Biallelic MAD2L1BP (p31comet) mutation is associated with mosaic aneuploidy and juvenile granulosa cell tumors

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp–/– mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.


SUPPLEMENTAL FIGURE 8
Summary of p31 comet interactions and functions.p31 comet -DC is unable to support onset of anaphase or homology directed repair of DSBs.IR, insulin receptor; n.d., not determined.

SUPPLEMENTAL TABLE 2
Results from MAD2L1BP mutation screening in patients with sporadic non-syndromic JGCT. a , known somatic pathogenic variant, b , coverage too low in some regions.
(A) Relative contribution of identified signatures in the two samples.(B) Signature decomposition for the sample of Patient 1a.(C) Signature decomposition for the sample of Patient 1b.Both mutational profiles are dominated by SBS30 (putative FFPE-damage signature).
signature using high quality somatic variants with MAF ³5%.(A) Relative contribution of identified signatures in the two samples.(B) Signature decomposition for the sample of Patient 1a.(C) Signature decomposition for the sample of Patient 1b.Quality filtering of somatic mutations revealed a contribution of signatures SBS5 and SBS11 in Patient 1b.etoposide.Cell viability of fibroblasts from controls and patients were treated for 72h with the indicated concentrations of etoposide normalized to DMSO vehicle.Error bars represent the s.d.(n = 4).Data were analyzed by two-tailed, unpaired Students t-test (****p<0.0001,***p<0.001;**p<0.01;*p<0.05).Each experiment was repeated 3 times.SUPPLEMENTAL FIGURE 5 Characterization of recombinant p31 comet -DC.(A) Recombinant p31 comet -DC and patient derived, endogenous p31 comet exhibit the same molecular weight in SDS-PAGE.Endogenous p31 comet from asynchronously growing, patient-derived fibroblasts was immunoprecipitated and analyzed by SDS-PAGE and immunoblotting side-byside with the indicated in vitro translated (IVT) p31 comet variants, from which the FLAG3-Tev2-tag had (or had not) been removed by treatment with TEV protease.(B) Cells that rely on p31 comet -DC progress slowly through mitosis.HeLaK cells transfected to replace endogenous p31 comet by the indicated FLAG3-Tev2-tagged variants were synchronously released from a RO3306-mediated G2-arrest and analyzed by flow cytometry (Figure 6B) and time-resolved immunoblotting.Note that cyclin A2, a SACindependent APC/C substrate, is degraded with normal kinetics in p31 comet -DC expressing cells indicating that entry into mitosis is not delayed.(Note also that Cdc27 is hyperphosphorylated in early mitosis and becomes visible as a sharp band only upon dephosphorylation in late M-phase.)SUPPLEMENTAL FIGURE 6 Functional characterization of p31 comet -DC in mitosis.(A) Replacing endogenous p31 comet by p31 comet -DC greatly prolongs metaphase.HeLaK cells transfected to replace endogenous p31 comet by the indicated FLAG3-Tev2-tagged variants were synchronously released from a taxol-mediated prometaphase arrest by the aurora B inhibitor ZM 447439 (ZM) and analyzed by time-resolved immunoblotting using the indicated antibodies.(B) p31 comet -DC cannot support the disassembly of SAC-induced Mad2 complexes.Cells from (A) were subjected to time-resolved Mad2-IPs followed by immunoblotting using the indicated antibodies.
truncated p31 comet cannot support homology-directed repair of DNA double strand breaks.(A) U2OS DR-GFP (HDR reporter) and U2OS EJ5-GFP (NHEJ reporter) cells transiently expressing HA-and estrogen receptor-tagged I-SceI (HA-ER-I-SceI) and Flag-tagged p31 comet variants were transfected with the indicated siRNAs, synchronized in G2-phase and supplemented -where indicated -with 4-hydroxytamoxifen (OHT) to direct the homing endonuclease into the nucleus and with ligase IV inhibitor SCR7 to block NHEJ.Two days later, cells were subjected to immunoblotting.(B) Samples from (A) were also analyzed by flow cytometry to determine the percentage of GFP-positive cells.Shown are averages (bars) of three independent experiments (dots).Note that overexpression of p31 comet -WT has a suppressive effect on NHEJ.SEP, SEPARASE, positive control for a protein required for HDR.

Rare homozygous variants identified in WES and present in Patient 1a and Patient 1b, but not their healthy sister.
Where applicable, diseases known to be associated with the respective genes are given.Red, disease gene, MAD2L1BP.Chr, chromosome; gnomAD, Genome Aggregation Database; homo, number of homozygous individuals annotated in gnomAD; rs, Reference SNP cluster ID. "Pathogenicity predictions": Number of applicable pathogenicity prediction programmes that suggest functional impairment/pathogenicity.