Clinical and genetic heterogeneity in patients with mosaic variegated aneuploidy: delineation of clinical subtypes

Mosaic variegated aneuploidy (MVA) is a rare autosomal recessive syndrome related to BUB1B gene mutations and characterized by multiple mosaic aneuploidies, cancer predisposition, and a distinct phenotype. We report on two mildly affected sibs with MVA syndrome but without BUB1B mutation. Both patients exhibited growth retardation, frontal bossing, triangular face and micrognathia but not microcephaly or cancer. Aneuploidies were assessed both in G-banded metaphases from lymphocyte cultures and in interphase nuclei from buccal cells by FISH. Screening of 23 exons and intron-exon boundaries of BUB1B was also carried out. These patients were then compared with other 19 MVA patients screened for BUB1B mutations. Around one half of the cultured lymphocytes from our patients had aneuploidies ranging from nullisomies to heptasomies; the most frequent abnormalities were trisomies (42%) and monosomies (28%). FISH results demonstrated more chromosomal losses than gains. Screening of BUB1B in our two patients failed to identify any mutation. A review of the 21/35 patients screened for BUB1B demonstrated three clinical pictures. Patients with monoallelic BUB1B mutations were severely affected with Dandy-Walker complex (7/8), cataracts (6/6), and Wilms' tumor (7/8); premature chromatid separation (PCS) was observed in 8/8 propositi and 7/7 carrier parents. Patients without BUB1B mutations were mildly affected with no evidence of cancer, Dandy-Walker malformation or cataract, and rarely (1/7) showed PCS. Finally, patients with biallelic BUB1B mutations showed a moderate phenotype. The distinct MVA clinical groups delineated here point to involvement of at least another mitotic spindle checkpoint gene in addition to the BUB1B gene.     

Prevalence of germline mutations in the spindle assembly checkpoint gene BUB1B in individuals with early‐onset colorectal cancer

Germline mutations in BUB1B, encoding BUBR1, one of the crucial components of the spindle assembly checkpoint (SAC), have been shown to cause variable phenotypes, including the recessive mosaic variegated aneuploidy (MVA) syndrome, which predisposes to cancer. Reduced levels of the wild‐type BUBR1 protein have been linked to the development of gastrointestinal neoplasms. To determine whether mutations in BUB1B are enriched in individuals with colorectal cancer (CRC), we performed amplicon‐based targeted next‐generation sequencing of BUB1B on germline DNA of 192 individuals with early‐onset CRC (≤50 years). None of the individuals was found to be homozygous or compound heterozygous for mutations in BUB1B. However, we did identify two rare heterozygous variants, p.Glu390del and p.Cys945Tyr, in patients who developed CRC at the ages of 41 and 43 years, respectively. Both variants were shown not to affect BUBR1 protein expression levels and protein localization. Since the p.Glu390del variant is located in the BUB3‐binding domain, we also performed immunoprecipitation to examine whether this variant affects the binding of BUB1 or BUB3 to BUBR1 but, compared to wild‐type BUBR1, no difference was observed. Our data suggest that mutations in BUB1B do not occur frequently in the germline of individuals with CRC and that BUB1B unlikely plays a major role in the predisposition to early‐onset CRC. Whether carriers of pathogenic BUB1B mutations, such as the parents of MVA syndrome patients, have an increased risk for cancer remains of interest, as studies in mice have suggested that haploinsufficiency of BUB1B may cause an increase in carcinogen‐induced tumors. © 2016 Wiley Periodicals, Inc.     

Mosaic variegated aneuploidy without microcephaly: implications for cytogenetic diagnosis

Mosaic variegated aneuploidy (MVA) is a rare condition characterized by multiple trisomies, rarely monosomies, and a non-specific phenotype including microcephaly, growth and mental retardation, mild malformations, and an increased risk of malignancy. We describe a patient with MVA in whom trisomy 19 mosaicism was originally suspected. The patient was the product of an uncomplicated term pregnancy and delivery. Significant findings were mental retardation, obesity, mild epicanthal folds, tapering fingers, relatively small hands and feet, alternating exotropia, nasal speech limited to short phrases, and generalized hypotonia. There is no family history for birth defects, mental retardation, or consanguinity. The initial peripheral blood chromosome study showed trisomy 19 in 4 of 31 metaphase cells. Because mosaic trisomy 19 is rare, the study was extended to 100 cells, wherein two cells with trisomy 8 were identified. A second blood karyotype was obtained and found to be 47,XX,+8[3]/47,XX,+19[3]/47,XX, +18[2]/47,XX,+9[1]/46,XX[91]. Skin fibroblast chromosome studies revealed a 46,XX karyotype in 120 cells examined. There was no evidence of premature centromere separation. Mutations in the BUB1B gene that encodes a key mitotic spindle checkpoint protein have been described in MVA; however, no mutations of this gene were identified in our patient. This case illustrates the importance of considering other possibilities when confronted with an extremely rare diagnosis such as mosaic trisomy 19. In addition, it shows the importance of not simply interpreting a low percentage of multiple aneuploidies as cell culture artifact, because an additional work-up to rule out MVA may be warranted since this diagnosis is associated with an increased risk of malignancy.     

