# Analysis of genetic predisposition to familial non-medullary thyroid cancer by whole genome sequencing

Christine Do Cao, Rachel Desailloud, Hélène Topolinski, Michel Crépin, Cathy Flament, Emmanuelle Leteurtre, Catherine Cardot-Bauters, Fredéric Frénois, Émilie Ait-Yahya, Fabrice Bonte, et al.

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## To cite this version:

Christine Do Cao, Rachel Desailloud, Hélène Topolinski, Michel Crépin, Cathy Flament, et al. Analysis of genetic predisposition to familial non-medullary thyroid cancer by whole genome sequencing. Annales d'Endocrinologie = Annals of Endocrinology, 2025, 86 (5), pp.102456. 10.1016/j.ando.2025.102456. hal-05257025

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Original article

# Analysis of genetic predisposition to familial non-medullary thyroid cancer by whole genome sequencing

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Christine Do Cao<sup>a</sup>, Rachel Desailloud<sup>b</sup>, Hélène Topolinski<sup>c</sup>, Michel Crépin<sup>d</sup>, Cathy Flament<sup>d</sup>, Emmanuelle Leteurtre<sup>e</sup>, Catherine Cardot-Bauters<sup>a</sup>, Fredéric Frénois<sup>f</sup>, Émilie Ait-Yahya<sup>g</sup>, Fabrice Bonte<sup>g</sup>, Julie Leclerc<sup>d,e</sup>, Pascal Pigny<sup>d,e,*</sup>

ARTICLE INFO

Keywords:

MST1R

Papillary thyroid carcinoma

Genetics

Familial forms

WGS

ABSTRACT

**Background.** – Several putative susceptibility genes for familial papillary thyroid carcinoma, or familial non-medullary thyroid carcinoma (FNMTC), have been identified. However, in most families the molecular basis for FNMTC remains elusive.

**Methods.** – Two families with ≥ 3 first-degree relatives in ≥ 2 generations and ≥ 2 unaffected relatives were selected. Germline DNA from 2 patients and 2 unaffected individuals per family were analyzed by whole genome sequencing. All individuals were genotyped for putative pathogenic variants by Sanger sequencing, and minor allele frequency (MAF) was determined in 179 PTC-free European controls and the GnomAD database. Methylation analysis of candidate genes was carried out by EPIC BeadChips or pyrosequencing on germline and tumoral DNA.

**Results.** – Heterozygous variants in cancer-related genes expressed in thyroid tissue and predicted to impact protein function were identified in patients from both families. In F8, the best candidate was the *P2RX5* p.Leu32Gln variant previously reported in another PTC family. However, its relatively high MAF in European controls (0.43%) casts doubt on its pathogenic role. In F17, the most relevant variant was c.345del in *MST1R* encoding the tyrosine kinase receptor RON. The variant is predicted to encode a truncated isoform. Further analysis of tumor and germline DNA showed no clear evidence of an expression switch from the long to the short oncogenic RON isoform.

**Conclusion.** – Our data provide novel insight on the genetics of FNMTC. The *P2RX5* p.Leu32Gln variant should be considered either as low-penetrant or not associated with PTC predisposition. *MST1R* appears as a new putative susceptibility gene for FNMTC that warrants further consideration.

© 2025 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC

BY license (http://creativecommons.org/licenses/by/4.0/).

# 1. Introduction

Papillary thyroid cancer (PTC) is the most frequent thyroid cancer, accounting for more than 85% of all cases [1]. PTC is a malignant epithelial tumor with follicular differentiation. Several histological subtypes have been described, the most frequent being the classi-

cal variant, the follicular variant and the tall-cell variant. Familial forms of PTC, referred to as familial non-medullary thyroid carcinoma (or FNMTC), are clinically defined by 2 or more first-degree relatives with PTC in the absence of external factors known to promote thyroid cancer (for example, ionizing radiation). FNMTC is rare, at 1–10% of all PTCs [2]. It is generally classified in 2 groups. In syndromic FNMTC, PTC is a minor component of a hereditary cancer syndrome such as Cowden syndrome or familial adenomatous polyposis. In this case FNMTC has a Mendelian monogenic mode of transmission. In isolated FNMTC, the genetic basis is complex and heterogeneous. There is evidence of autosomal dominant inheritance of very rare, highly penetrant pathogenic variants in

<footnote><sup>a</sup> Service d'endocrinologie et métabolisme, CHU de Lille, 59037 Lille, France</footnote>

<footnote><sup>b</sup> Service d'endocrinologie et métabolisme, CHU d'Amiens, 80054 Amiens, France</footnote>

