Multilocus imprinting disturbances in the structure of imprinting disorders in the Russian Federation

Imprinting disorders (ImpDis) are a consequence of violations of the mechanism of genomic imprinting, which regulates the expression of homologous alleles of genes of different parental origin in imprinted regions of chromosomes. A solitary ImpDis is characterized by a methylation anomaly within its specific imprinted region. Multilocus imprinting disturbances (MLID) are characterized by methylation abnormalities in several imprinted regions. Four classes of molecular anomalies have been established for ImpDis: deletions/duplications, uniparental disomies, epimutations, and mutations in imprinted genes. MLID are detected more often among patients with epimutations. The causes of MLID are pathogenic variants in genes encoding oocytic and zygotic factors of embryo development, such as NLRP2, NLRP5, NLRP7, KHDC3L, OOEP, PADI6, TLE6, UHRF1, ZFP57, ARID4A, ZAR1, ZNF445, TRIM28, involved in the life cycle of imprinting. The presence of causative variants in MLID-associated genes in the family of a patient with MLID significantly increases the risk of reproductive losses and the birth of children with ImpDis and MLID, as opposed to single-locus epimutation within one imprinted chromosomal region, which makes it important to follow-up on MLID among patients with single-locus epimutation. In the research, methylation-specific multiplex ligation-dependent probe amplification of regions 6q24.2; 7q32.2; 11p15.5; 14q32.2; 15q11.2; 19q13.43; 20q13.32 (MS-MLPA MLID) was applied to 209 DNA samples of patients both sexes. In a group of patients with previously confirmed ImpDis without CNVs (113 samples), previous results were confirmed in 113 cases, and MLID were additionally detected in 7 (6.2%) cases. The comparison of ImpDis’s spectrum between samples with previously confirmed ImpDis without CNVs and newly identified diagnosis shows an increase in the detection of the Temple syndrome from 1 (0.9%) case to 5 (15.2%) cases using МS-MLPA MLID. Among group of patients with previously unconfirmed diagnoses (25 samples), not only have we successfully confirmed diagnoses with molecular genetics in 6 (24.0%) cases, but also, we have identified methylation abnormalities different from the expected clinical syndromes in 2 (8.0%) cases, and MLID in 4 cases (16.0%). In the group of patients newly referred for the diagnosis (56 samples), clinical diagnoses were confirmed by molecular genetics in 17 (30.4%) cases, different molecular disorders were detected in 3 (5.4%) cases, MLID were detected in 1 case (1.8%). Based on the results of the assessment of the phenotype of patients with MLID, it is shown, that the phenotypic signs of MLID may coincide with the signs of known ImpDis, partially overlap with the signs of some known ImpDis and can be non-specific, which makes clinical diagnosis difficult. During the whole-genome analysis of patient with TNDM-MLID, previously undescribed homozygous variant NM_001109809.4.8(ZFP57): c.115delG (p.Val39fs) has been revealed and assessed as likely pathogenic (PVS1+PM2) according to ACMG criteria. Both parents are heterozygous carriers, which determines the risk of rebirth of a child with TNDM-MLID at 25%. Based on the results of the research, the algorithm for the diagnosis of ImpDis has been proposed, with MS-MLPA MLID as first-line test.

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