Centrosome amplification and aneuploidy in bone marrow failure patients

Patients with bone marrow failure are at risk for development of hematopoietic progenitor clones with abnormal numbers of chromosomes (aneuploidy) and leukemia. Numerical centrosome abnormalities or mutations in genes associated with the mitotic spindle checkpoint (BUB1 and MAD2) are two important mechanisms that can induce abnormal mitosis resulting in aneuploid daughter cells. To assess the role of these mechanisms, we used fluorescence in situ hybridization techniques to determine aneuploidy and centrosome copy number and PCR-SSCP to identify gene mutations of BUB1 and MAD2 in marrow cells of 25 patients. No mutations were found in BUB1 or MAD2 genes. However, we found that cells with more than two centrosomes exhibited aneuploidy for three or more chromosomes. We conclude that centrosome amplification may be associated with the development of a clonal population of potentially preleukemic aneuploid cells.     

Insufficiency of BUBR1, a mitotic spindle checkpoint regulator, causes impaired ciliogenesis in vertebrates

Budding uninhibited by benzimidazole-related 1 (BUBR1) is a central molecule of the spindle assembly checkpoint. Germline mutations in the budding uninhibited by benzimidazoles 1 homolog beta gene encoding BUBR1 cause premature chromatid separation (mosaic variegated aneuploidy) [PCS (MVA)] syndrome, which is characterized by constitutional aneuploidy and a high risk of childhood cancer. Patients with the syndrome often develop Dandy-Walker complex and polycystic kidneys; implying a critical role of BUBR1 in morphogenesis. However, little is known about the function of BUBR1 other than mitotic control. Here, we report that BUBR1 is essential for the primary cilium formation, and that the PCS (MVA) syndrome is thus a novel ciliopathy. Morpholino knockdown of bubr1 in medaka fish also caused ciliary dysfunction characterized by defects in cerebellar development and perturbed left-right asymmetry of the embryo. Biochemical analyses demonstrated that BUBR1 is required for ubiquitin-mediated proteasomal degradation of cell division cycle protein 20 in the G0 phase and maintains anaphase-promoting complex/cyclosome-CDC20 homolog 1 activity that regulates the optimal level of dishevelled for ciliogenesis.     

A homozygous CEP57 c.915_925dupCAATGTTCAGC mutation in a patient with mosaic variegated aneuploidy syndrome with rhizomelic shortening in the upper and lower limbs and a narrow thorax

Mosaic variegated aneuploidy syndrome (MVA) is a rare autosomal recessive disorder characterized by random chromosome gains and losses. Mutations in BUB1B and CEP57 genes have been involved in MVA. Here we report on a male child with MVA due to c.915_925dupCAATGTTCAGC mutation in the CEP57 gene. Our patient was homozygous for this mutation and he is the first case with rhizomelic shortening of both the upper and lower limbs and mild respiratory insufficiency due to a narrow thorax. It is also the second MVA Mexican family reported with this mutation that lives in the northwestern region of Mexico, suggesting a “local founding effect”. Additional cases are needed to better understand the MVA genotype-phenotype relationship.     

A double missense variation of the BUB1 gene and a defective mitotic spindle checkpoint in the pancreatic cancer cell line Hs766T

To determine sequence variations of the BUB1 and BUB1B genes in pancreatic cancer, the entire coding regions of the BUB1 and BUB1B genes were sequenced in pancreatic cancer cell lines and xenografts. Although only polymorphic alterations were found in the BUB1B gene, the aneuploid pancreatic cell line Hs766T had two novel missense variants (p.[Y259C;H265N]) in the BUB1 gene. These mutations were on the same allele, accompanied by a wild-type BUB1 allele. This change was not found in other samples, the literature, or 110 additional chromosomes from a reference population. Compared to two cell lines having microsatellite instability (MIN), the TP53 wild-type pancreatic cell line Hs766T had a defective mitotic spindle checkpoint, indicative of a cell line with chromosomal instability (CIN). Evidence that this checkpoint pathway can be abrogated by mutations in the BUB1 gene (Cahill et al., 1998) supports the suggestion the missense mutations of the BUB1 gene in the Hs766T cell line may contribute to its observed mitotic checkpoint defect.     