<footnote><sup>c</sup> Service d'endocrinologie diabétologie, CH de Béthune, 62408 Béthune, France</footnote>

<footnote><sup>d</sup> Service de biochimie, "Hormonologie, Métabolisme, Nutrition et Oncologie", CHU de Lille, 59037 Lille, France</footnote>

<footnote><sup>e</sup> CNRS, Inserm, UMR9020-U1277 - Cancer Heterogeneity Plasticity and Resistance to Therapies (CANTHER), CHU de Lille, University Lille, 59000 Lille, France</footnote>

<footnote><sup>f</sup> Faculté de médecine, ULR 7364 équipe RADEME, CHU de Lille, 59037 Lille, France</footnote>

<footnote><sup>g</sup> Équipe bio-informatique, CHU de Lille, 59037 Lille, France</footnote>

<footnote>* Corresponding author: Laboratoire de biochimie hormonologie, métabolisme, nutrition, oncologie, centre de biologie pathologie, CHU de Lille, 59037 Lille cedex, France.</footnote>

<footnote>E-mail address: pascal.pigny@chu-lille.fr (P. Pigny).</footnote>
susceptibility genes impacting different pathways. To date, more
than 20 candidate genes [3] have been described, and in most cases,
pathogenic variants are specific to 1 [4–6] or at most a few families
[7]. In the absence of functional data to validate their implication
in thyroid follicular carcinogenesis, this number may be overes-
timated, and these pathogenic variants might account for only a
small fraction of PTC heritability [8]. There are also data suggesting
a polygenic mode of inheritance [9] in which individual genetic risk
of PTC comprises the additive effect of several rare low-penetrant
nucleotide variants [8,10]. As recently reported [10], up to 11% of
PTC heritability in patients of European descent can be explained
by a combination of 5 single-nucleotide polymorphisms (SNPs).
Finally, there is still a high frequency of "gene-negative" FNMTC
families, which may result from the genetic heterogeneity men-
tioned above, but also from weak clinical definition: 2 cases is not
enough to define a familial form [2,11].

The present study applied whole genome sequencing (WGS) to identify the genetic basis of FNMTC in 2 families predicted to be the best candidates, having ≥ 3 first-degree relatives with PTC in ≥ 2 generations plus ≥ 1 relative with early age at onset or ≥ 1 affected male [2,12,13]. The presence of unaffected healthy relatives who could be used as a control group in the analysis was also a prerequisite.

**2. Materials and methods**

2.1. *Subject selection*

Out of 29 unrelated families with putative FNMTC (≥ 2 first-degree relatives with PTC) followed up in our center. 2 were selected for WGS analysis because they had ≥ 3 cases in ≥ 2 generations plus ≥ 1 case with age at onset < 30 years or ≥ 1 affected male. The F8 family (followed in the university hospital of Lille) had 3 patients with PTC and 5 unaffected relatives whereas the F17 family (followed in the university hospital of Amiens) had 4 patients with PTC and 1 unaffected individual (Table 1 and Fig. 1). Another relative in F17 had a thyroid vesicular adenoma. All patients and relatives provided written informed consent prior to genetic analysis, in accordance with French regulations. To determine variant allele frequency in a control population free of PTC, we used germline DNA from 179 patients of European origin (112 female and 67 male; mean age, 54 years) who had genetic testing for medullary thyroid carcinoma with absence of PTC on pathological examination [14].

## 2.2. DNA extraction and analysis

Blood samples were collected from patients and relatives. Genomic DNA was extracted from leukocytes using the chemicagen DNA Blood 4k Kit H24, as previously described [15]. Whole genome sequencing (WGS) was performed on selected patients (2 per family) and unaffected controls (see Fig. 1 for details) on an Illumina Nova Seq platform (Genewiz, Germany) with 150 bp paired-end reads. Sanger sequencing was performed on a Applied Biosystems 3730 XL genetic analyzer (ThermoFischer) as previously described [15].

After deparaffinization (deparaffinization solution, Qiagen), DNA was extracted from formalin-fixed paraffin-embedded (FFPE) thyroid tumor or non-tumoral samples using the QIAamp DNA FFPE Tissue kit (Qiagen). Loss of heterozygosity was screened for by comparing tumoral and germline DNA sequencing electrophoregrams.

For alternative internal *MST1R* promoter analysis, three primer
pairs were designed to amplify a genomic region encompass-
ing exon 9 to exon 11 (see Sup. data for details). PCR fragments
amplified from germline and PTC DNA were analyzed by Sanger
sequencing [15].

