.1965-1G>A, identified in a male patient diagnosed with CRC and 25 adenomatous polyps at age 40 with no relevant family history of cancer (Fig. 1), causes an out-of-frame deletion of 11 bases, produced by the disruption of the canonical acceptor site of exon 18 and usage of the next AG as novel splicing acceptor (r.1965_1975del; p.S655Rfs*32) (Additional file 1: Figure S1). The EBV-transformed lymphoblastoid cell line obtained from this carrier showed reduced BUB1 mRNA expression (p = 0.0020) and increased chromosome segregation errors (p = 0.0050) compared to control lymphoblastoid cell lines (Fig. 2a-b). The c.1965-1G>A cells showed reduced BUB1 levels at the kinetochores, where the SAC is active, compared to controls (30% reduction; p < 0.0001) (Fig. 2c-d).
Fig. 2

Functional assays for BUB1 c.1965-1G>A and c.2296G>A (p.E766K) and 3D structure of the proteins and location of the identified BUB1 and BUB3 germline mutations. a Quantitative analysis of levels of BUB1 mRNA demonstrating reduced BUB1 expression in the c.1965-1G>A cell line compared to controls. Data are normalized against HPRT expression. Error bars represent standard error mean (SEM) values. b Quantification of chromosome segregation errors in EBV-transformed cells from a control individual and the BUB1 c.1965-1G>A mutation carrier. Measurements were performed in triplicate and error bars represent SEM values. c Localization and d quantification of BUB1 levels at the kinetochores of nocodazole-arrested EBV-transformed cells. Each dot represents one cell and the level of BUB1 is normalized to the kinetochore (KT) intensity of CENP-C and is the average fold change of three experiments (±SEM) normalized to the values of control cells. Cells from the c.1965-1G>A carrier revealed reduced levels of BUB1 at the KT in comparison to the control. ** P < 0.001; *** P < 0.0001. e Crystallographic 3D structure of BUB1 (a.a. 736–1083) and location of p.E766K and p.P825S, and 3D model of BUB3 (a.a. 6–324) and location of p.T26I (current study) and p.K21N, p.R149Q and p.F264L [2]. f Protein domains of BUB1 (UniProtKB - O43683) and BUB3 (UniProtKB - O43684) and location of the identified mutations. In red, residues affected by mutations identified in this study; in blue, residues affected by the mutations identified by de Voer et al. [2]; in orange, variants identified by Broderick et al. [4]; and in green, variants detected by Shindo et al. [6]

BUB1 c.2296G>A (p.E766K), located in a β-strand outside the protein kinase domain, was identified in three CRC-affected members of an Amsterdam-positive CRC family (Fig. 1). Computational analyses predicted a destabilizing effect on the protein (Table 1). However, the EBV-transformed lymphoblastoid cell line obtained from the proband did not show reduced BUB1 expression (Fig. 2a) or reduced BUB1 levels at the kinetochores (Fig. 2c-d) when compared to controls. Assessment of chromosome segregation errors could not be performed.

BUB1 c.2473C>T (p.P825S) (rs748392521, MAFExAC: 0.004%), which affects a highly conserved amino acid (considering 13 species) and is located in the catalytic kinase domain of the protein, was identified in an individual diagnosed with CRC at age 44, with no immediate relatives affected with cancer but with a cousin diagnosed with CRC at age 44 whose mutation status could not be assessed (Fig. 1). The variant was predicted to affect the function and structure of the protein (Table 1, Fig. 2e and f). Sample unavailability prevented us from carrying out the specific functional studies in the patient-derived lymphoblastoid cell line.

Finally, BUB3 c.77C>T (p.T26I) was identified in patient diagnosed with prostate cancer at age 70 and two synchronous CRCs and 22 adenomatous polyps at age 73, belonging to a family fulfilling the Amsterdam I criteria. The variant, located in the WD40 repeat 1 of a seven-bladed beta-propeller fold, was predicted to be functionally deleterious and destabilizing (Table 1). The location of the mutated residue together with the previously reported BUB3 CRC-predisposing mutations is shown in Fig. 2e-f. Co-segregation results in other cancer-affected family members did not support a causal role of the variant in the aggregation of cancer in the family (Fig. 1). The in vitro functional studies could not be performed due to the unsuccessful transformation of the patient’s lymphocytes.

