Chromosomal localization of mutated genes in non-syndromic familial thyroid cancer

Familial non-medullary thyroid carcinoma (FNMTC) is a type of thyroid cancer characterized by genetic susceptibility, representing approximately 5% of all non-medullary thyroid carcinomas. While some cases of FNMTC are associated with familial multi-organ tumor predisposition syndromes, the majority occur independently. The genetic mechanisms underlying non-syndromic FNMTC remain unclear. Initial studies utilized SNP linkage analysis to identify susceptibility loci, including the 1q21 locus, 2q21 locus, and 4q32 locus, among others. Subsequent research employed more advanced techniques such as Genome-wide Association Study and Whole Exome Sequencing, leading to the discovery of genes such as IMMP2L, GALNTL4, WDR11-AS1, DUOX2, NOP53, MAP2K5, and others. But FNMTC exhibits strong genetic heterogeneity, with each family having its own pathogenic genes. This is the first article to provide a chromosomal landscape map of susceptibility genes associated with non-syndromic FNMTC and analyze their potential associations. It also presents a detailed summary of variant loci, characteristics, research methodologies, and validation results from different countries.


Introduction
Familial non-medullary thyroid carcinoma (FNMTC) refers to a form of thyroid cancer that occurs in families, where there are at least two cases of thyroid cancer of follicular epithelial cell origin in first-degree relatives, excluding individuals with a history of exposure to known thyroid-cancer-causing factors (1,2).It accounts for approximately 5% of all non-medullary thyroid carcinoma (1,2).Epidemiological studies have shown that first-degree relatives of FNMTC patients have a 4 to 10 times higher risk of developing the disease compared to the general population.If two individuals in a family are affected, the probability of it being hereditary is estimated at 31% to 38%.If three or more individuals are affected, the probability of it being hereditary is over 94% (3).
However, it is important to note that the identified susceptibility genes, such as MAP2K5 (15q23) (13)(14)(15), HABP2 (10q25.3)(16- 25), and the 19q13.2locus (TCO) (26-30), primarily exist within specific families and may not be universally applicable across different families.FNMTC exhibits strong genetic heterogeneity, with each family having its specific pathogenic genes.It is the combination of these genes that may constitute the true susceptibility gene pool for FNMTC.
Therefore, this article provides a comprehensive overview of the chromosomal landscape of susceptibility genes associated with nonsyndromic FNMTC (Figure 1).Additionally, it offers a detailed summary of research findings, including information on variant loci, variant characteristics, research methodologies employed, and validation results from various countries.

Susceptibility chromosomal loci associated with risk of nonsyndromic FNMTC
Initially, studies predominantly used SNP linkage analysis to identify susceptibility loci associated with FNMTC, but without specific genes.These loci are mostly related to specific types of NMTC.For instance, the discovery of the 1q21 locus is linked to papillary renal neoplasia (PRN) (31), the 2q21 locus is associated with follicular variant papillary thyroid carcinoma (fvPTC) (6), 4q32 locus was identified in 11 PTC patients and 2 families with undifferentiated thyroid cancer (7), 14q32 locus may be related to multinodular goiter (MNG) (32), and the 19p13.2microdeletion may be associated with thyroid cancer with oncocytic (TCO) features (26), and so on.However, except for specific subtypes, the aforementioned susceptibility loci have been rarely validated in other NMTC (7,26,30,30,33,34).For example, Suh (5) conducted a whole-genome SNP array analysis on 38 families.A significant correlation with chromosome locus 6q22 (the same chromosomal region as the PRN1 locus) and FNMTC was found, with an LOD score of 3.30.Similarly, Cavaco (33) performed SNP linkage analysis on 11 affected family members of Portuguese descent.The highest LOD score obtained was +4.41 at the FTEN locus on chromosome 8p23.1-p22.This suggests that the association between the chromosomal locus and FNMTC may not be universally applicable and could vary among different populations or families.
Significantly, the chromosomal locus at 19p13.2 has been implicated in numerous subsequent studies, exhibiting associations with various genes such as INSR (35), TIMM44 (36), NDUFA13/ GRIM-19 (37) and MYO1F (38).Several studies have reported eosinophilic alterations in cells associated with TCO features (27)(28)(29).These alterations include abnormal mitochondrial proliferation in pro-oxidant or cancer cells.It is also the locus with the highest number of associated genes for FNMTC identified to date.This provides valuable insights for the fine mapping of susceptibility genes related to specific types of thyroid cancer.Table 1 summarizes the chromosomal loci associated with FNMTC.