2.3. Bioinformatics analysis

Sequence reads were aligned to the human reference genome GRCh38/hg38 using the Dragen genome pipeline (Edico Genome Inc). Variant calling (SNPs and indels) and genotyping were performed with the Genome Analysis Toolkit (GATK), following the general guidelines for whole exome sequencing. Local realignment around indels was performed on alignments lacking duplicates, using the following GATK tools: RealignerTargetCreator, IndelRealigner and FixMateInformation. Base quality score recalibration (GATK BaseRecalibrator) was then applied using the known Sus scrofa 10.2 variants (VCF) from Ensembl release 72 as the known sites. Variant calling targeted exonic regions + 25 base pairs flanking each exon. Calling was performed using GATK UnifiedGenotyper with downsampling to 10x coverage threshold, at which at least 90% of the target bases are covered for most samples. The results were filtered using GATK Variant Filtration, filtering out variants which were covered by fewer than 5 reads [DP < 5], had low quality score [Q < 10], with low variant confidence over unfiltered depth of non-reference samples (QD) [QD < 1.5], which mapped to non-exome regions or to predicted mRNA (XM* according to the NCBI classification), or with MAF ≥ 0.5% in the gnomAD exome variants resource. Then, intersection of variants present in the 2 affected family members and absent in selected control subjects was carried out. Combined annotation dependent depletion (CADD) scores > 10 were used to select variants belonging to the top 10% of probable deleterious variants in the human genome [16]. Finally, a list was generated for variants mapped to the genes expressed in the thyroid (source: GTEx portal) and predicted to have a high or moderate impact on protein function. Only variants in genes associated with cancer after thorough literature analysis were further considered. All variants from the final list were validated by Sanger sequencing.

4.4. DNA methylation analysis

In total, 1000 ng of germline DNA was treated with sodium bisul-
fitte using the EZ DNA Methylation kit (Zymo Research), according to
the manufacturer's instructions. Elution was performed with 12 µL
M-Elution Buffer. DNA methylation status of the two CpG islands
from *MST1R* promoter was analyzed using the Illumina Infinium
Methylation EPIC 850K BeadChips (See Sup. Data for details). The
methylation score for each CpG was represented as a β value
according to the fluorescent intensity ratio [0: non-methylated to
1: completely methylated]. Raw intensity data (.idat files) were
assessed for quality using BeadArray Controls Reporter (Illumina).
Probe location and gene annotation used Illumina reference files
(GRCh37/hg19). β values were computed with Genome Studio soft-
ware (Illumina). 200 ng of tissue-extracted DNA was treated with
sodium bisulfite using the EZ DNA Methylation Gold kit (Zymo
Research), according to the manufacturer's instructions. Elution
was performed with 10 µL M-Elution Buffer.

