The role of imprinting genes’ loss of imprints in cancers and their clinical implications

Genomic imprinting plays an important role in the growth and development of mammals. When the original imprint status of these genes is lost, known as loss of imprinting (LOI), it may affect growth, neurocognitive development, metabolism, and even tumor susceptibility. The LOI of imprint genes has gradually been found not only as an early event in tumorigenesis, but also to be involved in progression. More than 120 imprinted genes had been identified in humans. In this review, we summarized the most studied LOI of two gene clusters and 13 single genes in cancers. We focused on the roles they played, that is, as growth suppressors and anti-apoptosis agents, sustaining proliferative signaling or inducing angiogenesis; the molecular pathways they regulated; and especially their clinical significance. It is notable that 12 combined forms of multi-genes’ LOI, 3 of which have already been used as diagnostic models, achieved good sensitivity, specificity, and accuracy. In addition, the methods used for LOI detection in existing research are classified into detection of biallelic expression (BAE), differentially methylated regions (DMRs), methylation, and single-nucleotide polymorphisms (SNPs). These all indicated that the detection of imprinting genes’ LOI has potential clinical significance in cancer diagnosis, treatment, and prognosis.


Introduction
Genomic imprinting has significant roles in individual growth, development, and cell differentiation in mammals (1).In this epigenetic process, a small group of genes, called imprinted genes, are expressed depending on their parental origin.Imprinting is manifested mainly as silencing of transcription when a gene is expressed by one parent and activation of transcription when it is expressed by the other parent (2).When the original imprint status of imprinted genes is lost, known as loss of imprinting (LOI), silenced alleles are abnormally activated, or active genes are suppressed.Such imprint disorders can affect growth, neurocognitive development, metabolism, and even tumor susceptibility.
Insulin-like growth factor 2 (IGF2) is among the most studied genes affected by LOI in cancers.The LOI of IGF2 gene was firstly demonstrated in wilms' tumor (WT), a renal malignancy of childhood with an embryonic origin (3).LOI of the IGF2 has also been found in some adult somatic tumors including colorectal cancer (CRC), renal cell carcinoma (RCC), stomach adenocarcinoma (STAD), and esophageal squamous cell carcinoma (ESCC) (4-7).The LOI of imprinted genes has gradually emerged as an early event in tumorigenesis, as well as being implicated in the development of tumors (8).Some studies have reported aberrant gene imprinting status in specific cancer types, whereas others have focused on the impact of these changes on tumors.
LOI affects tumorigenesis and progression mainly through conferring resistance to apoptosis and evasion of growth suppressors, sustaining proliferative signaling, inducing angiogenesis, and activating metastasis (Figure 1).For example, IGF2 overexpression caused by LOI leads to the activation of the AKT and extracellular-regulated kinase (ERK) pathways, which promotes tumorigenesis (including cell proliferation and resistance to apoptosis) and metastasis (mainly liver metastases in CRC) (9,10).Moreover, higher serum IGF2 concentration is associated with metastasis in CRC, and is an indicator of poor prognosis (9).In triple-negative breast cancer (TNBC), the LOI of potassium two-pore domain channel subfamily K member 9 (KCNK9) gene involving differentially methylated region (DMR) hypomethylation leads to overexpression of the gene, increasing mitochondrial membrane potential and anti-apoptotic effect (11,12).In human hepatocellular carcinoma (HCC), hypomethylation at CpG85 has been reported to lead to an increase in levels of an alternative RB1-E2B transcript and concomitant downregulation of the RB1 main transcript in confirmed retinoblastoma (Rb) LOI, resulting in the absence of the Rb pathway and the loss of its suppressor function (13).Inhibition of transforming growth factor-b (TGF-b) signaling increases the probability of malignancy (14).Hypermethylation of the DIRAS family GTPase 3 (DIRAS3) CpG has been found to lead to LOI, resulting in a decrease in its expression, blunting the Ras or phosphatidyl-inositol-3 Kinase (PI3K) pathway (15,16).Defects in these feedback mechanisms could enhance proliferative signaling (17).The LOI of maternally expressed 3 (MEG3) inactivates its expression, thereby enhancing angiogenesis and promoting tumorigenesis (18,19).These findings suggest potential actionable targets for LOI genes in cancers.
Disruption of the imprinting status also has implications for cancer diagnosis and prognosis.Studies have established diagnostic models using multiple imprinted genes based on the differences in allelic expression between normal, benign tumor, and cancerous tissues and have shown that these can function as efficient epigenetic biomarkers (20)(21)(22).Moreover, the presence of LOI at the delta-like non-canonical notch ligand 1 (DLK1) and MEG3 locus has been found to vary between two different histological subtypes of rhabdomyosarcoma (RMS) (23).Therefore, LOI detection represents a novel tool for cancer diagnosis.For instance, patients with esophageal adenocarcinoma (EAC) <65 years old with IGF2 LOI were found to have longer 5-year disease-free survival (DFS) (24), whereas in patients with CRC, the LOI was associated with higher overall mortality (4).These results demonstrate the importance of understanding the role of LOI in cancers and also illustrate the complexity arising from cancer tissue specificity.
More than 120 imprinted genes have been identified in humans (as displayed at geneimprint, http://www.geneimprint.com).However, there has been a lack of studies summarizing which imprinted genes are associated with cancers.Therefore, in this review, we used a systematic literature search strategy (Supplementary Figure 1A) to identify a total of 297 studies (after elimination of duplicate records).The two authors cross-checked the remaining articles, resulting in a The molecular mechanism of LOI affecting cancers.total of 105 articles to be included in the review (the process is summarized in Supplementary Data 1, search strategy and selection criteria).These articles comprised results for 13 single genes (including two gene clusters) in 26 types of cancers and 12 combined forms of multi-gene LOI testing in 16 imprinted genes.Thus, our review provides a basis and prospective reference for the co-detection of imprinted genes and the selection of suitable biomarkers to establish novel clinical models in the future.Imprinted genes are regulated by imprinted cluster-associated DMRs that play a critical part in maintaining parent-specific gene expression patterns known as imprinted control regions (ICRs).LOI is due to aberrant methylation in the DMRs of imprinted genes, usually loss of methylation maintenance, which produces aberrant transcripts that lead to activation of normally silent alleles.Methods for the detection of LOI have been established based on these mechanisms, such as the detection of biallelic expression (BAE), detection of DMR methylation, and detection of single-nucleotide polymorphisms (SNPs).In addition, 17 LOI detection methods are also summarized and grouped into three categories according to their principles, which will be helpful for selecting the appropriate LOI detection method in cancers.