RNA-associated susceptibility genes of FNMTC
RNA-associated susceptibility genes refer to genes that play important roles in the synthesis, modification, and regulation of RNA, and are associated with disease susceptibility.Currently, several RNA and genes associated with FNMTC have been identified, as shown in Table 2.
Firstly, the researchers found that miR-886-3p was downregulated by 3-fold in FNMTC compared to SNMTC, while miR-20a was down-regulated by 4-fold in FNMTC compared to SNMTC.Overexpression of miR-886-3p was found to inhibit the expression of DNA replication gene CDC6 and focal adhesion genes PIP5K1C, PXN, and ZYX.This overexpression of miR-886-3p resulted in the inhibition of cell proliferation and invasion, keeping the cells in the S phase.On the other hand, miR-20a is predominantly found in undifferentiated thyroid carcinoma and was shown to down-regulate the expression of LIMK1, a gene associated with reduced cell invasion (39,40).
Papillary thyroid carcinoma susceptibility candidate (PTCSC) gene families belong to long non-coding RNA (lnc-RNA) that have been implicated in the development of papillary thyroid carcinoma (41).Currently, studies have shown that PTCSC1, PTCSC2, and PTCSC3 are associated with the occurrence of FNMTC.
PTCSC1, also known as AK023948, is a gene that has been found to interact with DHX9 and the PI3K regulatory subunit p85-b (PIK3R2).The activation of AK023948 through this interaction leads to the phosphorylation and activation of protein kinase AKT.However, it is important to note that this specific association between AK023948 and breast cancer cell growth has not been explained or explored in the context of other studies on FNMTC (46).
An interaction between rs965513, a susceptibility SNP for FNMTC, PTCSC2 has been observed.The SNP rs965513 in the 9q22 region play a crucial role in the genetic predisposition to PTC.And the [AA] risk genotype of rs965513 is associated with decreased expression of FOXE1, PTCSC2 unspliced transcript in unaffected thyroid tissue.The decrease in gene abundance is associated with dedifferentiation, which promotes malignant transformation (42,43).Furthermore, MYH9 has been found to  Overview of the chromosomal landscape of susceptibility genes associated with non-syndromic non-medullary thyroid carcinoma (NMTC).
bind to the lncRNA PTCSC2, regulating the p53 signaling pathway by inhibiting FOXE1 expression (45).PTCSC3, located downstream of SNP rs944289 at a distance of approximately 3.2 kb, exhibits characteristics of tumor suppressor genes (44).Rogounovitch et al. (47) confirmed the association of the T allele of SNP rs944289 with the risk of both PTC and benign thyroid nodules in the Japanese population.

Candidate genes identified by WES and susceptibility genes identified by GWAS
GWAS is a method used to detect genetic variations and polymorphisms across the whole genome in multiple individuals.GWAS aims to identify genetic variations that are most likely to influence a specific trait by examining the genotype-phenotype relationship.So far, GWAS studies from various populations have revealed several susceptibility genes for FNMTC research (8-10, 35, 48, 49) (Tables 3, 4).
Although GWAS studies have identified numerous risk SNPs, FNMTC cannot currently be explained by single-gene variants.Therefore, researchers have started using polygenic risk scores (PRS) to assess the risk of developing thyroid cancer.
For example, a study utilizing GWAS data from the United States, Iceland, and the United Kingdom proposed a 10-SNP PRS assessment model (rs12129938, rs11693806, rs6793295, rs73227498, rs2466076, rs1588635, rs7902587, rs368187, rs116909374, and rs2289261), which could be applied for personalized assessment of thyroid cancer susceptibility (54).A study analyzed the PRS of 12 thyroid cancer-associated SNPs (rs11693806, rs2466076, rs1588635, rs368187, rs116909374, rs12129938, rs6793295, rs73227498, rs7902587, rs2289261, and rs56062135) in childhood cancer survivors of European population.The findings showed that for each one standard deviation increase in PRS (1.57) (55).Furthermore, another study compared the high PRS tertile with the low PRS tertile and found that the PRS of six SNPs (rs6759952, rs13059137, rs7834206, rs72616195, rs1369535, rs11175834) increased the risk of thyroid cancer by 3.9 times (56).These studies suggest that utilizing PRS can better assess an individual's risk of developing thyroid cancer.However, it is important to note that PRS is still evolving, and further validation and refinement are necessary before widespread clinical implementation.(Table 3).
In addition to the aforementioned PRS method, Asta et al. ( 92) introduced the familial cancer variant prioritization pipeline (FCVPP), which is a comprehensive approach for analyzing germline genomes in Mendelian cancer pedigrees.This pipeline involves variant calling, quality control, frequency screening, segregation analysis, and bioinformatics assessment.By considering these steps and ensuring accurate pedigree information, researchers can enhance the likelihood of identifying novel genes associated with Mendelian types of cancer.Building upon this, Abhishek et al. (93) introduced FCVPPv2, an enhanced pipeline for prioritizing variants in pedigrees that incorporates multiple tools to assess variant deleteriousness and intolerance scores.The pipeline also encompasses the evaluation of noncoding regions using various datasets and tools.Application of the pipeline on a papillary thyroid cancer family revealed one variant in the CPXM1 gene (c.G1717A:p.G573R), suggesting its potential involvement in tumorigenesis (Table 3).However, further functional characterization is required to validate its role in cancer predisposition.Overall, FCVPPv2 provides a comprehensive approach for predicting high-risk cancer predisposing variants in familial cancer pedigrees.
WES is also widely used for the screening of candidate genes.However, most of these loci have not been mentioned or functionally validated in other studies, as indicated in Table 3.
For example, Yang et al. (52), identified 10 germline mutations were found in 8 genes, including APC (L292F/A2778S), BRAF (D22N), MSH6 (G355S/A36V), MSH2 (L719F), MEN1 (G508D), BRCA1 (SS955S), BRCA2 (G2508S), and GNAS insertions.The mutation rate of these eight genes was found to be 53.2% in the FNMTC group and 31.3% in the SNMTC group.However, it is important to note that the sample size might have contributed to the lack of statistical significance in distinguishing FNMTC from       Although this class only involves the analysis of candidate genes at the level of bioinformatics, it may also have certain significance for expanding the FNMTC gene library.In the future, more verification is needed to confirm the contribution of these genes.