PCR amplification was performed in a final volume of 25 µL
containing 0.2 µM of each primer, 1 X Multiplex PCR Master Mix
(Qiagen) and 1 µl of bisulfite-treated DNA. Following a denatura-
tion step at 95 °C for 15 minutes, 45 cycles of denaturation at 95 °C
for 20 seconds, annealing at 60 °C for 20 seconds and extension at
72 °C for 20 seconds were carried out, followed by a final exten-
sion at 72 °C for 5 minutes. Eight µl of the PCR product underwent
pyrosequencing on a PyroMark Q24 system (Qiagen), according
to the manufacturer's instructions (see Sup. Data for details). The
ratio of cytosines to thymines for targeted CpG sites, which corre-
sponds to the ratio of methylated to unmethylated cytosines, was
determined on PyroMark Q24 software (Qiagen).
<table><caption>**Table 1**
Clinical and pathological features of members of F8 and F17.</caption><thead><tr><th>Member ID</th><th>Age at PTC onset</th><th>Status</th><th>Gender</th><th>Histology</th><th>Year of surgery</th><th>Tumor Stage (TNM UICC classification)</th><th>Tumor size (mm)</th><th>Slide re-review</th><th>Age at clinical evaluation (relatives)</th><th>Somatic analysis</th><th>Ultrasonography (US) Thyroid data (relatives)</th></tr></thead><tbody><tr><td colspan="12"><strong>F8 family</strong></td></tr><tr><td>I.1</td><td>47</td><td>Affected</td><td>F</td><td>Micro-PTC with adipose tissue invasion</td><td>2009</td><td>pT1</td><td>4</td><td>No</td><td></td><td>NA</td><td></td></tr><tr><td>I.2</td><td>50</td><td>Affected</td><td>F</td><td>PTC vesicular variant with 30% tall cells + PTC classic variant</td><td>2013</td><td>pT1b(m)N0 (UICC 7th Ed 2009)</td><td>9 + 14</td><td>No</td><td></td><td>BRAF p.V600E</td><td></td></tr><tr><td>I.3</td><td></td><td>Unaffected</td><td>M</td><td></td><td></td><td></td><td></td><td></td><td>61</td><td></td><td>EU-TIRADS2 nodules</td></tr><tr><td>I.4</td><td></td><td>Unaffected</td><td>F</td><td></td><td></td><td></td><td></td><td></td><td>40</td><td></td><td>Total thyroidectomy for benign goiter</td></tr><tr><td>II.1</td><td></td><td>Unaffected</td><td>F</td><td></td><td></td><td></td><td></td><td></td><td>26</td><td></td><td>2 micronodules (&lt; 5 mm)</td></tr><tr><td>II.2</td><td>21</td><td>Proband</td><td>F</td><td>PTC classic variant</td><td>2007</td><td>pT2N1</td><td>NA</td><td>No</td><td></td><td>ETV6: NTRK3 Transcript fusion</td><td></td></tr><tr><td>II.3</td><td></td><td>Unaffected</td><td>F</td><td></td><td></td><td></td><td></td><td></td><td>30</td><td></td><td>Normal US</td></tr><tr><td>II.4</td><td></td><td>Unaffected</td><td>F</td><td></td><td></td><td></td><td></td><td></td><td>30</td><td></td><td>Normal US</td></tr><tr><td colspan="12"><strong>F17 family</strong></td></tr><tr><td>I.1</td><td>50</td><td>Affected</td><td>F</td><td>Multifocal PTC (4 lesions)</td><td>2007</td><td>pT2bN0M0</td><td>30 + 2 + 6 + 7</td><td>No</td><td></td><td>NA</td><td></td></tr><tr><td>I.4</td><td></td><td>?</td><td>F</td><td></td><td>2020</td><td></td><td></td><td>Yes</td><td>63</td><td></td><td>Vesicular adenoma (6.5 cm)</td></tr><tr><td>II.1</td><td>39</td><td>Affected</td><td>M</td><td>PTC classic variant</td><td>2017</td><td>pT3</td><td>45</td><td>No</td><td></td><td>NA</td><td></td></tr><tr><td>II.2</td><td>42</td><td>Affected</td><td>F</td><td>PTC classic variant + PTC vesicular variant</td><td>2018</td><td>pT1b(m)N0M0</td><td>14 + 5</td><td>No</td><td></td><td>BRAF WT NRAS p.Q61R</td><td></td></tr><tr><td>II.3</td><td></td><td>Unaffected</td><td>F</td><td></td><td></td><td></td><td></td><td></td><td>44</td><td></td><td>EU-TIRADS3 nodule (16 mm)</td></tr><tr><td>III.1</td><td>18</td><td>Proband</td><td>M</td><td>PTC (vesicular and trabecular variant) with aggressive features on histology: vascular invasion, peri-thyroid lymph-node metastasis</td><td>2017</td><td>pT3N1b (UICC 7th Ed 2009)</td><td>NA</td><td>No</td><td></td><td>BRAF WT PPP1R21: ALK transcript fusion</td><td></td></tr></tbody></table>

NA: not available.
<table><caption>**Table 2**
Exome analysis of F8 family.</caption><thead><tr><th>CADD-phred</th><th>Gene</th><th>HGVSc</th><th>HGVSp</th><th>Consequence</th><th>IMPACT</th><th>Thyroid expression<sup>a</sup></th><th>Cancer-related gene<sup>b</sup></th><th>Varsome</th><th>PolyPhen</th><th>SIFT</th><th>gnomad. exome_All. Freq.</th><th>gnomad. exome_non_cancer_AF</th></tr></thead><tbody><tr><td>37</td><td>SLC9B1</td><td>NM_001100874.2: c.1318A>T</td><td>NP_001094344.1: p.Lys440Ter</td><td>Stop gained</td><td>HIGH</td><td>Low</td><td>2</td><td>VUS</td><td></td><td></td><td>4.08047E-05</td><td>4.33715E-05</td></tr><tr><td>25.8</td><td>P2RX5</td><td>NM_002561.3: c.95T>A</td><td>NP_002552.2: p.Leu32Gln</td><td>Missense</td><td>MODERATE</td><td>Low</td><td>13</td><td></td><td>Probably damaging (0.998)</td><td>Deleterious (0)</td><td>0.00426938</td><td>0.00423815</td></tr><tr><td>25.4</td><td>SH3BP5L</td><td>NM_030645.1: c.595C>T</td><td>NP_085148.1: p.Arg199Trp</td><td>Missense</td><td>MODERATE</td><td>High</td><td>0</td><td></td><td>Possibly damaging (0.883)</td><td>Deleterious (0.01)</td><td>0.000515389</td><td>0.000499577</td></tr><tr><td>19</td><td>ZBTB24</td><td>NM_014797.2: c.301G>A</td><td>NP_055612.2: p.Ala101Thr</td><td>Missense</td><td>MODERATE</td><td>Medium</td><td>9</td><td></td><td>Benign (0.009)</td><td>Tolerated (0.18)</td><td>0.000174959</td><td>0.000181473</td></tr></tbody></table>