Imprinted genes' loss of imprint in cancers
LOI often occurs in many imprinted genes in malignancies, involving either a single imprinted gene LOI in one type of cancer or a specific cancer with multiple imprinted genes' LOI simultaneously.Single genes, especially oncogenes or protooncogenes, may undergo alternations in expression when LOI occurs, subsequently affecting their biological functions in specific cancer types.Importantly, some of these genes are regulated in clusters.The chromosomal locations and regions that regulate imprinting and expression offer the potential for new therapeutic targets to be developed.Table 1 provides a summary of LOI sites, expression levels, and clinical significance of 13 single imprinted genes in 26 types of cancers, as well as epigenetically mediated mechanisms of carcinogenesis, based on the literature.In addition, multi-gene testing has shown that most gene combinations are grouped in clusters or in similar positions; some of these combinations have already been used to establish tumor diagnostic models, showing impressive potential for direct clinical applications.These gene combinations could also provide an index for future diagnostic models.Table 2 lists 12 gene combinations identified by multi-gene LOI testing, of which 3 have been established as cancer diagnostic models.

IGF2-H19 locus
IGF2 and H19, located on chromosome 11p15.5 in humans, are a mutually imprinted pair of genes that share a common regulatory locus (Figure 2A) (117).The IGF2 gene consists of 10 exons, and its expression is driven by five promoters (p0-p4) that possess different transcriptional activities both pre-and postnatally.In some cancer cells, four promoters (p0, p2, p3, and p4) whose IGF2 mRNA transcripts are imprinted contribute significantly to total IGF2 expression (118)(119)(120).Human H19 expressing a long non-coding RNA (lncRNA) contains six exons and two promoters.H19 DMR, also known as imprinting control region 1 (ICR1), is located between IGF2 and H19 and contains the binding sites for the epigenetic master regulator CTCF (121,122).ICR1, IGF2 promoter-specific DMRs 0, 1, and 2, which partially overlap the IGF2 intronic and exonic sequences, the IGF2 enhancer region downstream from H19, and imprinting factor zinc finger protein 57 (ZFP57) jointly play a crucial part in maintaining normal imprinting and expression of these two genes in mammals (123)(124)(125).
In most healthy adults, IGF2, which encodes proteins that promote fetal growth, is expressed only by the paternal allele (maternal ICR1 hypomethylation), whereas H19, which encodes an lncRNA with growth inhibitory properties, is expressed only by the maternal allele (paternal ICR1 hypermethylation) (126).This balance of expression of different parental alleles is broken when LOI occurs, routinely exhibiting opposing methylation states and biological functions, especially in the majority of patients with tumors.IGF2 LOI associated with hypermethylation of ICR1 and hypomethylation of IGF2 DMRs is prevalent and increases gene expression levels in the majority of cancers (Figure 2B) (4,33).Moreover, ICR1 hypomethylation is also considered to be characteristic of H19 LOI and regularly results in the upregulation of H19 mRNA expression in human bladder cancer (Figure 2C) (29).
IGF2 undergoes normal imprinting changes, can act synergistically with multiple signaling pathways, and participates in physiological processes (autophagy, oncogenesis, and glycemic metabolism) of patients.It is well known that IGF2/IGF1R binding exerts cellular autophagy mediated by inhibiting the PI3K-Akt-mTOR signaling pathway in the CRC (127).Activated glycogen synthase kinase-3b (GSK3b) can inhibit B-cell lymphoma-2 (Bcl-2) as a mediating event to stimulate autophagy (128,129).A recent study demonstrated that IGF2 LOI cancer stem cells (CSCs) were generally more prone to tumor formation and had higher levels of autophagy (CD133 with high expression and p62 with low expression) compared with maintenance of imprinting (MOI) cells in patients with CRC (46).Low expression of miRNA-195 in patients with CRC increased IGF2/IR-A binding, which more strongly promoted Akt expression and phosphorylation than IGF2/IGF1R, further decreasing GSK3b phosphorylation (46,130).Overexpression of IGF2 related to LOI and receptor tyrosine kinase genes including DDR1, ERBB2, and FGFR1 have implicated the IGF2-INSR pathway in sphere formation of solitary fibrous tumor (SFT) (63).Hypoglycemia was also observed in SFT patients with IGF2 LOI.
Several studies have assessed the clinical value of imprinted genes in tumors.LOI and ICR/DMR methylation and alterations in expression levels due to LOI are relevant to clinical parameters, especially those related to survival and mortality.LOI of IGF2 was first identified in WT, which is a hereditary malignant embryonic tumor of infants (3), with a relatively older age at diagnosis of         IGF2 LOI has been reported to be associated with a fivefold increased risk of adenoma formation and higher overall mortality in CRC (4,47).IGF2 LOI appeared to predispose RCC patients to low-grade and low-stage tumors (5) and was more likely to occur in advanced STAD (6).Patients with ESCC with IGF2 LOI showed a higher degree of lymph node involvement, metastasis, and shorter survival times (7,61).However, patients with EAC with IGF2 LOI were found to have a longer 5-year DFS (24).These not only show the importance of paying attention to LOI in cancers but also illustrate the complexity arising from cancer tissue specificity.Finally, H19 LOI has been found to be present in patients with head and neck carcinoma, and patients with high expression of H19 appeared to be more likely to experience relapse (31).