Identified susceptibility genes associated with FNMTC
In the FNMTC susceptibility gene map, most of the genes have been validated through other pedigrees or functional studies, but there is controversy surrounding many genes.Some of them may be susceptibility genes for specific subtypes of NMTC, while others may only belong to specific certain populations.This once again highlights that the genetic susceptibility of FNMTC is not determined by a single gene.Therefore, the following text and Table 4 provide detailed information.

1p13.2 WDR77
WD repeat domain 77 (WDR77), also known as methyltransferase-like protein 50 and androgen receptorassociated protein p44, was studied by Zhao et al. through WES in two unrelated Chinese pedigrees with FNMTC.In one pedigree, a missense mutation (p.R198H) was found in exon 6 of WDR77, while in the other pedigree, a splice site mutation (c.619 + 1G>c) was identified at the 5' end of intron 6 (76).
WDR77 forms a complex with protein arginine methyltransferase 5 (PRMT5) to regulate the formation of histone H4 arginine 3 dimethylation (H4R3me2) in cells.The functional activity of WDR77 protein is dependent on its subcellular localization.For example, in early developing prostate epithelial cells, WDR77 is located in the cytoplasm and plays a crucial role in cell proliferation (94, 95).Functional studies have shown that mutations in WDR77 disrupt the formation of the PRMT5/WDR77 complex, resulting in reduced H4R3me2 levels within cells.Knockdown of the WDR77 gene leads to an increased proliferation rate in thyroid cancer cells (76).

q41 BROX
Pasquali et al. conducted WES analysis on five FNMTC pedigrees and identified two pedigrees with loss-of-function mutations in the BROX gene.In one pedigree, a frameshift deletion mutation (chr1:222892283 NM_001288579:c.119delG:p.Arg40fs) occurred in the BROX gene, while in another pedigree, a missense mutation (chr1:222886144 NM_144695:c.2898C>T)was observed (77).However, there is currently no direct evidence of a functional effect associated with the second variant.BROX encodes human Brox, which contains a Bro1 domainlike sequence involved in the endosomal sorting of cargo proteins and degradation processes in lysosomes, such as integrin and EGFR (96)(97)(98).The researchers hypothesized that a BROX mutation is sufficient to alter the degradation of EGFR, leading to the accumulation of EGFR and subsequently abnormal cell growth (77).

1q42.3 NID1
WES was performed on a Brazilian family with a history of PTC.Previously reported FNMTC susceptibility genes were examined, and novel candidate genes were identified using PhenoDB.No variants in known FNMTC susceptibility genes cowith the disease.However, a missense variant (c.1971T>G:p.Ile657Met) in the NID1 gene co-segregated with the disease and had a low allele frequency.In silico analysis suggested its deleteriousness, and NID1 expression was observed in PTC cells but not normal thyroid tissue.TCGA data showed higher NID1 expression associated with the risk of recurrence and lateral neck lymph node metastasis in PTCs.The NID1 variant in this study shows potential as a novel FNMTC predisposing gene (78).