Cytogenetic analysis of non-transformed lymphocytes obtained from BUB1 c.1965-1G>A, BUB1 c.2296G>A (p.E766K), BUB3 c.77C>T (p.T26I) mutation carriers and from unrelated controls, did not show differences in the number of metaphases with chromosome number alterations. Notably, complex chromosomal translocations were identified in one of 41 metaphases of the BUB1 c.1965-1G>A carrier, while no chromosomal translocations were detected in any of the 91 control metaphases analyzed (Additional file 1: Table S2).

No reminiscent traits of the mosaic variegated aneuploidy syndrome or variegated aneuploidy in lymphocytes were found in any of the studied carriers, not even in the carrier of BUB1 c.1965-1G>A (p.S655Rfs*32). However, the EBV-transformed lymphocytes from the latter revealed chromosome segregation errors, which may lead to aneuploidy in the next cell cycle. It is unclear what the cell fate of the missegregated cells is, as the subsequent chromosomal instability might be detrimental. De Voer et al. identified mosaic aneuploidy in all three BUB1/BUB3 mutation carriers they studied, while only two of the three carriers showed dysmorphic features [2]. On the other hand, no aneuploidies were observed in a 54 year-old Dupuytren patient without family history of CRC and a 1.7 Mb deletion of chromosome 2q13, which includes BUB1 [5]. These findings suggest that BUB1/3 monoallelic mutations may or not cause mosaic aneuploidy and/or phenotypic affectation.

The role of BUB1 and BUB3 in familiar cancer has been topic of debate in the last years. Recently, Broderick et al. scrutinized the exomes of 863 familial/early-onset CRC cases and 1604 cancer-free controls in order to validate the proposed hereditary CRC genes, including BUB1 and BUB3 [4]. Neither the herein identified variants nor the ones identified by De Voer et al. [2] were detected in the exome study. While only one novel/rare missense variant predicted to be deleterious (none stop-gain, frameshift or splice-site variants) was identified in BUB1 in the 863 cases (0.11%), and none in BUB3, several (4 in BUB1 (0.25%) and 2 in BUB3 (0.12%)) novel/rare disruptive variants were identified in controls. Of note, the frequency of disruptive (stop-gain, frameshift and splice acceptor/donor) mutations in controls identified by Broderick et al. is remarkably higher than the frequency of disruptive mutations annotated in large population-based browsers (BUB1: ExAC, 0.065% (40 carriers in 60,703 individuals); GnomAD, 0.063% (88/138,044). BUB3: ExAC, 0.006% (4/59,569); GnomAD, 0.008% (11/123,084)) [4]. Taking into account the two largest series analyzed ([4] and present study), 0.14% (2/1392; 1/863 and 1/529, respectively) of familial and/or early-onset CRC cases would be explained by functionally relevant germline mutations in BUB1 (0% in BUB3), compared to a population frequency of 0.063–0.065%. Nevertheless, despite the demonstrated functional effect of several BUB1/BUB3 variants, their causal implication in colorectal carcinogenesis, either alone or in combination with other mutations/variants in other genes, is yet to be proven.

To date, 11 CRC families and 1 pancreatic ductal adenocarcinoma family with different novel/rare germline mutations in BUB1 or BUB3 have been reported (Additional file 1: Table S3; Fig. 2e-f) [2, 4, 6]. Of the 12 variants, 7 were proven deleterious based on functional experimental evidence and/or on their splice-site or frameshift nature, one did not show any effect, and the remaining 4 missense variants were not subjected to functional studies. Six of the 8 mutation carriers (7 families) in whom functionally relevant mutations have been identified were diagnosed with CRC at young ages (range: 29–40 years), one of them together with the presence of 25 adenomas. The increased risk to other tumor types, such as lung cancer, remains uncertain until additional functionally relevant mutation carriers with the disease are identified.

Based on the evidence gathered to date, we conclude that the paucity of functionally-relevant cancer-predisposing mutations in BUB1 and BUB3 do not support the need for germline genetic testing of these genes in familial CRC for diagnostic purposes.

Notes

Funding

This work was supported by the Spanish Ministry of Economy and Competitiveness, co-funded by FEDER funds – a way to build Europe – (grants SAF2016–80888-R to LValle, SAF2015–68016-R to GC and MP, and SAF2013–45836-R to XSP]; Carlos III National Health Institute [PI16/00563 to CL and CIBERONC]; the Government of Catalonia [Pla estratègic de recerca i innovació en salut (PERIS) and 2014SGR338]; the Scientific Foundation Asociación Española Contra el Cáncer; and EU COST Action BM1206. PM holds a Juan de la Cierva postdoctoral fellowship from the Spanish Ministry of Economy and Competitiveness, and RMdV, a Dutch Cancer Society (KWF) Fellowship (KUN14–6666).

