Pathogenic correlation between mosaic variegated aneuploidy 1 (MVA1) and a novel BUB1B variant: a reappraisal of a severe syndrome

Background The BUB 1 mitotic checkpoint serine/threonine kinase B (BUB1B) gene encodes a key protein in the mitotic spindle checkpoint, which acts as a surveillance mechanism, crucial for the maintenance of the correct chromosome number during cell deviation. Mutations of BUB1B gene are linked to mosaic variegated aneuploidy 1 (MVA1) syndrome, a rare autosomal recessive disorder characterized by widespread mosaic aneuploidies, involving different chromosomes and tissues. MVA1 is clinically characterized by intrauterine growth restriction, post-natal growth retardation, and severe neurologic impairment including microcephaly, developmental delay/intellectual disability, epileptic seizures, and generalized hypotonia. Malignancies are also serious sequelae associated with the disorder. We reported on a case of two-year-old Italian girl with MVA1 who shows severe neurologic impairment, microcephaly and epileptic seizures. Materials and methods Clinical data collection and genetic diagnosis of the patient were assessed. Mutational analysis covers the chromosomal microarray analysis, the gene methylation pattern studied using the methylation-specific multiplex ligation-dependent probe amplification, and the family-based Whole Exome Sequencing (WES). A literature research based on reported cases of MVA and premature chromatid separation was also included. Results Karyotyping has revealed 12% of mosaics in the patient who carries a novel variant in BUB1B gene (c.2679A > T, p.Arg893Ser) detected by WES. Thirty-one cases of MVA1 including the present report, and four prenatally diagnosed cases with MVA1 were selected and inspected. Conclusion Clinical and genetic findings reported in the girl strongly suggest a new MVA1 genotype–phenotype correlation and lead to a reappraisal of a severe syndrome. Diagnosis and in-depth follow-up provided worthwhile data. Supplementary Information The online version contains supplementary material available at 10.1007/s10072-022-06247-w.


Background
The BUB1 mitotic checkpoint serine/threonine kinase beta (BUB1B) gene encodes for the protein BubR1 (budding uninhibited by benzimidazole-related-1). This protein has a central regulatory role of mitosis through phosphorylation of members of mitotic checkpoint complex (MCC), which subsequently bind to CDC20 protein preventing the activation of the anaphase promoting complex/cyclosome (APC/C), to ensure proper chromosome segregation in the metaphase before the progression to anaphase stage of mitosis [1]. The BubR1 protein is essential for the primary cilium formation and the characterization of mutations has been recently enclosed in the group of ciliopathies [2]. Mosaic variegated aneuploidy (MVA) is a group of rare autosomal recessive disorders characterized by premature chromatid separation (PCS) in > 50% metaphase cells and a variety of mosaic aneuploidies. MVA1 type 1 (OMIM 257,300) is caused by homozygous or compound heterozygous mutations in the BUB1B gene (OMIM 602,860) located in chromosome 15q1 and is clinically characterized by microcephaly, developmental delay/intellectual disability (DD/ID), epileptic seizures, generalized hypotonia, intrauterine growth restriction (IUGR), and postnatal growth retardation. Moreover, MVA1 may be associated with severe congenital malformations, and a high risk of malignancies such as rhabdomyosarcoma, Wilms tumor, myelodysplasia, and variable types of leukemia [3]. Herein, we report a case of 2-year-old girl who showed pre-and post-natal growth retardation, minor facial features, severe microcephaly with protruding metopic suture, epileptic seizures, generalized hypotonia, and congenital ovarian cyst. The genetic and genomic screening detected respectively in the patient 12% mosaic aneuploidies and a novel likely pathogenic variant (c.2679 A > T, p.Arg893Ser) of BUB1B gene. Clinical data were consistent with the diagnosis of MVA1 syndrome. A highly likely genotype/phenotype correlation between MVA1 and the novel BUB1B variant has been advanced.