Expression of BUB1 protein in gastric cancer correlates with the histological subtype, but not with DNA ploidy or microsatellite instability

During mitosis, the spindle checkpoint delays the onset of anaphase until all chromosomes have attached properly to the mitotic spindle, preventing chromosome missegregation. BUB (budding uninhibited by benzimidazole) 1 is one of the key components of this checkpoint. BUB1 mutations are rare in cancer tissues and no mutations have been identified in gastric cancer. In mice, immunodepletion of BUB1 abolished the spindle checkpoint. Thus, aberrant expression of BUB1 protein could impair mitotic checkpoint function, resulting in aneuploidy, a common phenomenon in gastric cancer. In the present study, an antibody was generated against BUB1 and its expression was studied in gastric cancer tissue sections (n = 80) by immunohistochemistry. Nuclear BUB1 expression was found in all gastric cancer cases. The proportion of tumour cells expressing BUB1 was significantly greater in diffuse-type than in intestinal-type gastric carcinoma (p < 0.001). No correlation was found between BUB1 expression and deoxyribonucleic acid (DNA) ploidy, microsatellite instability or any other histopathological parameters investigated. To the authors' knowledge, this is the first study of BUB1 protein expression in gastric cancer tissues. Different BUB1 protein expression levels in intestinal- and diffuse-type gastric cancer may provide further evidence of a potential link between different genetic pathways and morphological phenotype in gastric carcinogenesis. However, further studies are needed to establish whether there is an association between BUB1 protein expression level and mitotic spindle checkpoint function in gastric cancer.     

Syndrome of microcephaly, Dandy-Walker malformation, and Wilms tumor caused by mosaic variegated aneuploidy with premature centromere division (PCD): report of a new case and review of the literature

We report a male infant with multiple congenital anomalies and mosaic variegated aneuploidy; a rare cytogenetic abnormality characterized by mosaicism for several different aneuploidies involving many different chromosomes. He had prenatal-onset growth retardation, microcephaly, dysmorphic face, seizures, hypotonia, feeding difficulty, and developmental delay. In addition, he developed bilateral Wilms tumors. Neuroradiological examination revealed Dandy-Walker malformation and hypoplasia of the cerebral hemisphere and pons. Cytogenetic analysis revealed various multiple numerical aneuploidies in blood lymphocytes, fibroblasts, and bone marrow cells, together with premature centromere division (PCD). Peripheral blood chromosome analysis from his parents also showed PCD, but no aneuploid cells. The clinical phenotype and multiple aneuploidies of the patient may be a consequence of the homozygous PCD trait inherited from his parents. Comparison with previously reported cases of multiple aneuploidy suggests that mosaic variegated aneuploidy with PCD may be a clinically recognizable syndrome with major phenotypes being mental retardation, microcephaly, structural brain anomalies (including Dandy-Walker malformation), and possible cancer predisposition. Received: January 21, 1999 / Accepted: February 26, 1999     

Child with mosaic variegated aneuploidy and embryonal rhabdomyosarcoma

We report on a 7-year-old boy with mosaic variegated aneuploidy (MVA) who developed embryonal rhabdomyosarcoma of the soft palate. This patient is the 11th case report of MVA and represents further documentation of the true existence of this rare mitotic mutant. Clinical findings share similarities to those previously described patients including microcephaly and growth retardation as the two most common abnormalities. Notably, mental retardation is not universally present. Results of serial cytogenetic analyses performed on somatic and neoplastic tissues are reviewed and compared with those of other previously reported patients. We postulate that mosaic variegated aneuploidy is causally related to the development of rhabdomyosarcoma in our patient. This is the first report of a patient with MVA who developed cancer and suggests that these patients may be at risk for malignancy and require long-term follow-up and cancer surveillance. Am. J. Med. Genet. 82:20–24, 1999. © 1999 Wiley-Liss, Inc.     