Availability of data and materials

Not applicable

Authors’ contributions

LValle supervised the study. LValle, PM, RMdV, XSP and GC contributed to the study concept and design. PM, RMdV, RO-S, SR-P, TP, FS, GA, RV-M, AB, LVreede, ME, AV and LValle performed experiments and/or acquired data. MP, MN, SI, SG, JB, CL, MU and GC contributed to the acquisition of samples and clinical data. PM, RMdV, RO-S, SR-P, TP, RV-M, AB, LVreede, AV, GJPLK, XSP, GC and LValle contributed to the analysis interpretation of data. LValle, RdV and PM drafted the manuscript. All authors critically reviewed the manuscript for important intellectual content and approved its final version.

Ethics approval and consent to participate

Informed consent was obtained from all subjects and the study received the approval of the Ethics Committees of the Bellvitge Biomedical Research Institute (IDIBELL) (PR073/12).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

12943_2018_762_MOESM1_ESM.doc (292 kb)
Additional file 1: Methods and Supplementary information (DOC 291 kb)

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Pilar Mur
    • 1
    • 2
    • 3
  • Richarda M. De Voer
    • 4
    • 5
  • Rubén Olivera-Salguero
    • 1
  • Sandra Rodríguez-Perales
    • 6
  • Tirso Pons
    • 7
    • 8
  • Fernando Setién
    • 9
  • Gemma Aiza
    • 1
  • Rafael Valdés-Mas
    • 3
    • 10
  • Angelo Bertini
    • 6
  • Marta Pineda
    • 1
    • 2
    • 3
  • Lilian Vreede
    • 4
  • Matilde Navarro
    • 1
    • 2
    • 3
  • Silvia Iglesias
    • 1
    • 2
    • 3
  • Sara González
    • 1
    • 2
    • 3
  • Joan Brunet
    • 1
    • 2
    • 3
    • 11
  • Alfonso Valencia
    • 12
    • 13
  • Manel Esteller
    • 3
    • 9
    • 13
    • 14
  • Conxi Lázaro
    • 1
    • 2
    • 3
  • Geert J. P. L. Kops
    • 5
  • Miguel Urioste
    • 15
  • Xose S. Puente
    • 3
    • 10
  • Gabriel Capellá
    • 1
    • 2
    • 3
  • Laura Valle
    • 1
    • 2
    • 3
  1. 1.Hereditary Cancer Program, Catalan Institute of OncologyIDIBELL, Hospitalet de LlobregatBarcelonaSpain
  2. 2.Program in Molecular Mechanisms and Experimental Therapy in Oncology (Oncobell)IDIBELL, Hospitalet de LlobregatBarcelonaSpain
  3. 3.Centro de Investigación Biomédica en Red de Cáncer (CIBERONC)SalamancaSpain
  4. 4.Department of Human Genetics, Radboud Institute for Molecular Life SciencesRadboud University Medical CenterNijmegenThe Netherlands
  5. 5.Hubrecht Institute – KNAW (Royal Netherlands Academy of Arts and Sciences)UtrechtThe Netherlands
  6. 6.Molecular Cytogenetics Group, Human Cancer Genetics ProgramSpanish National Cancer Research Center (CNIO)MadridSpain
  7. 7.Structural Biology and Biocomputing ProgramSpanish National Cancer Research Center (CNIO)MadridSpain
  8. 8.Department of Immunology and OncologyNational Centre for Biotechnology (CNB-CSIC)MadridSpain
  9. 9.Cancer Epigenetics and Biology Program (PEBC)Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de LlobregatBarcelonaSpain
  10. 10.Department of Biochemistry and Molecular Biology, Instituto Universitario de Oncología del Principado de AsturiasUniversidad de Oviedo and CIBERONCOviedoSpain
  11. 11.Hereditary Cancer Program, Catalan Institute of OncologyIDIBGiGironaSpain
  12. 12.Life Science DepartmentBarcelona Supercomputing Centre (BSC-CNS)BarcelonaSpain
  13. 13.Institució Catalana de Recerca i Estudis Avançats (ICREA)BarcelonaSpain
  14. 14.Physiological Sciences Department, School of Medicine and Health SciencesUniversity of Barcelona (UB)BarcelonaSpain
  15. 15.Familial Cancer Clinical Unit, Human Cancer Genetics ProgramSpanish National Cancer Research Centre (CNIO) and Center for Biomedical Network Research on Rare Diseases (CIBERER)MadridSpain

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