Patients' medical report
A 9-month-old female first came to the Department of Pediatrics, University Hospital Vittorio-Emanuele-Policlinico, Catania, Italy, for clinical checkup. Family history was irrelevant. The mother referred that at the age of 7 months of gestation, fetal ultrasound displayed the presence of a right ovarian cyst. Two weeks later, another ultrasound examination was done and revealed the presence of ascitic fluid with vanishing of the ovarian cyst. She reported normal fetal movements throughout the pregnancy. At the time of gestation, the ages of the little girl's mother and father were 30 and 36 years old respectively. A programmed caesarean section was carried out with the birth of a female newborn with the following anthropometric measurements: weight 2190 g (3rd percentile), length 47 cm (50th percentile), and occipito-frontal circumference (OFC) 28 cm (< 3rd percentile). Apgar scores were 7 and 9 at 1 and 5 min respectively. The newborn was admitted to local Neonatal Intensive Care Unit (NICU) where she was oxygenated by intubation for four days. She was fed by nasogastric tube due to poor suckling reflex and refusal of alimentation. No signs of ascites were detected on a new abdominal ultrasound. At the age of 2 months and a half, she had an acute respiratory infection with severe respiratory distress, which required endotracheal intubation kept for 2 days. During the first months of life, she showed generalized hypotonia, feeding difficulty, and frequent episodes of vomiting. At 7th month, she had an epileptic seizure manifesting by fixed gaze and head rotation on the right side lasted few minutes and recovered by rectal midazolam. The EEG record was non-informative. Two days later, a new seizure came out with the same characteristic of the previous one and treatment with valproate was started. At 8th month, she was admitted to the Hospital of North Italy and discharged with a diagnosis of axial and segmental hypotonia, epileptic seizures, microcephaly with brain MRI of simplified cortical gyral pattern, and wide corpus callosum hypoplasia. At physical examination, the weight was 6800 g (3rd percentile), length 70 cm (50th), and OFC 38.5 cm (> 4th percentile). She showed minor facial features, microcephaly, developmental delay, generalized hypotonia, and growth retardation. The anterior fontanelle was open 0.5 × 0.5 cm and flat. Tendon reflexes were normally present. She was unable to hold her head up by arm traction maneuver. Routine laboratory analysis, electrolytes, plasma and urinary amino acid, organic acid, urinalysis, thyroid markers, sialo-transferrin, plasma purine, and total cholesterol were within normal limits. TORCH screen, inflammatory markers and otoacoustic emissions screening were normal. On abdominal ultrasound examination, no anomalies were found in the liver, spleen, kidneys, pancreas, or ovaries. ECG and echocardiogram were normal. EEG showed a background disorganization with the presence of rare spike and waves. Treatment with valproate and levetiracetam was continued. The little girl has been serially followed up in ambulatory care. During this period, she had three tonic clonic seizures of short duration, the last one was associated with fever. She presented recurrent stereotyped upper arm waving movements, raising and lowering upper limbs, beating the hands on the tables or other surfaces. The movement anomalies were seen during the day and with less frequency during sleep. At the current age of 2 years old, she has been newly admitted to the Institution. Her anthropometric measurements were weight 12 kg (25th-50th percentile), height 85 cm (50th percentile), and OFC 41.5 cm (< 4th percentile). At clinical examination, she showed minor facial features with notably small head and slightly protruding metopic suture, slanting forehead, short nose with round tip, right epicanthal fold, hypotelorism, down slanting palpebral fissures, small mouth, and dyschromic teeth. At the neurological examination, she showed generalized hypotonia, difficulty in holding her head up, and inability to sit up without support. The anterior fontanelle closed. Patellar reflexes were normally present. Routine laboratory analyses were normal. ECG, echocardiogram, fundus examination, and hearing exploration were also normal. Video-EEG at awake and during sleep showed paroxysmal multifocal spike and waves particularly evident in the bilateral frontal regions (Fig. 1a,b). No other seizures were recorded. Physiotherapy and antiepileptic treatment with valproate and levetiracetam were maintained.