2q35 DIRC3
DIRC3 (Disrupted in Renal Cancer 3) was already known to associate hereditary renal cancer and was believed to have tumor suppressor activity (99).In a GWAS study involving 561 Icelandic thyroid cancer cases and 40,013 controls, rs966423 variant of the DIRC3 was found to be associated with thyroid cancer risk and levels of thyroid-stimulating hormone for the first time (57).Multiple reports have shown the prognostic significance of the rs966423 variant and its pathogenic effects in cases of DTC (35,(57)(58)(59)(60). Subsequent replication studies identified additional variants, rs6759952 (8,35) and rs12990503 (35).However, Mankickova et al. (10) were unable to establish an association between rs966423 and thyroid cancer in European populations.More recently, GWAS analysis discovered novel variants, 11693806 (49) located near DIRC3, and rs16857609 (61), that are associated with European ancestry.63) conducted a study on thyroid cancer in Chinese populations.They found that TERT rs2736100 was significantly associated with thyroid cancer in the Chinese cohort.Other study in populations of European ancestry confirmed this association (rs2736100).Additionally, they identified rs10069690 as the variant with the strongest association in this region and suggested that these two variants likely represent the same underlying association signal, with rs10069690 providing a slight refinement in the European ancestry study groups compared to the Chinese population (49).

Ge et al. (
In an article that examined 75 FNMTC families, the promoter region of the TERT gene was sequenced.Somatic mutations in the TERT promoter, as well as RAS and BRAF mutations, were evaluated, along with the entire EIF1AX gene in 54 familial cases of thyroid tumors.The study found that FNMTC is associated with short telomeres, an increased copy number of the TERT gene, and elevated telomerase activity in both germline and somatic cells. Additionally, the study suggested that TERT promoter and EIF1AX mutations are infrequently involved in the development of FNMTC.However, TERT mutations were more frequently observed in late-onset FNMTC cases compared to BRAF mutations, indicating a potential correlation between TERT mutations and the late onset of FNMTC (62).

5q31.3 SPRY4
Marques et al. (79) performed WES on leukocyte DNA from six affected members of FNMTC family.Shared genetic variants were identified through bioinformatic analysis and validated using Sanger sequencing.After filtering, a specific variant (c.701C>T, p.Thr234Met) in the SPRY4 gene was identified as the most promising.In vitro experiments showed that this variant increased cell viability and colony formation, indicating enhanced proliferation and clonogenic capacity.Analysis suggested that the SPRY4 variant acted through the mitogen-activated protein kinase/ extracellular signal-regulated kinase pathway, with increased sensitivity to a MEK inhibitor in thyroid cancer cells.

7q31.33 POT1
POT1, also known as GLM9, CMM10, and HPOT1, is involved in telomere maintenance.Mutations in POT1 were first reported in familial melanoma, where some family members also developed thyroid cancer (80, 100).Capezzone et al. found that peripheral blood samples from familial PTC patients exhibited an imbalance in the telomere-telomerase complex, characterized by shortened telomeres and TERT gene amplification (101).Richard et al. used functional variant approaches to confirm the association between lowfrequency intronic regulatory POT1 variants in survivors of childhood cancer and subsequent development of malignant thyroid tumors.Intronic variations in POT1 may affect key proteins involved in telomere maintenance and genomic integrity (102).
Recently, Srivastava et al. ( 81) reported a pedigree with FNMTC, in which a novel mutation (p.Val29Leu) was identified in the POT1 gene among family members.In vitro experiments showed that the p.Val29Leu mutation in the POT1 gene exhibited increased telomere length, indicating loss of function caused by the mutation and resulting in telomere dysfunction.However, Orois et al. (82) did not find potential pathogenic mutations in the POT1 gene through WES in four FNMTC pedigrees from Spain.

8p12 NRG1
The NRG1 gene encodes neuregulin 1, a signaling protein involved in the development of both malignant and benign thyroid tumors.Additionally, it plays a role in protecting against oxidative damage, and influencing cell proliferation (57,59,103,104).