<footnote><sup>a</sup> Classification is based on the rank of the tpm value for the gene under consideration (provided by the GTEx portal [Broad Institute]), in relation to all the tpm values of the genes expressed in the thyroid. High: tpm value ≥ 75th percentile; Medium: tpm value ≥ median; Low: tpm value < median.</footnote>

<footnote><sup>b</sup> Number of articles retrieved after searching PubMed with the keywords "gene and cancer".</footnote>
### Family F8

<figure><img src="image_5.png" /></figure>

### Family F17

<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Pedigree status of the 2 families with respect to thyroid cancer, germline DNA availability, WGS analysis and *MST1R* genotype (F17). Circles and squares represent female and male members, respectively. The proband is indicated by a black arrow. Deceased individuals are indicated by diagonal lines. *: germline DNA available; #:</figcaption></figure>

germline DNA analyzed by WGS; +/- and -/-: *MST1R* c.345del status

Papillary thyroid carcinoma (PTC);

Vesicular / Vesicular adenoma

## 3. Results

We sequenced 8 germline DNA samples with an average 27X depth and achieved 80% coverage at 20X depth. In F8 (Fig. 1), after bioinformatic analysis, a total of 19 variants common to affected patients II.2 and I.2 and absent in unaffected relatives II.3 and II.4, were identified by WGS. The 4 nucleotide variants with the highest CADD score are shown in Table 2. Only 3 were in genes previously related to cancer and expressed in the thyroid tissue: *SLC9B1*, *P2RX5* and *ZBTB24*. However, using in-silico predictors, the *ZBTB24* p.Ala101Thr variant was classified as likely benign, whereas the *SLC9B1* p.Lys440* variant was classified as a variant of unknown significance (VUS). Thus, we decided to focus on the *P2RX5* variant, since it was previously found in patients with FNMTC [17]. Interestingly, protein modeling showed that replacement of leucine 32, which is located at the start of the transmembrane domain 1 of *P2RX5*, by a glutamine can destabilize the protein structure by introducing a negative charge (Sup. Data 1). Sanger sequencing was carried out in all members of F8 and showed that the nucleotide variant co-segregated with PTC. The p.Leu32Gln variant was not found in the proband in 20 of our families with putative FNMTC, suggesting that it was specific to F8. The MAF of this variant is

0.43% in control exomes in the Gnom AD data base (Table 1). We also detected it in 1 out of 179 controls free of PTC, corresponding to a frequency of 0.55%.

For WGS analysis in F17 (Fig. 1), controls comprised the unaffected relative II.3 plus the 2 unaffected individuals of F8 in the variant intersection. The unaffected status of subject I.4 in F17 was questionable due to a vesicular thyroid adenoma (see Table 1). At the end of the analysis pipeline, a total of 51 variants predicted to have a high or moderate impact on protein function were identified in patients I.1 and III.1 and absent in controls. All high-impact variants and the 10 moderate-impact variants with the highest CADD are shown in Table 3. These 18 variants were in genes expressed in the thyroid. Four variants classified by bioinformatic tools as likely benign or concerning genes unrelated to cancer were not further considered. In the other 14 genes, on careful literature analysis, only *MST1R*, which encodes the tyrosine kinase (TK) receptor RON, was previously associated with thyroid cancer [18,19].

