Comprehensive molecular and clinical findings in 29 patients with multi-locus imprinting disturbance

Abstract

Background: Multi-locus imprinting disturbance (MLID) with methylation defects in various differentially methylated regions (DMRs) has recently been identified in approximately 150 cases with imprinting disorders (IDs), and deleterious variants have been found in genes related to methylation maintenance of DMRs, such as those encoding proteins constructing the subcortical maternal complex (SCMC), in a small fraction of patients and/or their mothers. However, integrated methylation analysis for DMRs and sequence analysis for MLID-causative genes in MLID cases and their mothers have been performed only in a single study focusing on Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) phenotypes.

Results: Of 783 patients with various IDs we have identified to date, we examined a total of 386 patients with confirmed epimutation and 71 patients with epimutation or uniparental disomy. Consequently, we identified MLID in 29 patients with epimutation confirmed by methylation analysis for multiple ID-associated DMRs using pyrosequencing and/or methylation-specific multiple ligation-dependent probe amplification. MLID was detected in approximately 12% of patients with BWS phenotype and approximately 5% of patients with SRS phenotype, but not in patients with Kagami-Ogata syndrome, Prader-Willi syndrome, or Angelman syndrome phenotypes. We next conducted array-based methylation analysis for 78 DMRs and whole-exome sequencing in the 29 patients, revealing hypomethylation-dominant aberrant methylation patterns in various DMRs of all the patients, eight probably deleterious variants in genes for SCMC in the mothers of patients, and one homozygous deleterious variant in ZNF445 in one patient. These variants did not show gene-specific methylation disturbance patterns. Clinically, neurodevelopmental delay and/or intellectual developmental disorder (ND/IDD) was observed in about half of the MLID patients, with no association with the identified methylation disturbance patterns and genetic variants. Notably, seven patients with BWS phenotype were conceived by assisted reproductive technology (ART).

Conclusions: The frequency of MLID was 7.5% (29/386) in IDs caused by confirmed epimutation. Furthermore, we revealed diverse patterns of hypomethylation-dominant methylation defects, nine deleterious variants, ND/IDD complications in about half of the MLID patients, and a high frequency of MLID in ART-conceived patients.

Keywords: Array-based methylation analysis; Imprinting disorders; MS-MLPA; Multi-locus imprinting disturbance; Pyrosequencing; Subcortical maternal complex; Whole-exome sequencing.

Fig. 1

Study flowchart. Dx, diagnosis; BWS, Beckwith-Wiedemann syndrome; SRS, Silver-Russell syndrome; TS14, Temple syndrome; KOS, Kagami-Ogata syndrome; PWS, Prader-Willi syndrome; AS, Angelman syndrome; PHP1B, pseudohypoparathyroidism type 1B; TNDM, transient neonatal diabetes mellitus; SGA, small for gestational age; MLID, multi-locus imprinting disturbance; DMR, differentially methylated region; LOM, loss of methylation; EPIC, array-based methylation analysis using Infinium MethylationEPIC Kit (Illumina); WES, whole-exome sequencing; Pt, patient; Mo, mother

Fig. 2

Results of methylation analyses using MS-MLPA, pyrosequencing, and array-based methylation analysis. The color-coded background in the Analysis row indicates the degree of coincidence of the locus of evaluated CpG sites in the DMR between MS-MLPA or pyrosequencing and array-based methylation analysis using Infinium MethylationEPIC Kit (Illumina): yellow for complete coincident, light yellow for partial coincident, and green for no coincident. DMR, differential methylated region; B, Beckwith-Wiedemann syndrome; P, pseudohypoparathyroidism type 1B; O, overgrowth; S, Silver-Russell syndrome; T, Temple syndrome; TN, transient neonatal diabetes mellitus; W, Prader-Willi syndrome; SG, small for gestational age; MS-MLPA, methylation-specific multiple ligation-dependent probe amplification; Pyro, pyrosequencing; EPIC (3SD), array-based methylation analysis using analysis method 1; EPIC (CH-t), array-based methylation analysis using analysis method 2

Fig. 3

Heatmap of array-based methylation analysis using analysis method 1. The heatmap indicates 56 aberrant DMRs out of 78 examined DMRs. Each row represents a DMR; each column represents a patient. Germline DMRs are shown on a gray background, secondary DMRs on a light green background, and unclassifiable DMRs on a white background. Methylation disturbances of DMRs are classified into seven categories based on the degree. B, Beckwith-Wiedemann syndrome; P, pseudohypoparathyroidism type 1B; O, overgrowth; S, Silver-Russell syndrome; TS, Temple syndrome; TN, transient neonatal diabetes mellitus; W, Prader-Willi syndrome; SG, small for gestational age; N, neurodevelopmental delay and/or intellectual developmental disorder; A, assisted reproductive technology; T, monozygotic monochorionic diamniotic twins; V, variants of uncertain significance; L, likely pathogenic; DMR, differential methylated region; Chr, chromosome; MML, median methylation level of CpG sites in the DMR; SD, standard deviation; mean, mean MML of 16 healthy controls