9q22 FOXE1/TTF-2
The FOXE1 gene, located on chromosome 9q22.33,encodes the FOXE1 transcription factor (also known as thyroid transcription factor 2, TTF-2).This gene is involved in regulating the expression of genes such as thyroglobulin and thyroid peroxidase, and it plays an essential role in the formation and development of the thyroid gland.
Regarding the association of FOXE1 with FNMTC, several studies have been conducted to investigate its potential role.In one study (64), the SNP rs1867277 variation at the 5'UTR was found to regulate FOXE1 transcription.Another study (65, 67) tested the FOXE1 gene in 60 Portuguese FNMTC families and 80 SNMTC cases and identified ten germline variants in the promoter and coding sequence of the gene.One variant, c.743C > G (p.A248G), showed gene-disease co-segregation in one FNMTC family and was detected in a disseminated PTC case.
In another study (66), a total of 672 patients from 133 FNMTC progeny were tested at 11 known candidate loci, including the chromosomal locus 9q22.33 near FOXE1.The SNPs rs965513 and rs10759944 at 9q22.33 showed the most consistent association with FNMTC.However, Bonora et al. (66) reported that among the previously mentioned rs944289 (68) and rs1867277 (64) SNPs, only rs1867277 was consistently associated with FNMTC.Furthermore, validation from 95 FNMTC cases carrying three risk alleles of related SNPs (rs965513, rs10759944, rs1867277) at the 9q22.33 locus did not find any deleterious missense mutations in the entire coding sequence of FOXE1.Therefore, the aforementioned FOXE1-associated SNPs were not consistently shown to be associated with FNMTC.
A study conducted on the Icelandic population and people of European ancestry (67) showed that two simultaneous polymorphic changes (rs944289 and rs965513) near the FOXE1 gene increased the risk of PTC and follicular thyroid carcinoma (FTC) development.Overall, it should be noted that not all literature supports a direct association between the FOXE1 gene and FNMTC.

10q25.3 HABP2
Gara et al. ( 16) performed exome sequencing on seven FNMTC patients from one family, in which the HABP2 G534E mutation was found to promote tumorigenesis, growth, invasion, and loss of tumor suppression.Weeks et al. (17) conducted Sanger sequencing on 37 Australian families with FNMTC to validate the presence of the HABP2 G534E mutation.Additionally, WES was performed on 59 participants from 20 families to validate the HABP2 mutation.However, no explainable HABP2 G534E mutation was detected in the Australian family.Furthermore, studies conducted in regions including Europe and the Middle East have also failed to replicate this data (17)(18)(19)(20)(21)(22)(23)(24).
In another study by Kern et al. (24), Sanger sequencing was performed on 20 patients with familial PTC from 11 European families.They identified c.1601G > A (p.Gly534Glu) mutations in two patients at the heterozygous level and c.364C > T (p.Arg122Trp) mutations in three patients.The minor allele frequencies (MAF) for these mutations were 5.0% and 7.5%, respectively.Therefore, these findings do not support the pathogenicity of the HABP2 c.1601G > A (p.Gly534Glu) variant.However, the study highlighted the presence of a novel mutation, c.364C > T (p.Arg122Trp), which was not found in another study (25) involving 32 Italian FNMTC families that did not identify the HABP2 R122W variant.
Finally, additional HABP2 variants (rs2286742 and rs3740530) were identified, which have the potential to increase the risk of PTC in a recessive model, respectively (71).
Therefore, considering potential ethnic variations across different regions, further investigation is warranted in a broader cohort of patients with familial PTC to thoroughly evaluate its pathogenicity.