Discussion
The patient presented with pre-and post-natal growth retardation, severe primary microcephaly with pointed forehead, minor facial features, epileptic seizures partially responsive to treatment, generalized hypotonia, corpus callosum hypoplasia, and prenatal ovarian cyst. Clinical presentation associated with genetic findings revealing mosaic aneuploidies by karyotyping, and a novel heterozygous  (Fig. 2) are consistent with a diagnosis of MVA1 syndrome. A likely correlation between clinical features presented by the patient the novel BUB1B variant seems to be highly probable. Subjects affected by MVA1 syndrome show a quite homogeneous and typical pattern of phenotypic manifestations as also observed in the present case. Kajii, Ikeuchi [3] reported five patients and reviewed other five cases from literature illustrating that all of these patients presented with similar clinical and cytogenetic findings, among which (a) more than 50% of mitotic lymphocytes with total PCS, (b) mosaic variegated aneuploidy in all the patients, (c) pre-and post-natal growth retardation, and profound developmental delay, (d) severe microcephaly, (e) hypoplasia of the brain with Dandy-Walker complex or other malformations of the posterior fossa, (f) drugs unresponsive clonic epileptic seizures, and (g) both parents with 3% or more mitotic lymphocytes in total PCS. As suggested by this study, less frequent features included minor facial features, bilateral cataracts, microphthalmia, cleft palate, ambiguous genitalia in males, skin abnormalities, and malignancies.
In the present study, the contribution of disease association for new low-penetrance BUB1B variant is determined. The revealed mutation in the patient and in her healthy father was predicted to be likely deleterious affecting the highly conserved catalytic domain of serine/threonine kinase subunit that may cause the loss of BUB1B kinase activity involved in the regulation of the signaling cascade of the spindle assembly checkpoint (SAC) components [29,30], and other chromatin substrates, such as histones [31,32] (Fig. 3). In addition, the variant seems to overlap with an active promoter region encoding for the readthrough transcripts of BUB1B-PAK6 gene, which product is the same as the downstream gene (PAK6) and was considered as a candidate gene for epileptic encephalopathy [4]. This can explain the epileptic seizures found in the patient. Furthermore, the overall framework emerged from ChIP-seq data (Fig. 4) indicates that the detected variant includes a conserved cluster of transcription factor binding site (TFBS) predicted to be targets for transcription factors implicated in the genomic stability. Therefore, the sequence variant may disrupt the gene expression and transcription regulatory motifs for CTCF, RAD21, and SMC3 proteins. Their co-distribution is suggestive to mediate the higher-order chromatin organization.     [20] Chaker et al. 2017 [21] Kato et al. 2017 [22] Ayaz et al. 2018 [23] Present study   The role of the insulator/repressor transcription factor CTCF and cohesins RAD21 and SMC3 has been investigated in the chromosomal instability (CI) leading to several tumors [5][6][7]. Recent studies on the clinical evaluation of VUS variants in some rare neurodevelopmental disorders have confirmed that the interpretation and the classification of VUS is influenced by the epigenetic signatures of the chromatin or changes in DNA methylation profile. Thus, the different methylation patterns of the entire affected gene may assist in the discrimination between a benign and pathogenic VUS in the same carriers [5,6]. Considering the mosaic aneuploidies found in a small subset of cells, although the cytogenetic investigation was performed only in white blood cells and not repeated, aberrant chromosomal disjunctions of the reported case are representative of the causal mechanism leading to a defective cell division typically associated to mutations in BUB1B gene. A genotype-phenotype correlation can be assumed, despite cannot be fully proven for the limits of the heterozygosity, is compatible with the observed clinical framework that is mostly coincidental and overlapped with other known cases of literature affected by BUB1B mutations.

Conclusion
The new BUB1B variant (c.2679A > T; p.Arg893Ser) should be enclosed as a potential risk of MVA1. When MVA diagnosis has been established after carrying out all relevant radiological and laboratory investigations, the management plan should be posed for the patients for early detection of malignancies. In infants or very young children, we suggest alerting parents about the nature of such disorder, and to keep in touch with the physician when some new clues arise. Abdominal ultrasound, fundus examination, and hematological checkup are advised every 3 months. Pathogenic mechanisms underlying MVA syndrome and the progression of related complications remain unclear. Up to now, one of the best described molecular events that can be attributed to MVA1 pathogenesis is the failure of mitotic checkpoint and ciliary functions caused by BUB1B mutations, which are indeed associated with a more severe disease phenotype and increased cancer risks. Therefore, follow-up of the patients and screening for malignancy is mandatory.

Data collection process
According to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, publications of reported cases of mosaic variegated aneuploidy   Funding Open access funding provided by Università degli Studi di Catania within the CRUI-CARE Agreement.
Data availability All data generated or analyzed during this study are included in this published article and may be released upon application to the corresponding author who can be contacted at ppavone@unict.it.

Declarations
Ethics approval and consent for publication The study was conducted ethically in accordance with the World Medical Association Declaration of Helsinki and approved by ethics Committee of the University of Catania, Italy (Ethical Committee Catania 1 Clinical Registration n. 95/2018/PO). Written informed consent was obtained from the parents.

Competing interests
The authors declare no competing interests.
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