Mosaic variegated aneuploidy with multiple congenital abnormalities: Homozygosity for total premature chromatid separation trait

Separation of chromatids of all mitotic chromosomes, here called total premature chromatid separation (total PCS), was observed in 67 to 87.5% of repeated cultures of peripheral blood lymphocytes from two unrelated infants. Also noted was a variety of mosaic aneuploidies, especially trisomies, double trisomies, and monosomies, to be called mosaic variegated aneuploidy. The infants both showed severe pre- and postnatal growth retardation, profound developmental retardation, uncontrollable seizures, severe microcephaly, hypoplasia of the brain, Dandy-Walker anomaly, abnormal facial appearance, and bilateral cataract. Patient 1, a girl, in addition had a cleft palate, multiple renal cysts, and Wilms tumor of the left kidney. Whereas patient 2, a boy, had ambiguous external genitalia. They both died within 2 years of age. In the two families of the infants, their parents and three other members showed 2.5 to 47% lymphocytes with total PCS but without mosaic variegated aneuploidy or phenotypic abnormalities. Another 10 relatives studied showed 0 to 1% cells with total PCS and so were judged negative for the total PCS trait. It was deduced that the total PCS trait in the two families was transmitted in an autosomal-dominant fashion, and the two affected infants were homozygous for the trait. Am. J. Med. Genet. 78:245–249, 1998. © 1998 Wiley-Liss, Inc.     

Overexpression of BUBR1 is associated with chromosomal instability in bladder cancer

BUBR1, a mitotic checkpoint protein, is a key component of the mitotic spindle checkpoint machinery. Defective BUBR1 has been proposed to contribute to chromosomal instability (CIN). To elucidate the relationship of BUBR1 expression with CIN, expression of BUBR1, numbers of centrosomes, numerical aberrations of chromosomes, and DNA ploidy were examined, and BUBR1 expression status was compared with clinicopathological parameters in 104 human urothelial bladder carcinomas. Expression of BUBR1 and numbers of centrosomes were assessed by immunohistochemistry. Numerical aberrations of chromosomes 7, 9, and 17 were evaluated by fluorescence in situ hybridization. Cancers with a large intercellular variation in centromere copy number were designated as CIN cancers. Tumors with BUBR1 overexpression were associated with CIN, DNA aneuploidy, and centrosome amplification. Array CGH revealed that BUB1B amplification and loss rarely occurred, indicating that the overexpression of BUBR1 in these bladder cancers was independent of BUB1B copy number. Overexpression of BUBR1 significantly correlated with higher histological grade, advanced pathological stage, and high cell proliferation. Overexpression of BUBR1 predicted tumor recurrence and disease progression. These data suggest that overexpression of BUBR1 is potentially a new tumor marker for estimating biological characteristics of bladder cancer.     

Spindle proteins Aurora A and BUB1B, but not Mad2, are aberrantly expressed in dysplastic mucosa of patients with longstanding ulcerative colitis

Background

Long term ulcerative colitis (UC) increases the risk of colorectal cancer (CRC). DNA aneuploidy is a common feature of both dysplastic and non‐dysplastic colonic epithelia from patients with longstanding UC, and is regarded as an early sign of possible malignant transformation. The spindle proteins Aurora A, BUB1B and Mad2 have been implicated as contributors to aneuploidy and carcinogenesis.

Aims

To investigate the role of these spindle proteins in relation to DNA aneuploidy and during the progressive morphological changes in ulcerative colitis associated colorectal cancer (UCCRC).

Methods

Tissue microarrays were made from 31 colectomy specimens from patients with longstanding UC. Expression of Aurora A, BUB1B and Mad2 was investigated by immunohistochemistry and their relation to ploidy status, mucosal morphology and Ki67 levels was explored.

Results

Expression of Aurora A and BUB1B was significantly associated with the progressive morphological changes of UCCRC. In the progression from non‐dysplastic to dysplastic mucosa, Aurora A expression decreased while BUB1B expression increased. There was an increasing incidence of aneuploidy with progression towards cancer; expression of all spindle proteins was associated with the level of Ki67 but not with aneuploidy.

Conclusion

Due to the significant differences in Aurora A and BUB1B expression in dysplastic compared non‐dysplastic mucosa, these proteins may serve as putative biological markers for the progressive morphological changes in UC associated carcinogenesis. The close relationship to Ki67 levels reflect that spindle proteins are expressed in tissues with a high proliferative rate; a role for these proteins in the development of aneuploidy was not found.Ulcerative colitis (UC) is a chronic disease which is associated with an increased risk of developing colorectal cancer (CRC). The overall prevalence of CRC in patients with UC is 3.7%,¹ and the risk of cancer increases with duration of the disease.² UC associated colorectal cancer (UCCRC) is the cause of death in every sixth patient with UC.³ Surveillance colonoscopy with biopsies is the current screening method to detect dysplastic lesions in patients with UC. However, mucosal dysplasia as a marker for cancer has several implications,⁴ and there exist conflicting reports concerning the significance of dysplasia as a precancerous lesion.^5,6 Furthermore, surveillance colonoscopy has a poor success rate in preventing cancer in patients with UC,⁷ and current guidelines on CRC screening state that there is no evidence that endoscopic surveillance reduces cancer mortality in patients with UC.⁸ Obviously there is a need for improved screening methods and there is an ongoing search for biological markers to detect patients with UC at greatest risk of developing cancer. One such marker is DNA aneuploidy that is present in more than half of colorectal adenocarcinomas developing in UC,⁹ and aneuploidy has been shown to precede mucosal dysplasia.^10,11,12,13 A compromised spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA after mitosis, has been suggested to contribute to aneuploidy and carcinogenesis.¹⁴ Mad2 (mitosis arrest‐deficient 2) and BUB1B (budding uninhibited by benzimidazole) are vital parts of this checkpoint, and together they control the separation of sister chromatids. The Aurora family of threonine kinases, and especially Aurora A (STK6/15, ARK1, Aurora 2), has received attention for its role in the development of aneuploidy,¹⁵ and has been shown to induce centrosome amplification and aneuploidy in malignant human cell lines.¹⁶ Hence, in this study we wanted to investigate the role of Aurora A, BUB1B and Mad2 proteins in the development of aneuploidy and during the progressive morphological stages of UC associated carcinogenesis.     