11q22.3ATM 17q21.31 BRCA1 22q12.1 CHEK2
Ionizing radiation has been documented to directly increase the risk of thyroid and breast cancer by causing DNA double-strand breaks, thereby promoting carcinogenesis.The process of DNA damage repair is influenced by genetic polymorphisms and mutations in the ATM-BRCA1-CHEK2 pathway.In mammalian cells, DNA double-strand breaks activate the ATM kinase, which phosphorylates and activates CHEK2.This activation leads to the phosphorylation of BRCA1, initiating DNA repair.If the repair fails, apoptosis may occur (72).
Wojcicka et al. conducted a study and found that certain genetic variations, such as ATM D1853N, BRCA1 E1038G, and CHEK2 I157T, were associated with an increased susceptibility to PTC.They also discovered that specific single-nucleotide polymorphisms (SNPs), namely rs17879961 in CHEK2 and rs16941 in BRCA1, were significantly associated with PTC susceptibility.However, they did not find an association between ATM rs1801516 and PTC (72).
In another study, it was suggested that polymorphic changes in the ATM gene increased the risk of sporadic PTC and DTC in Caucasians (105, 106).Gu et al. (73) investigated the relationship between polymorphic changes in ATM and the increased risk of PTC in an Asian population.Among the four studied polymorphisms (rs664677, rs373759, rs4988099, rs189037), only rs373759 was found to be associated with an increased risk of PTC development.Strong linkage disequilibrium was observed among three ATM SNPs (rs373759, rs664143, and rs4585).The presence of the ATM haplotype (C-G-T) +/-was associated with a lower risk of PTC compared to the absence of this haplotype (C-G-T) -/-, suggesting the potential role of ATM genetic polymorphisms in thyroid cancer development in the Korean population (74).
Siołek et al. (90) demonstrated a significant correlation between carrying CHEK2 mutations (such as 1100delC, IVS21G > A, del5395, and I157T) and an increased risk of PTC.To further investigate this relationship, they genotyped 468 PTC patients and 468 age-and sex-matched cancer-free controls.Among the PTC cases, 73 out of 468 (15.6%) carried CHEK2 mutations, while only 28 out of 460 (6.0%) of the controls had these mutations.Zhao et al. (91) conducted a study involving whole genome sequencing of from two PTC patients in the same family.As a result, a new heterozygous germline mutation in CHEK2 (c.417C→A) was identified in all affected members of the family.
Functional analysis revealed that the CHEK2 c.417C > A variant introduces a premature termination codon (Y139X), resulting in the production of a truncated protein.This loss-of-function variant led to reduced p53 phosphorylation and decreased abundance of the p53 protein.In addition to the Y139X variant, two rare missense variants (R180C and H371Y) were also detected in the CHEK2 gene.

12q14 SRGAP1
An SNP-linked analysis of 38 families suggested that three variants (rs781626187, rs797044990 and rs114817817) in the SRGAP1 gene might serve as potential candidate genes for FNMTC (75).These variants could potentially lead to the loss of gene function, resulting in the inactivation of the CDC42 protein.This, in turn, can impact the regulation of intracellular signaling networks, influencing various signaling pathways and promoting tumorigenesis (107).Additionally, the identified variants were not found in population studies conducted in Ohio or Poland.However, a different SNP (rs2168411) of the SRGAP1 gene demonstrated an association with PTC in both the Ohio and Poland populations (75).

14q12 TINF2
The study involved genome sequencing analysis of a large family with cases of PTC and melanoma.Screening for coding variants in the shelterin genes was conducted in 24 families using WES.A frameshift mutation in the TINF2 gene (TINF2 p.Trp198fs) was identified, showing complete co-segregation with PTC and melanoma in the key family.This mutation was unique to this family.Reduced TINF2 expression and disrupted binding to TERF1 were observed in individuals with the mutation.These individuals also exhibited significantly longer telomeres compared to unaffected family members and healthy controls.In addition, rare missense and synonymous variants in TINF2 and ACD were found in some families (84, 85).
Ngan et al. (86) conducted targeted DNA sequencing of the SRGAP1 gene in 20 patients with PTC who had a history of MNG, as well as in 284 patients with PTC without a history of MNG.Among the latter group, four patients with PTC were found to have the NKX2-1/TTF-1 mutation pattern.
Functional experiments have indicated that mutations in this gene, such as the TTF-1 A339V mutation, can stimulate thyroid cell proliferation independently of thyroid-stimulating hormone.These mutations have also been shown to activate STAT3 and Akt signaling pathways and increase the expression of cyclin D2.These findings suggest that the TTF-1 A339V mutation may be associated with the development of PTC.However, these results have not been consistently confirmed in other experiments (108).Gudmundsson et al. (47,68) discovered that downstream of the NKX2-1 gene, there is a risk allele T for the SNP rs944289.Liao et al. (109) conducted genetic testing on a Chinese family and found that the T allele of rs944289 is a risk allele for members of this family to develop MNG and PTC.This association was also confirmed by other countries (10,35,58,69).Another genome-wide study conducted by Takahashi et al. (70) focused on Belarusians who were exposed to radiation from the Chernobyl incident at 18 years of age or younger.However, this study did not find an association between radiation-associated PTC and the SNP rs944289 located at the 14q13.3locus.Other relevant variants, such as rs34081947 and rs368187, were initially identified in a GWAS conducted on European populations (49).
In addition to rs944289, Gudmundsson also proposed that the variants (rs116909374 on 14q13.3)were significantly associated with thyroid cancer.On chromosome 14q13.3,the nearby gene MBIP and NKX2-1/TTF-1 are important candidates to consider for their roles in thyroid development (57).