Dlk1-MEG3 locus
The human DLK1 gene resides in the chromosomal 14q32 region, positioned with MEG3, with which it constructs an imprinted gene cluster (NCBI reference sequence: NC_000014.9).The paternally expressed protein-coding DLK1 gene is composed of 5 exons, whereas MEG3 with 13 exons maternally expresses an lncRNA.At the DLK1-MEG3 locus, it is regulated by both the ICR and MEG3 DMR containing the CTCF binding DNA sequence, which lies among the two genes (131,132).Aronson et al. revealed that a hierarchical and unidirectional regulation existed between the ICR and MEG3 DMR, and the dominant ICR was established as a dichotomous control element that maintained imprinting through allele-specific restriction of the DNA (de)methylation mechanism (Figure 3A) (133).
DLK1 and MEG3 are methylated on the paternal allele, but unmethylated on the maternal allele, which regulates their expression in healthy individuals (134).However, in some cancer patients, the parental alleles are expressed in an imbalanced manner and usually exhibit opposite methylation states and expression.DLK1 LOI (ICR and MEG3 DMR hypermethylation) manifests as biallelic DLK1 expression and MEG3 silencing, whereas MEG3 LOI shows ICR and MEG3 DMR hypomethylation and the opposite expression trend (Figures 3B, C).In addition to LOI, allelic switching (opposite single allele expression) accompanied by gains or losses of DNA methylation primarily on IG-DMR at the DLK1-MEG3 locus had also been discovered in some patients with HCC (18).MEG3 copy number loss was found only in patients with nasopharyngeal carcinoma (NPC) whose LOI manifested as DMR hypermethylation (74).These results indicate that genetics and epigenetics may synergistically influence the vast majority of tumors.
Similar to IGF2 and H19, DLK1 and MEG3 also perform diverse biological functions in cancers.LOI was found to upregulate DKL1 mRNA expression; however, knocking down its expression would inhibit proliferation and tumorigenicity in embryonal carcinoma (EC) (26).DLK1 appears to exert a cancer-promoting role.Conversely, in glioma (GBMLGG), lower expression of MEG3 promotes not only oncogenesis, but also malignant behavior such as proliferation, migration, and tumorigenicity (135).When restored to normal expression levels, MEG3 played a tumor suppressor role suppressed by inducing a significant downward adjustment of focal adhesion kinase (FAK), vimentin, and inhibitory phosphorylation of non-receptor tyrosine kinase (SRC).Furthermore, MEG3 restoration increased levels of b-actin (an important skeletal protein), caveolin-1 (a negative growth regulator), and connexin-43, as well as activating N-myc downstream-regulated gene 1 (NDRG1), which has previously been shown to inhibit metastasis and migration in CRC (136).MEG3 also increased expression of p53 and a potent cyclindependent kinase inhibitor called p21, which might explain the observed enhancement of G1/S cell cycle arrest, and stimulated E3 ubiquitin ligase MDM2 production, which could represent suppressed NPC metastasis through the p53-MDM2-Slug pathway (74).
With respect to clinical applications, LOI of imprinted genes combined may appear to be useful for differentiating tumor subtypes.It has also been shown that both embryonal and alveolar rhabdomyosarcomas (ERMS and ARMS, respectively) show LOI for the DMR of the IGF2-H19 locus, while ERMS consistently shows LOI of the DMR at the DLK1-MEG3 locus (23).

Single genes' loss of imprint 2.2.1 Rb, KCNK9, PEG3, and P73
Apart from the best-known genetic changes in the form of heredity, such as mutations, genomic instability, loss of heterozygosity (LOH) and copy number aberrations (CNAs) leading to the inactivation of oncogenes or proto-oncogenes, epigenetic change can also cause this phenomenon.In contrast to clustered genes, single-gene LOI exhibits BAE or dysregulation of aberrant transcripts.Alteration of an imprinting control center may lead to abnormal expression of oncogenes or tumor suppressor genes, causing different effects on promoting and suppressing cancer.
On the one hand, LOI genes that promote cancer comprise Rb, KCNK9, and paternally expressed gene 3 (PEG3).The Rb gene, a retinoblastoma susceptibility gene, was the first tumor suppressor gene to be cloned and have its full sequence determined.Anwar et al. identified that LOI (CpG85 hypomethylation) is also a novel pathway for the inactivation of Rb in HCC (13).The Rb gene expresses only the maternal gene, while the paternal gene expresses the abnormal transcript (RB1-E2B) that starts at the CpG85 island.In the absence of imprinting, levels of RB1-E2B will increase, eventually leading to decreased expression of the main transcript RB. Patients with CpG85 hypermethylation have shorter overall survival (the median survival rates for hypermethylation and normal/hypomethylation are 34 and 156 weeks, respectively).KCNK9 LOI was found due to DMR hypomethylation, which leads to overexpression of its gene product, increasing mitochondrial membrane potential and anti-apoptosis in TNBC (11,12).Hypermethylation of the PEG3 promoter leads to LOI and decreased PEG3 mRNA expression, increasing b-catenin levels, promoting proliferation, and inhibiting p53-dependent apoptosis in human GBMLGG (84).On the other hand, the LOI gene that inhibits cancer is P73.The increased expression of P73, including that resulting from LOI, could be a partial compensatory mechanism for defective p53 in ESCC (77).

