Absence of aneuploidy and gastrointestinal tumours in a man with a chromosomal 2q13 deletion and BUB1 monoallelic deficiency

Abstract

Chromosomal instability is a potentially critical step in the development of colorectal cancer. The budding uninhibited by benzimidazole 1 (BUB1) gene is a highly conserved protein that plays a critical role at the spindle assembly checkpoint during cell division. BUB1 mutations function in a dominant-negative fashion and have been implicated in causing dysfunctional kinetochore attachments, premature chromatid separation, accelerated mis-segregation of whole chromosomes and aneuploidy. BUB1 mutations have been observed in patients with colorectal cancers. We report a remarkable case of BUB1 haploinsufficiency owing to a 1.7 Mb deletion of chromosome 2q13 in a 54-year-old man with no prior history of carcinoma. These mutant alleles were observed in both tissue from the hand and peripheral blood. Aneuploidy was not observed on cytogenetic analysis. These findings highlight the insufficiency of BUB1 haploinsufficiency to directly stimulate tumourigenesis, and suggest that other factors may be more critical to this process.

Background

Clinical and basic science investigations have shown that chromosomal instability is an important step in carcinogenesis, and is more likely than microsatellite instability to be associated with colorectal cancers.^{1 2} The benzimidazole 1 BUB1 gene encodes a highly conserved protein Bub1 (budding uninhibited by benzimidazole 1) that plays a prominent role in the functioning of the spindle assembly checkpoint, a critical checkpoint of cell division.² Bub1 localises and interacts closely with adenomatous polyposis coli (APC) as part of a mitotic checkpoint multimer complex at chromosomal kinetochores. Alterations in BUB1 are responsible for dysfunctional kinetochore attachments, premature chromatid separation, accelerated mis-segregation of whole chromosomes and aneuploidy—all implicated in driving gastrointestinal (GI) tumourigenesis. Biallelic alterations in BUB1 result in insufficient levels to maintain the crucial interaction with APC.³ Mutations to BUB1 are also thought to function in a dominant-negative fashion, given that the mutant gene has been shown to confer chromosomal instability to chromosomally stable cell lines.⁴ Additionally, in mice in which expression of Bub1 is gradually reduced, spontaneous tumours developed with increased incidence in a dose-dependent fashion.⁵ In humans, haploinsufficiency of BUB1 has been reported in a patient with the presence of microsatellite-stable colon carcinoma at 37 years of age, in which an increased level of aneuploidy was also observed in peripheral lymphocytes.⁶ Some have advocated the clinical determination of spindle-assembly checkpoint status, primarily through BUB1 genotyping, to guide diagnostic and therapeutic surveillance of GI neoplasias. We report a case of a man with BUB1 haploinsufficiency with no history of carcinoma or aneuploidy.

Case presentation

While performing an aCGH screen on diseased tissue from a series of patients with Dupuytren's disease, we encountered in a 54-year-old Caucasian male patient a 1.7 Mb deletion in chromosome 2q13 containing the BUB1 gene. He had an unremarkable medical and surgical history notable only for Dupuytren's disease treated with surgical fasciectomy. He was married and had no children. Routine evaluation with upper GI endoscopy and colonoscopy were both unrevealing except for a small 5 mm polyp in the rectum, which was biopsied and found to be benign. The patient has nine siblings, none of whom have any history of GI problems. Both of his parents also had no history of gastrointestinal pathology and were both deceased. His father died of a cerebrovascular accident and his mother of renal failure.

Investigations

DNA extraction

Genomic DNA was isolated from the patient's Dupuytren's disease-affected tissue of the hand and from peripheral lymphocytes using QIAamp DNA Mini Kit (Qiagen Inc., Valencia,  California, USA), according to the manufacturer's standard protocol. Genomic DNA was also isolated from the patient's father's peripheral lymphocytes using identical methods.

Oligonucleotide array comparative genomic hybridization  analysis

The labelling of patient and control DNAs, hybridisation onto an oligonucleotide array slide (G4449A SurePrint G3 Human CGH 4×180K Oligo Microarray Kit, each array contains 173 341 60-mer oligonucleotide probes, Agilent Technologies Inc, Santa Clara, California USA), posthybridisation wash, image capture and signal feature extraction were performed as described earlier.⁷ Genome-wide copy number variation was analysed using Agilent's DNA Analytical (V.4.0) with the built-in ADM-2 algorithm set at threshold value of 6, a cut-off value of 0.25, and a filter of six continuous probes. All base pair positions for detected genomic imbalances were designated according to the March 2006 Assembly (NCBI36/hg18) in the UCSC Human Genome browser (http://genome.ucsc.edu/).

