Loss-of-function maternal-effect mutations of PADI6 are associated with familial and sporadic Beckwith-Wiedemann syndrome with multi-locus imprinting disturbance

Abstract

Background: PADI6 is a component of the subcortical maternal complex, a group of proteins that is abundantly expressed in the oocyte cytoplasm, but is required for the correct development of early embryo. Maternal-effect variants of the subcortical maternal complex proteins are associated with heterogeneous diseases, including female infertility, hydatidiform mole, and imprinting disorders with multi-locus imprinting disturbance. While the involvement of PADI6 in infertility is well demonstrated, its role in imprinting disorders is less well established.

Results: We have identified by whole-exome sequencing analysis four cases of Beckwith-Wiedemann syndrome with multi-locus imprinting disturbance whose mothers are carriers of PADI6 variants. In silico analysis indicates that these variants result in loss of function, and segregation analysis suggests they act as either recessive or dominant-negative maternal-effect mutations. Genome-wide methylation analysis revealed heterogeneous and extensively altered methylation profiles of imprinted loci in the patients, including two affected sisters, but not in their healthy siblings.

Conclusion: Our results firmly establish the role of PADI6 in imprinting disorders. We report loss-of-function maternal-effect variants of PADI6 that are associated with heterogeneous multi-locus imprinting disturbances in the progeny. The rare finding of two siblings affected by Beckwith-Wiedemann syndrome suggests that in some cases, familial recurrence risk of these variants may be high. However, the heterogeneous phenotypes of the other pedigrees suggest that altered oocyte PADI6 function results in stochastic maintenance of methylation imprinting with unpredictable consequences on early embryo health.

Keywords: Beckwith-Wiedemann syndrome; DNA methylation; Genomic imprinting; Infertility; Maternal-effect variants; Multi-locus imprinting disturbance; PADI6; Subcortical maternal complex.

Fig. 1

Genetic characterization of the three families under study. Pedigree and corresponding PADI6 variants of families 1 (a), 2 (b), and 3 (c). Black filled symbol represents individual with BWS features, and black central dot unaffected carriers of PADI6 variants. Weeks of gestation (gw) are reported for the aborted fetus in family 2. MLID status and the number of DMRs with altered methylation are also reported

Fig. 2

PCA of imprinted DMR methylation. DNA methylation of the imprinted DMRs of the four probands, their mothers and siblings, and 12 age-matched healthy controls was compared by PCA. Symbol code: circles represent members of family 1, triangles members of family 2, rhombi members of family 3, and squares the controls. Color code: probands are represented in dark blue, mothers in pink, siblings in light blue, controls in gray

Fig. 3

DNA methylation analysis of imprinted DMRs. Heatmap showing imprinted DMR methylation levels of the probands of families 1, 2, and 3 (F1_III-1, F1_III-2, F2_II-3, F3_II-2); their mothers (F1_II-2, F2_I-4, F3_I-2) and siblings (F2_II-2, F3_II-1, F3_II-3); and 12 age-matched control individuals, organized by hierarchical clustering. Clustering is based on CpG methylation levels of 736 probes overlapping with 43 imprinted DMRs, containing at least three CpGs. Maternally methylated germline DMRs are in dark pink; maternally methylated secondary DMRs are in light pink. Paternally methylated germline DMRs are in dark blue; paternally methylated secondary DMRs are in light blue. The KCNQ1OT1:TSS-DMR and the H19/IGF2:IG-DMR diagnostic of BWS are highlighted in green. The VTRNA2-1 DMR has been reported to be polymorphic [24]

Fig. 4

Position of the pathogenic variants in the PADI6 protein. The variants associated with infertility of the carrier women are represented with black rhombi, and variants associated with MLID in the offspring with orange or red rhombi, if described in present or previous studies, respectively

Fig. 5

Effect of the frameshift variant on PADI6 protein structure. a Alignment of the C-terminal residues of the wild-type PADI6 with those of the protein resulting from the T669Kfs*85 variant. b Model of human wild-type PADI6 in dimeric form. The amino acids that are colored are the sites were mutations occur. The model is represented as a cartoon, and the two chains are colored in light or dark gray, respectively. The 26-aa carboxy-terminal region of the protein that is substituted by a longer peptide in the frameshift variant T669Kfs*85 is colored in blue; the side chains of M477 (yellow), P632 (orange), and P694 (red) are shown as balls-and-sticks