Diagnostic models GNAS, GRB10, SNRPN, and HM13
Traditional cytology and histopathology, imaging examination, and use of serum biomarkers have contributed tremendously to the early detection of cancer, but accurate diagnostic assessment of nodules and early-stage cancers with insufficient evidence of tumor morphology or abnormal metabolism remains a great clinical challenge at present (137)(138)(139)(140).However, epigenetics may compensate for this deficiency.There is already clear evidence that epigenetic changes during carcinogenesis often precede morphological changes (141,142).To provide reference for more accurate tumor-specific diagnosis and precise personalized treatment in clinical settings, we summarize 12 combined forms of multi-gene LOI testing in Table 2, of which 3 types of combinations have been established as cancer diagnostic models.Some researchers have successfully exploited a novel method, quantitative chromogenic imprinted gene in situ hybridization (QCIGISH), targeting non-coding intron nascent RNA, to directly observe BAE, multiallelic expression (MAE), and total expression (TE) at transcription sites of imprinted genes in the nucleus to select these appropriate imprinted genes for the construction, optimization, and validation of tumor diagnostic models (20).First, a diagnostic model for 10 different solid cancer types (bladder, breast, colorectal, esophageal, gastric, lung, pancreatic, prostate, skin, and thyroid cancers) was built using imprinted genes' GNAS complex locus (GNAS), growth factor receptor bound protein 10 (GRB10), and small nuclear ribonucleoprotein polypeptide N (SNRPN) with a total sensitivity of 94%, a specificity of 92%, and an accuracy of 93% (20).Next, based on the above preliminary model, a more specific diagnostic model for grading lung cancer (LC) was also established using GNAS, GRB10, SNRPN, and histocompatibility minor 13 (HM13).This diagnostic model was highly effective in the diagnosis of both different subtypes of LC and small lung nodules, with an overall sensitivity of 99.1%, a specificity of 92.1%, and an area under the curve (AUC) of 0.99 (21).Lastly, a thyroid cancer (TC) diagnostic model through imprinted genes SNRPN and HM13 has achieved an overall diagnostic sensitivity of 100%, a specificity of 91.5%, a positive predictive value (PPV) of 96.5%, a negative predictive value (NPV) of 100%, and a diagnostic accuracy of 97.5% in a prospective validation (22).
In sum, these findings provide considerable benefits and ideas for screening or predicting appropriate tumor markers, comprehensive clinical risk assessment, and finding new epigenetic therapeutic targets.This fully reflects the importance and non-negligibility of tumor epigenetics.