The *MST1R* c.345del variant is predicted to lead to synthesis of a truncated RON receptor (p.Gly116Valfs*33) (Fig. 2A). The 4 F17 patients with PTC were heterozygous for this nucleotide variant on Sanger sequencing. It was absent in the unaffected individual II.3 but present at a heterozygous level in I.4, who had a vesic-
<table><caption>**Table 3**
Exome analysis of F17 family.</caption><thead><tr><th>CADD-phred</th><th>Gene</th><th>HGVSc</th><th>HGVSp</th><th>Consequence</th><th>IMPACT</th><th>Thyroid expression<sup>a</sup></th><th>Cancer-related gene<sup>b</sup></th><th>Varsome</th><th>PolyPhen</th><th>SIFT</th><th>gnomad. exome_All. Freq.</th><th>gnomad. exome_non_cancer_AF</th></tr></thead><tbody><tr><td>36</td><td>ZNF226</td><td>NM_001032372.2: c.100C&gt;T</td><td>p.Arg34Ter</td><td>Stop gained</td><td>HIGH</td><td>High</td><td>1</td><td>VUS</td><td></td><td></td><td>8.35262E-05</td><td>8.02094E-05</td></tr><tr><td>31</td><td>RBM19</td><td>NM_016196.4: c.921+2T&gt;C</td><td></td><td>Splice donor</td><td>HIGH</td><td>High</td><td>5</td><td>VUS</td><td></td><td></td><td>3.97772E-06</td><td>4.22187E-06</td></tr><tr><td>13.92</td><td>DIS3</td><td>NM_014953.5: c.2875T&gt;C</td><td>p.Ter959Gln ext Ter14</td><td>Stop lost</td><td>HIGH</td><td>Medium</td><td>102</td><td>Likely benign</td><td></td><td></td><td>0.00138254</td><td>0.00133571</td></tr><tr><td rowspan="3">10</td><td>ZNF264</td><td>NM_003417.4: c.299del</td><td>p.Glu100 GlyfsTer14</td><td>Frameshift</td><td>HIGH</td><td>Medium</td><td>5</td><td>Not classified</td><td></td><td></td><td>3.9841E-06</td><td>4.22826E-06</td></tr><tr><td rowspan="2">STARD9</td><td>NM_020759.3: c.3899_3900 insACCCCATT</td><td>p.Glu1304 ProfsTer10</td><td>Stop gained and Frameshift</td><td>HIGH</td><td>Medium</td><td>11</td><td>Likely benign</td><td></td><td></td><td>0.00128917</td><td>0.00126808</td></tr><tr><td>NM_002447.4: c.345del</td><td>p.Gly116 ValfsTer33</td><td>Frameshift</td><td>HIGH</td><td>Medium</td><td>101</td><td>Not classified</td><td></td><td></td><td>0.000274404</td><td>0.000239462</td></tr><tr><td>35</td><td>GOLGA8N</td><td>NM_001282494.2: c.1168C&gt;T</td><td>p.Gln390Ter</td><td>Stop gained</td><td>HIGH</td><td>Medium</td><td>0</td><td>Not classified</td><td></td><td></td><td>nf</td><td>nf</td></tr><tr><td>10</td><td>NPIPB11</td><td>NM_001310137.5: c.1641del</td><td>p.Pro548 ArgfsTer49</td><td>Frameshift</td><td>HIGH</td><td>Medium</td><td>0</td><td>Not classified</td><td></td><td></td><td>nf</td><td>nf</td></tr><tr><td>24.2</td><td>AIMP1</td><td>NM_004757.3: c.819G&gt;T</td><td>NP_004748.2: p.Gln273His</td><td>Substitution</td><td>MODERATE</td><td>High</td><td>30</td><td>Possibly damaging</td><td>Deleterious</td><td></td><td>7.97906E-06</td><td>4.23488E-06</td></tr><tr><td>15.65</td><td>ZNF234</td><td>NM_006630.2: c.1790C&gt;T</td><td>NP_006621.1: p.Thr597Ile</td><td>Substitution</td><td>MODERATE</td><td>Medium</td><td>1</td><td>Possibly damaging</td><td>Deleterious</td><td></td><td>0.000834738</td><td>0.000839545</td></tr><tr><td>25.9</td><td>NIPAL2</td><td>NM_024759.1: c.646G&gt;A</td><td>NP_079035.1: p.Ala216Thr</td><td>Substitution</td><td>MODERATE</td><td>High</td><td>2</td><td>Possibly damaging</td><td>Deleterious</td><td></td><td>0.00112886</td><td>0.00108791</td></tr><tr><td>29.1</td><td>TRPM1</td><td>NM_002420.5: c.470C&gt;T</td><td>NP_002411.3: p.Ser157Phe</td><td>Substitution</td><td>MODERATE</td><td>Low</td><td>75</td><td>Probably damaging</td><td>Deleterious</td><td></td><td>0.0032295</td><td>0.00310149</td></tr><tr><td>25.1</td><td>NYAP2</td><td>NM_020864.1: c.61C&gt;G</td><td>NP_065915.1: p.Gln21Glu</td><td>Substitution</td><td>MODERATE</td><td>Low</td><td>1</td><td>Probably damaging</td><td>Deleterious</td><td></td><td>0.000366091</td><td>0.000333123</td></tr><tr><td>27.8</td><td>PLEC</td><td>NM_000445.4: c.5714G&gt;A</td><td>NP_000436.2: p.Arg1905Gln</td><td>Substitution</td><td>MODERATE</td><td>High</td><td>136</td><td>Probably damaging</td><td>Deleterious</td><td></td><td>0.000356633</td><td>0.00036234</td></tr><tr><td>26.3</td><td>PHLDB1</td><td>NM_015157.3: c.3332C&gt;T</td><td>NP_055972.1: p.Thr1111Met</td><td>Substitution</td><td>MODERATE</td><td>High</td><td>33</td><td>Probably damaging</td><td>Deleterious</td><td></td><td>3.98479E-06</td><td>4.22919E-06</td></tr><tr><td>28.3</td><td>PIBF1</td><td>NM_006346.2: c.1793T&gt;G</td><td>NP_006337.2: p.Leu598Arg</td><td>Substitution</td><td>MODERATE</td><td>High</td><td>32</td><td>Probably damaging</td><td>Deleterious</td><td></td><td>0.00137314</td><td>0.00132979</td></tr><tr><td>27.9</td><td>IFT27</td><td>NM_006860.4: c.500C&gt;T</td><td>NP_006851.1: p.Ala167Val</td><td>Substitution</td><td>MODERATE</td><td>High</td><td>4</td><td>Probably damaging</td><td>Deleterious</td><td></td><td>0.000552693</td><td>0.000519079</td></tr><tr><td>27.9</td><td>MGAM2</td><td>NM_001293626.1: c.1855T&gt;C</td><td>NP_001280555.1: p.Cys619Arg</td><td>Substitution</td><td>MODERATE</td><td>Low</td><td>9</td><td>Probably damaging</td><td>Deleterious</td><td></td><td>0.000331296</td><td>0.000320508</td></tr></tbody></table>

