Abstract Germline mutations leading to aneuploidy are rare, and their tumor-promoting properties are mostly unknown at the molecular level. We report here novel germline biallelic mutations in MAD1L1, encoding the spindle assembly checkpoint (SAC) protein MAD1, in a 36-year-old female with a dozen of neoplasias. Functional studies demonstrated lack of full-length protein and deficient SAC response, resulting in ~30 to 40% of aneuploid blood cells. Single-cell RNA analysis identified mitochondrial stress accompanied by systemic inflammation with enhanced interferon and NFκB signaling both in aneuploid and euploid cells, suggesting a non–cell autonomous response. MAD1L1 mutations resulted in specific clonal expansions of γδ T cells with chromosome 18 gains and enhanced cytotoxic profile as well as intermediate B cells with chromosome 12 gains and transcriptomic signatures characteristic of leukemia cells. These data point to MAD1L1 mutations as the cause of a new variant of mosaic variegated aneuploidy with systemic inflammation and unprecedented tumor susceptibility. __________________________________________________________________ MAD1L1 mutations found in an individual with high tumor susceptibility and a systemic inflammation response to aneuploidy. INTRODUCTION Aneuploidy is a frequent finding in spontaneous abortions and a near-universal characteristic of tumor cells ([54]1). However, germline mutations leading to chromosomal instability are rare in humans and lead to clinical and genetically heterogeneous syndromes ([55]2). The hallmark of these patients is the presence of mosaic aneuploidies, mainly trisomies and, more rarely, monosomies, and a variegated distribution of affected chromosomes in different cells and tissues throughout the body. Human diseases characterized by chromosomal instability are typically associated to mutations in genes related to cell division. Mutations in genes associated with centrosome and microtubule dynamics, chromosome morphology, and kinetochore functions affect the majority of patients with primary microcephaly ([56]3), and alteration of different cohesin subunits and associated regulators results in a variety of pathologies known as cohesinopathies ([57]4). Aneuploidy, however, is only occasionally observed in these pathologies ([58]4). On the other hand, mosaic variegated aneuploidy (MVA) typically correlates with mutations in components of the spindle assembly checkpoint (SAC), a multigene network that, together with other biological processes, controls the accurate chromosome segregation into daughter cells during cell division and permits ordered cell cycle progression from metaphase to anaphase ([59]5, [60]6). The SAC monitors the complete bipolar attachment of kinetochores on chromosomes to microtubules of the mitotic spindle and delays anaphase entry in the presence of unattached kinetochores. This is achieved through the formation of the mitotic checkpoint complex (MCC), composed of BUBR1, mitotic arrest deficiency 2 (MAD2), BUB3 and CDC20, and the subsequent inhibition of the anaphase-promoting complex/cyclosome (APC/C). SAC deficiency results in chromosome segregation in the presence of unattached chromosomes, generating daughter cells with abnormal chromosome numbers. In patients, these alterations are associated with a heterogeneous and nonspecific phenotype that typically involves pre- and postnatal growth retardation, eye and facial anomalies, variable developmental delay, and intellectual disability ([61]7–[62]9). Despite the widespread presence of aneuploidy in human tumors, to what extent chromosomal instability plays oncogenic or tumor suppressor roles is unclear ([63]1, [64]10). Cancer predisposition is not a common feature in patients with microcephaly or cohesinopathies. MVA is associated with a higher increase in some childhood malignancies, such as rhabdomyosarcoma, Wilms tumor, and leukemia, although these neoplasias are only observed in a subset of patients with MVA, and specific MVA-associated mutations have been suggested to impair tumor progression ([65]9, [66]11). Common features of patients with MVA are therefore intellectual disability and growth retardation, with or without microcephaly and tumors. We describe here the first germline biallelic mutations in MAD1L1 as a novel cause of aneuploidy in an individual with no intellectual disability and an unprecedented number of neoplasias. The MAD1L1 product MAD1 is an essential component of the mitotic checkpoint originally identified as a protein required for mitotic arrest in the presence of mitotic poisons ([67]12, [68]13). MAD1 recruits the MCC component MAD2 to unattached kinetochores, thus promoting the formation of the MCC and APC/C inhibition ([69]14–[70]16). Lack of Mad1l1 is lethal in the mouse during early development ([71]17), and no germline pathogenic mutations in the MAD1L1 gene have been described in humans. The proband displays biallelic mutations that are present in her parents in a heterozygous manner. The proband developed 12 neoplasias including five malignant tumors before the age of 36 years. Cellular studies suggested defective SAC function associated to high levels of aneuploidy (30 to 40%) in peripheral blood cells. Single-cell transcriptomic studies allowed the first analysis of the molecular consequences of aneuploidy in an individual with germline chromosomal instability. These studies suggested the presence of a non–cell autonomous inflammatory response to aneuploidy and identified small populations of cells with selected aneuploidies and specific functional or premalignant properties of physiological or clinical relevance. RESULTS Identification of germline biallelic mutations in MAD1L1 The proband, a female, was born in 1986 after an uncomplicated term pregnancy of a 26-year-old mother and a 30-year old father, both healthy and not consanguineous. Apgar was 9 of 10. Birth weight was 2510 g (3rd < P < 10th centile), with a length of 48 cm (10th < P < 25th centile). Developmental delay and slightly psychomotor retardation were apparent during the first months of life ([72]Table 1). Several café-au-lait spots were noted before the 6 months of life. At the age of 2 years, she had a stage III embryonal rhabdomyosarcoma of the left auditory canal that was treated with chemotherapy and radiotherapy. The impairment of her growth curve was interpreted as a consequence of the radiotherapy treatment, and growth hormone therapy was indicated. In 2001, several bone masses suggesting enchondromatosis were seen in the femur, humerus, and ulna. In addition, in 2001, a stage IB clear cell cervical carcinoma with ectocervix and endocervix involvement was diagnosed, without evidences of human papillomavirus infection, and treated with hysterectomy, bilateral adnexectomy, brachytherapy, and external radiotherapy. The proband had not been exposed to diethylstilbestrol in utero. In 2006, a pleomorphic adenoma of left parotid gland was surgically removed. One year later, she suffered a left mastoidectomy with left parotidectomy due to a low-grade fusiform cell sarcoma. During the period 2006–2010, several dysplastic nevi, a mammary lipoma, and a pilomatrixoma were removed. In 2010, a hemithyroidectomy was performed by a multinodular goiter. In 2012, a polypectomy of one adenoma of the colon harboring an intramucosal adenocarcinoma was carried out, and 2 years later, a pT3N0M0 rectal adenocarcinoma was resected. Another tubular adenoma was removed in the control colonoscopy in 2014. Table 1. A comparison of phenotypes in individuals with germline aneuploidy. CRC, colorectal carcinoma; ERMS, embryonic rhabdomyosarcoma; WT, Wilms tumor. Gene N of individuals Major phenotype Age* Aneuploidy^† Cancer Other neoplasias Representative references