15q21.1 DUOX2
Ohye et al. (11) utilized WES to identify a novel germline mutation in the DUOX2 gene associated with FNMTC.This mutation, known as DUOX2 Y1203H, affects the function of DUOX2, which is responsible for hydrogen peroxide (H2O2) production in the thyroid gland.H2O2 is essential for iodide tyrosine residue oxidation and the synthesis of thyroxine and triiodothyronine by thyroid peroxidase.
The study found that DUOX2 Y1203H is a functionally active mutation, and it was observed to be highly expressed in normal thyroid tissue, potentially increasing the risk of tumorigenesis.Notably, the expression of DUOX2 was found to be elevated in patients harboring the rs965513 variant, suggesting a potential link between dysregulated H2O2 metabolism mediated by DUOX2 and hereditary thyroid carcinoma with either high or low penetrance.However, a study on 33 Italian FNMTC pedigrees did not find these mutations (110).

15q23 MAP2K5
Ye et al. ( 13) conducted a study utilizing WES in 33 patients with FNMTC and further validated their results in an additional 44 intra-family patients.Through this analysis, they identified five candidate genes: MAP2K5, ZNF500, MUC6, IGSF3, and FRG1.
An interesting aspect to consider is that the pathogenesis of FNMTC diverges from the classical MAP2K1/2-ERK1/2 signaling pathway typically associated with sporadic cases.In FNMTC, there is a distinct activation of the MAP2K5-ERK5 pathway, which subsequently causes changes in gene expression downstream.These alterations ultimately drive the malignant transformation of thyroid epithelial cells.
Additionally, a separate study targeting MAP2K5 DNA assays in Italian families did not yield any positive results (14, 15).

16p13.3 SRRM2
This study (88) involved a sample of 6 FNMTC patients from a single family, which was then validated by including an additional 138 FNMTC families.The study also screened for the presence of the FNMTC susceptibility gene SRRM2 in 1170 sporadic PTC cases from Ohio, as well as 1404 healthy controls.
The data obtained from this study suggest that the S346F mutation in the SRRM2 gene may play a role in PTC development.This mutation affects the splicing process and leads to variable splicing of downstream target genes.A total of 7 SRRM2 mutations were identified in 7 PTC cases, while no mutations were found in the 1404 controls.However, it is worth noting that these mutations were not detected in the 138 FNMTC families analyzed.

19p13.2 GRIM-19/NDUFA13
There is a cluster of genes involved in mitochondrial metabolism, including GRIM-19.In a study of 52 thyroid tumors, researchers searched for GRIM-19 mutations.They found somatic missense mutations in three sporadic Hurthle cell carcinomas and a germline mutation in a Hurthle cell papillary carcinoma.No mutations were detected in non-Hurthle cell carcinomas or blood donor samples.One of the sporadic Hurthle cell papillary carcinomas with a GRIM-19 mutation also had a RET/PTC-1 rearrangement.Interestingly, no GRIM-19 mutations were identified in familial Hurthle cell tumors, suggesting that GRIM-19 may not be the TCO gene.The GRIM-19 mutations may contribute to the development of sporadic or familial Hurthle cell tumors by affecting GRIM-19's role in mitochondrial metabolism and cell death (37).

19p13.2 TIMM44
This particular study (36) focused on 8 families affected by oncocytic thyroid carcinomas.The researchers conducted a systematic screening of 14 candidate genes and observed their localization in regions associated with affected members of the families with oncocytic thyroid carcinomas.
Through this screening, the study identified two new variants in TIMM44.These variants were located in exon 9 and exon 13 of the gene, specifically at 19p13.2.TIMM44 is known as a mitochondrial endosomal transporter involved in the import of nuclear-encoded proteins into mitochondria.Interestingly, these TIMM44 variants were found to co-occur with the oncocytic thyroid carcinomas phenotype within the affected families.

19p13.2 MYO1F
A studies (38) revealed a novel heterozygous mutation in exon 5 of the MYO1F gene (c.400G> A, NM: 012335; p.Gly134Ser) that maps to the linkage site.In addition to this mutation, another variant was found in exon 7 among the 192 FNMTC families studied.
This variant leads to an increase in mitochondrial mass and significantly elevates reactive oxygen species levels.Furthermore, it confers a significant advantage in terms of colony formation, invasion, and anchorage-independent growth.Moreover, there is another variant in exon 7 that causes exon skipping, which is predicted to alter the ATP-binding domain in MYO1F.

19q13.33 NOP53
A study (12) conducted WES of one family and analyzed an additional 44 FNMTC families using Sanger sequencing.In this study, a shared germline variant in NOP53, known as Asp31His (rs78530808, with a minor allele frequency of 1.8%), was identified in three non-syndromic FNMTC families.
Functional studies of NOP53 in thyroid cancer cell lines have demonstrated its oncogenic function.However, considering the relatively high frequency of this variant in the general population, it suggests that NOP53 may not be a causative gene but rather a low penetrance gene associated with FNMTC.