nf: not found.

<footnote><sup>a</sup> Classification is based on the rank of the tpm value for the gene under consideration (provided by the GTEx portal [Broad Institute]), in relation to all the tpm values of the genes expressed in the thyroid. High: tpm value ≥ 75th percentile; Medium: tpm value ≥ median; Low: tpm value < median.</footnote>

<footnote><sup>b</sup> Number of articles retrieved after searching PubMed with the keywords "gene and cancer".</footnote>
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** A. Schematic representation of full-length RON isoform (FL-RON) showing the different protein domains: Sema, ligand-binding domain; TIG, Ig-like-plexin-transcription factors domain; Pkinase-Tyr: Tyrosine kinase domain. Position of the proteolytic cleavage site and of the deletion identified in F17 patients are shown. (adapted from cBioPortal). B. Frequency of the somatic mutations in 7 putative predisposition genes identified in the F17 in PTC samples from the Cancer Genome Atlas Program (TCGA, n = 492) plus the poorly differentiated cancer samples from MSK (n = 121) according to cBioPortal [Cerami E, et al. *Cancer Discov* 2012;2:960 and Gao J, et al. *Sci Signal* 2013;6:pl1].</figcaption></figure>

ular thyroid adenoma (Fig. 1). The *MST1R* variant was absent in the 179 controls free of PTC, in agreement with a reported MAF of 0.051% in the non-Finnish European population. The *MST1R* variant was also not found in the 20 probands in our series of unrelated PTC kin. Sanger sequencing of thyroid tumor DNA in subjects III.1 and II.2 revealed the alternative variant, without loss of the wild type allele. Absence of LOH was also observed in the vesicular adenoma of I.4 (Sup. Data 2).

Since a previous study showed that the methylation status of *MST1R* promoter affects the expression of the full-length RON isoform (FL-RON) in tumor cells [20], methylation levels at the 2 CpG islands (CGI1 and CGI2, as defined in the UCSC database) located in *MST1R* exon 1 (Sup. Data 3) were determined both in leukocyte DNA and in thyroid tumoral DNA in F17 patients. We also examined methylation status in papillary thyroid adenocarcinomas in the TCGA-THCA collection; only data obtained in tumors from white patients younger than 30 years (n = 47) were used, in order to be comparable with our data.