Detecting methods of LOI genes in cancers
Various methods have been used in the detection of imprinted gene LOI in the past three decades.In the 105 studies listed in Table 3, restriction fragment length polymorphism PCR (RFLP-PCR) was the most frequently used method, used up to 84 times (75/105) from 1993 to 2020.This was followed by bisulfite sequencing PCR (BSP) (25/105, 2003-2021) and pyrosequencing (8/105, 2007-2014).LOI arises from abnormal methylation of the DMR of imprinted genes (usually loss of methylation maintenance), which produces double alleles (aberrant transcripts leading to silencing of a normally active allele).LOI can also be discriminated based on SNPs.According to the detection objects used, the 17 methods for this purpose can be categorized into three types: (I) detection of BAE: hot-stop PCR, nest PCR, QCIGISH, RFLP-PCR, real-time quantitative reverse transcription PCR, reverse transcription PCR, and pyrosequencing; (II) detection of DMR methylation: BSP, bisulfite PCR-Luminex, combined bisulfite restriction analysis, Illumina 450 K arrays, pyrosequencing, methylation-specific PCR, NOMe-sequencing, RFLP-PCR, and the MassARRAY EpiTYPER; and (III) detection of SNPs: SNuPE assays, RNA sequencing (RNA-seq), and DNA sequencing.To make the results more credible and convincing, there is a growing trend towards the simultaneous use of multiple analytical methods with the same or different principles and away from the use of single or single-principle methods in some studies.
Sequencing techniques based on sulfite treatment are widely used; however, despite their convenience, their drawbacks are also increasingly obvious.Sulfite treatment may lead to severe degradation of the input DNA owing to harsh reaction conditions, which is a common problem with most sequencing methods.Chemical enzymes compensate for this defect (143).For instance, a combination of chemical enzymes such as APOBEC3A (A3A) or engineered APOBEC3C (eA3C) and sequencing technologies has achieved consistent and reliable results (144,145).This highlights the potential of multidisciplinary combinations to lead to new approaches.
Notably, several high-throughput techniques are being used for genomic methylation and allele-specific expression (ASE), showing great promise for the analysis and detection of imprinted gene LOI in cancers.The demand for comprehensive descriptions of DNA methylation patterns has led to a diversity of DNA methylation profiling technologies, including reduced representation bisulfite sequencing (RRBS) based on bisulfite conversion, methylated DNA binding domain sequencing, methylated DNA immunoprecipitation sequencing (MeDIP-seq) based on affinity enrichment, and methylation-sensitive restriction enzyme sequencing (MRE-seq) based on endonuclease digestion that targets genomic distribution (146).Recent studies have shown that utilizing the complementary properties of MeDIP-seq and MRE-seq can provide a rapid comparative analysis of the entire methylome at a fraction of the cost of whole-genome bisulfite sequencing (WGBS) (the gold standard method for detecting methylation at single-base resolution) with higher accuracy and reproducibility than either individual method (147-149).Analysis of existing RNA-seq datasets can be used to identify ASE of imprinted genes beyond evaluation of gene expression, thereby detecting the LOI of imprinted genes (150).However, when heterogeneous populations of cells, such as cancer samples, are analyzed, only single-cell measurements allowed the detection of widespread LOI events (151).Therefore, the use of effective and appropriate data analysis methods to analyze single-cell transcriptomic data will provide a major advantage in the analysis of tumor epigenetic aberrations.For example, BrewerIX, a standardized approach for the analysis of known imprinted genes, can be used to analyze RNA-seq data from single breast cancer cells to identify LOI of imprinted genes (151).Differential allelic expression using single-cell data (DAESC), a powerful method for differential ASE analysis using single-cell RNA sequencing (scRNA-seq) from multiple individuals, is capable of analyzing genes with differential ASE in pancreatic endocrine cells from patients with type 2 diabetes and controls, taking into account the effect of allelic switching, although it is not suitable for estimating cancer cells (152).These In conclusion, the presence or absence of LOI in cancers can be determined by using multiple methods of the same type vertically, two or more different types of methods horizontally, or even methods that combine multiple disciplines, making the results more accurate and reliable.Furthermore, the establishment of standardized data analysis methods for high-throughput technologies, in addition to combining multiple approaches, will help to uncover more potential imprinted genes and LOI, thereby facilitating the discovery of context-specific regulatory effects in cancers.As sequencing costs decrease, these methods will also be appealing in clinical practice.