Pathway and process enrichment analysis
We performed pathway and process enrichment analysis on the given gene list above using the following ontology source from Metascape (version v3.5.20230501, https://metascape.org/):KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical Pathways, CORUM, WikiPathways, and PANTHER Pathway.All genes in the genome have been used as the enrichment background.Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 are collected and grouped into clusters based on their membership similarities.The most statistically significant term within a cluster is chosen to represent the cluster.
According to Figure 2A, we have found that the currently reported genes are mainly involved in key processes related to carcinogenesis, such as negative regulation of DNA metabolic process, endocrine system development, cell activation, and regulation of cell-substrate adhesion in GO Biological Processes.Additionally, key pathways associated with thyroid cancer, such as DNA damage response (only ATM dependent) and EGF/EGFR signaling pathway, were identified in WikiPathways.
To enhance the representation of term relationships, we have created a network plot using a subset of enriched terms.In this network, terms with a similarity greater than 0.3 are connected by edges.Our selection process involved choosing terms with the best p-values from each of the 20 clusters.We ensured that each cluster had no more than 15 terms and that the total number of terms did not exceed 250.The resulting network is visualized using Cytoscape (111), where each node represents an enriched term.Nodes are initially colored based on their cluster ID and then further differentiated by their p-value (Figure 2B).

Protein-protein interaction enrichment analysis
For each given gene list, protein-protein interaction enrichment analysis has been carried out with the following databases: STRING6, BioGrid7, OmniPath8, InWeb_IM9.Only physical interactions in STRING (physical score > 0.132) and BioGrid are used.MCODE_1 shows the three best-scoring terms (GO:0051053, negative regulation of DNA metabolic process, Log10(P)= -11.6;GO:0032205, negative regulation of telomere maintenance, Log10

Discussion
Currently, no significant loci have been successfully identified in the research on FNMTC, indicating that the genetic causal model of FNMTC may be complex and mostly validated only in individual pedigrees, without genetic replication in other pedigrees.This may be related to various factors, with racial differences being one possible reason.Another important factor is the genetic heterogeneity of inherited diseases, where different gene variations can lead to the same disease, or in other words, a single factor does not determine a particular disease.
The strength of this article lies in its first-time summary of the chromosomal landscape map, which facilitates researchers to visually identify gene clustering loci.We also provide a summary of the localization and methodologies of susceptibility genes from different countries.The integration presented in this study may provide new insights for further research.
However, the limitation of this study is that FNMTC susceptibility genes are family-specific pathogenic genes, and each family may have its unique mechanisms of disease development.Currently, there is no precise localization of the causative genes for this condition.
It is indeed worth discussing the potential avenues for future research in the field of FNMTC, such as the development of new assessment methods like PRS and FCVPP.Researchers may uncover novel insights into the molecular mechanisms underlying FNMTC by investigating gene expression patterns, studying individual cells, and examining copy number variations in the future.These investigations could potentially lead to the identification of biomarkers for early detection, as well as the development of targeted therapeutic approaches.

Conclusion
This review provides an overview of the recent advancements in non-syndrome FNMTC susceptibility gene research, offering comprehensive theoretical support for subsequent investigations in this field.To explore the pathogenesis of FNMTC, more FNMTC pedigrees need to be included to expand and improve the FNMTC gene database.For non-syndromic FNMTC patients, early detection, diagnosis, and treatment may be an effective way to prevent disease progression.

2 (
FIGURE 2 (A) Bar graph of enriched terms across input gene lists, colored by p-values; (B) Network of enriched terms: colored by cluster ID, where nodes that share the same cluster ID are typically close to each other; colored by p-value, where terms containing more genes tend to have a more significant p-value.

TABLE 1
Susceptibility chromosomal loci associated with risk of non-syndromic FNMTC.

TABLE 3
Candidate genes identified by whole exome sequencing (WES) and susceptibility genes identified by genome-wide association studies (GWAS).

TABLE 4
Identified susceptibility genes associated with Familial non-medullary thyroid carcinoma.

TABLE 4 Continued
(50)bly, three of these genes (PIK3CB, CAV2, and KANK1) have been linked to tumorigenesis through the PI3K/Akt signaling pathway.Sarquis et al.(50)performed WES on three FNMTC pedigrees in Brazil and identified seven potential FNMTC susceptibility genes.The three FNMTC pedigrees had a total of 11 patients with an average age of diagnosis at 41.5 years.