Cell cycle and centromere FISH studies in premature centromere division

Background  

Mitotic configurations consistent in split centromeres and splayed chromatids in all or most of the chromosomes or premature centromere division (PCD) have been described in three categories. (1) Low frequency of PCD observed in colchicines-treated lymphocyte cultures from normal individuals. (2) High frequency of PCD with mosaic variegated aneuploidy. (3) High frequency of PCD as a sole chromosome abnormality observed in individuals with no recognizable clinical pattern. We report four members of a family with the third category of PCD.     

Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus

To engineer cucumber mosaic virus (CMV-Ix) into a gene vector, genome component RNA 3 of the virus was modified and split into two sub-components, RNA 3A and RNA 3B. In RNA 3A, the open reading frame of the movement protein (MP) was replaced by a reporter gene encoding the green fluorescent protein (GFP), to monitor virus replication and movement. In RNA 3B, the coat protein (CP) gene was eliminated and a multiple cloning site (MCS) was created for foreign gene insertion. Each sub-component alone is defective and relies on its companion sub-component to restore full RNA 3 function. The vector system was evaluated for its ability to deliver and express the bacterial beta-glucuronidase (GUS) gene and a modified bean yellow mosaic virus coat protein (BYMV-CP) gene in Nicotiana benthamiana plants. Results showed that the engineered virus was able to move from cell to cell in the inoculated leaf and enter the minor veins of the inoculated leaf. Foreign gene expression was detected in the inoculated leaves. However, intermolecular recombination between RNA 3A and 3B occurred frequently, preventing efficient systemic expression of the foreign gene(s). Modifications and further evaluations are being undertaken to improve the gene delivery system.     

BUB1 mRNA is significantly co-expressed with AURKA and AURKB mRNA in advanced-stage ovarian serous carcinoma

The objective of this study was to investigate the expression and clinical role of the spindle checkpoint kinase budding uninhibited by benzimidazole 1 (Bub1) in primary and metastatic advanced-stage ovarian serous carcinoma. BUB1 mRNA expression was analyzed in 178 tumors (88 effusions, 38 primary carcinomas, and 52 solid metastases) from 144 patients with advanced-stage disease using quantitative real-time polymerase chain reaction (PCR). Bub1 protein expression by Western blotting was studied in 63 carcinomas (30 effusions and 33 solid lesions). BUB1 mRNA expression at different anatomic sites was studied for association with clinicopathologic parameters, including chemotherapy resistance and survival. BUB1 mRNA was universally expressed in serous carcinomas, irrespective of anatomic site. BUB1 mRNA levels were uniformly low in six ovarian surface epithelium specimens analyzed for comparative purposes. Bub1 protein was expressed in 22/30 effusions and 28/33 solid lesions. BUB1 mRNA expression was significantly higher in chemo-naïve primary carcinomas and solid metastases compared to specimens obtained following neoadjuvant chemotherapy (p?<?0.001) and was unrelated to chemotherapy exposure in effusions nor to chemoresponse or survival at any anatomic site. BUB1 mRNA levels in both effusions and solid lesions were strongly related to the mRNA levels of AURKA and AURKB previously studied in this cohort (p?<?0.001 for both). Bub1 is widely expressed in primary and metastatic OC, suggesting a biological role in this cancer. BUB1 mRNA levels are lower following chemotherapy exposure in solid lesions, though its presence is unrelated to clinical behavior including response to chemotherapy and survival. BUB1 is co-expressed with AURKA and AURKB suggesting biological relationship between these spindle cell components.