Discussion
The established association between LOI and microsatellite instability (MSI) seems to provide a new epigenetic view of cancer susceptibility (40,91), although this is complex, given the expression of imprinted genes in a parent-of-origin-specific manner.For the imprinted gene Rb, allele mutations from different parents have different effects on tumor susceptibility in hereditary retinoblastoma: if the mutation is of paternal origin, the offspring has a 12% chance of developing retinoblastoma, whereas when the mutation is of maternal origin, the offspring have a 75% chance of developing retinoblastoma (16, 153).Beyond embryonicderived blastomas, epigenetic alterations in imprinted genes, often presenting as LOI, have been found in various somatic cancers.In addition, LOI of imprinted genes has been increasingly implicated in malignant behavior.The detection of LOI thus has potential clinical significance in cancer diagnosis, treatment, and prognosis.
Here, we have summarized 13 single-gene LOI in cancers, identifying the relevant detection sites and cancer types and considering whether they promote or inhibit functions in cancers.This provides a convenient index for co-detection of imprinted gene LOI in specific types of cancer.Moreover, as recent studies have found that aberrant gene imprinting patterns can occur together with cancer-associated CNAs (154) or allelic switching (77), we have also included these types of change in our analysis of studies (Table 1).Although the role of aberrant imprinting patterns in tumors is unquestionable, few studies have considered CNAs (1/70) or allelic switching (2/70) when reporting methylation profiles.Therefore, we suggest increasing the investigation of CNAs or allelic switching in future research to improve the accuracy of functional research on LOI genes.Coupled genes may be either clustered, as in the IGF2-H19 locus or DLK1-MEG3 locus, or non-clustered in specific cancers.In the analysis of loci for multi-gene detection panels, 12 combined forms of multi-genes were included, of which 3 gene combinations have been established as cancer diagnostic models.It is possible that more patterns may be found in the future based on the characteristics of imprinted genes in clustered LOI.There is also evidence to suggest that both the imprinting state and expression can be uncoupled in clustered genes.For instance, IGF2 LOI was not found to be coupled with downregulation of H19 expression in HCC (98,99); in RMS, although H19 LOI was present, the imprinting state of IGF2 was maintained (110).These cases not only illustrate the complexity arising from cancer tissue specificity but also indicate an independent control mechanism for imprinting.
Notably, the role of imprinting gene LOI may vary among different tumors.For instance, the protein encoded by p73 is structurally and functionally similar to that encoded by p53, a tumor suppressor.In p53-defect ESCC, p73 was found to have elevated expression and LOI, which is speculated to be a substitute mechanism for the tumorsuppressing function (79).However, in RCC, LOI or switching of allelic expression of p73 is associated with cancer development (77).On the other hand, even if a gene undergoes LOI, its downstream pathways may differ in different tumor types.In CRC, LOI of IGF2 can enhance cell autophagy through the PI3K/Akt/mTOR pathway, whereas it might promote tumor formation through the IGF2-INSR pathway in SFT (46,63).These findings suggest that it will be necessary for the future design of targeted LOI therapies to consider mutations of key factors in downstream pathways in different tumor types.
In the detection of imprinted genes' LOI in cancers, although DNA methylation status changes are characteristic of LOI, their detection is distinct from that of overall DNA or promoter region methylation.Therefore, the focus should be on DMR/ICR only.In HCC, global loss of methylation and increased methylation at DLK1 and MEG3 DMR/ICR-specific sites have been simultaneously observed (18).Detection methods for LOI have evolved from qualitative to quantitative, from detecting overall CpG islands to single CpG site, and to more simplified procedures (Supplementary Figure 1B).Although we have summarized the mature LOI methods currently used in tumor detection based on the literature, when considering the depth of sequencing, sample requirements, and mutation detection, high-throughput methods such as wholegenome sequencing, whole-exon sequencing, and single-cell sequencing have great application prospects for LOI detection of imprinted genes in cancers (32,155).
Both blood samples and tissue samples are suitable for the detection of LOI.IGF2 LOI has been found in the blood and tissues of both patients with CRC and healthy controls and may be a valuable predictive marker of an individual's risk of carcinoma (39,40,47).Although blood samples are more clinically accessible, tissue samples were more commonly used in the studies reviewed here (63/70 for single-gene detection, 34/35 for multiple-gene detection).This may be because in adult cancer patients, only the imprinted genes in cancer cells are LOI, while those in somatic cells maintain their imprint.With the development of enrichment methods for circulating tumor cells, use of tumor-derived exosomes in liquid biopsies, and advances in circulating cell-free DNA (cfDNA) methylation detection methods, blood samples have greater application prospects (156-158).Blood tests may therefore be of great informative value for large-scale LOI testing in cancersusceptible populations.
(2022NSFSC1584), and the Foundation of Xinglin Scholar Research Program Project of Chengdu University of TCM (QJRC2022048).

3
FIGURE 3 Schematic comparison of normal and loss of imprinting for human DLK1-MEG3 gene cluster.(A) Dark green boxes: DLK1 exons, light green boxes: DLK1 introns, dark blue rectangles: ICR (CpG Island CGI and TRE work independently on different alleles to restrict the activities of TETs and DNMTs), yellow quads in CGI: conserved tandem repeat array, black circle: methylated, white circle: unmethylated, orange trapezoid: demethylated enzyme TETs, blue cloud: methylated enzyme DNMTs, black green rectangle: transcription element ZFP57, dark yellow boxes: MEG3 exon, light yellow box: MEG3 introns.(B) Green solid arrows: parent-specific transcripts of DLK1, red letter x: absence.(C) Yellow solid arrows: parent-specific transcripts of MEG3.

TABLE 1
Single-gene LOI in cancers.

TABLE 2
Multiple-gene LOI in cancers.

TABLE 3
Detection methods of LOI in cancers.