Cytogenetic analysis

Conventional cytogenetic investigations were performed according to standard methods on cultured lymphocytes from the proband and his father. Chromosome spreads were processed for G-banding.

Real-time quantitative polymerase chain reaction  assay

RT-qPCR was performed in triplicate using the M×3000P QPCR System (Agilent Technologies Inc, Santa Clara,  California, USA) to confirm copy number alterations identified by microarray analysis. The Real-time quantitative polymerase chain reaction RT-qPCR reactions were prepared using Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies Inc) following the manufacturer's protocol. Five primer pairs for the targeted genes MALL, BUB1 and ZC3H6 within the deleted region and the locus in the neighbouring region and one primer pair for the reference gene MALL at 2q13 were designed to assess gene copy number of the 2q13 region.

On aCGH screen, the patient was noted to have a deletion on chromosome 2q13 in a region containing BUB1 as well as several other genes including ACOXL, BCL2L11, ANAPC1, MERTK and FBLN7 (Figure 1). The patient underwent conventional cytogenetic analyses revealing a normal karyotype and chromosomal G-banding. RT-qPCR confirmed the presence of this 1.7 Mb deletion in DNA from the skin contracture. A follow-up RT-qPCR analysis on DNA extracted from the patient's peripheral blood detected the same 2q13 deletion pattern. Combined results from aCGH and RT-qPCR involving five primer pairs (P1–P5) further defined the deletion break points between 110 337 690and 111 111 335 in the centromeric side and between 112 819 206 and 112 820 146 in the telomeric side. This clearly demonstrates a monoallelic-insufficiency of BUB1 (Figure 2). However, the chromosome 2q13 deletion was not detected in the patient's father's peripheral blood.

Figure 1.

Results of array comparative genomic hybridization  and Real-time quantitative polymerase chain reaction for the 2q13 region with aCGH chromosome and gene views showing a 1.7-Mb deletion (Chr2: 111 115 515–—112 819 206) of chromosome 2q13 (shaded in red).

Figure 2.

Real-time quantitative polymerase chain reaction results of the 2q13 region. RT-qPCR was used to confirm the deletion in the patient and showed no corresponding deletion in the patient's father. Genes within the deleted intervals and the flanking regions were targeted with RT-qPCR probes in the patient (P), patient's father (F) and a normal control (C). Data represent mean ±SEM. A relative quantity of 1 and 0.5 indicates the presence of two copies and one copy in the genome, respectively.

Outcome and follow-up

Patient is healthy at 1 year follow-up and remains without any GI symptoms.

Discussion

Our report suggests that haploinsufficiency of BUB1 is alone insufficient to drive development of aneuploidy or colorectal cancer detectable using conventional screening methodologies. Aneuploidy was not observed in our patient with BUB1 haploinsufficiency. Though studies have demonstrated causality between BUB1 mutations and chromosomal instability,⁴ our observations suggest that other factors in addition to BUB1 insufficiency may be required to promote the development of aneuploidy. The role of BUB1 in the development of colorectal cancer remains unclear. Additionally, it has not been established whether genomic instability is the instigating factor for the adenoma–carcinoma sequence, or rather arises as a result of the tumourigenic process.¹ Thus, a more detailed understanding of the BUB1 pathway genomic is needed before intervention can be advocated on the basis of BUB1 haploinsufficiency. A more cautious and complete understanding of the basis of chromosomal instability of the cancer genome needs to be achieved before new diagnostic, prognostic and therapeutic modalities can carry the promise of more effective medical treatment strategies for colon cancer.

The BUB1 gene is an exciting area of cancer biology research. Microdeletions of 2q13 involving the BUB1 gene have been recognised as recurrent genomic aberrations in phenotypically normal family members and control individuals mediated by regional highly homologous low copy repeats or segmental duplications via non-allelic homologous recombination.⁸ Evolutionarily, chromosome 2 is the result of a telomere–telomere fusion point of two ancestral chromosomes, located to the 2q13 band.⁹ At this fusion point, an array of TTAGGG sequence repeats within and between proximal 2p and 2q can trigger DNA rearrangements. This region of segmental duplications is extremely unstable and prone to breakage, fragility and recombination.¹⁰ The significance of these chromosomal characteristics is not well understood, though the highly conserved nature of BUB1 argues for the critical role of Bub1 in the process of cell division. More studies are needed to better characterise the role of BUB1 in tumourigenesis and elucidate its upstream and downstream interactions.

Learning points.

• BUB1 haploinsufficiency alone appears to be insufficient to directly stimulate tumourigenesis.

• BUB1 genotyping may not be necessary to guide diagnostic and therapeutic surveillance of gastrointestinal neoplasias.

• BUB1 mutations are associated with chromosomal instability, though other factors may be required to promote development of aneuploidy.