At germline level, using EPIC methylation BeadChips, CGI2 (CpG located at 49,940,126 and 49,940,353 [hg19/GRCh37] was shown to be hypermethylated both in controls (patients with PTC but without the *MST1R* c.345del) and in 3 F17 patients (Sup. Data 3). In contrast, 4 CpGs in CGI1 were not methylated, in both patients and controls.

Interestingly, analysis of methylation data for papillary thyroid adenocarcinomas in the TCGA-THCA collection (n = 47) found that the 2 CpGs in CGI2 were constantly hypermethylated whereas 1 CpG in CGI1, at chr3: 49,941,378, showed variable methylation, ranging from 5.8 to 33.4%. The methylation status of this CpG was therefore analyzed by pyrosequencing in DNA from PTC samples

of patients II.2 and III.1, the vesicular adenoma of I.4 and a control PTC from a patient without the c.345del germline variant. There was no hypermethylation of the 2 CpGs in CGI1, located at chr3: 49,941,376 and 49,941,378, in any PTC sample from F17 members, on comparison with corresponding germline DNAs or with control PTC (Sup. Data 3). Thus, there was no evidence of decreased FL-RON expression in tumor tissue in the F17 family. Besides FL-RON, a shorter oncogenic isoform called SF-RON has been described [21]. SF-RON retains part of the extracellular domain but all the trans-membrane and TK domain of the receptor and is constitutively activated. Expression depends on an alternative promoter located between *MST1R* intron 8 and exon 11. We screened for abnormality in this region (chr3: 49,934,066 to 49,933,470) by Sanger sequencing of 3 overlapping fragments: 1 distal, covering exon 9 to the beginning of exon 10, and 2 proximal: one long, covering intron 9 to the beginning of exon 11, and a shorter one ending in intron 10 (see Sup. Data). The distal region was successfully amplified from both germline and thyroid tissue DNA of F17 patients I.4, II.2 and III.1 as well as from PTC of F8 patient I.2 acting as control (*MST1R* wild type). Sanger sequencing showed normal nucleotide sequences. The long 415 bp proximal region was successfully amplified and displayed a normal sequence using any germline DNA or DNA from the thyroid vesicular adenoma of F17 patient I.4. In contrast, no PCR fragments were obtained using any somatic PTC DNA. Thus a new reverse primer, located in intron 10 (Sup. Data), was designed and allowed PCR amplification of a shorter 324 bp proximal region in the 3 PTC DNAs. No interpretable nucleotide sequences were obtained with the DNA extracted from the PTC of F17 III.1, whereas Sanger sequencing showed a normal nucleotide sequence in the PTC of F17 II.2 and the F8 I.2 control.
## 4. Discussion

In France, the incidence of PTC increased sharply over the period 1990-2018, and is now around 5.0/100,000/year in males and 17.5/100,000/year in females [22]. Based on these data and assuming a mean disease duration of 26.5 years, prevalence in France is around 0.13% in men and 0.46% in women. This estimate is in agreement with a nationwide analysis in Taiwan which reported 0.16% prevalence of non-medullary thyroid carcinoma (NMTC) [23]. If we assume that the prevalence of PTC in the general population is 0.46%, that 10% of cases are familial [2], and that these are linked to one highly penetrant pathogenic variant in a predisposition gene, then the maximum frequency of this pathogenic variant in the general population should be around 0.046%. Given that there is genetic heterogeneity in PTC, with over 20 predisposition genes reported, the actual frequency of each causal pathogenic variant in the general population is even lower. This is an important consideration when interpreting WGS results.

In the present study, we performed WGS with a calling process restricted to exons in 2 families with the highest likelihood of genetic background according to Charkes' rule [2]; both had ≥ 3 affected members in ≥ 2 generations, which reduces the risk of selecting a sporadic case to less than 6% [2]. Moreover, in both families the probands were characterized by early age at onset (≤ 25 years), which is another trend seen in familial cancer [24]. In spite of data suggesting that familial forms of PTC are more aggressive [25], all PTC cases in the present study, including multifocal cases, had good prognosis with limited lymph-node extension and with cure after conventional treatment by surgical and radioactive iodine ablation. Despite using these selection criteria, WGS plus exome analysis did not identify any particular strong susceptibility gene for PTC in the F8 family. We found the *P2RX5* p.Leu32Gln variant, previously identified by Sarquis et al. in a Brazilian family with 3 patients with PTC [17]. However, its allele frequency is 0.426% in all populations in the GnomAD exome database, and 0.596% in the non-Finnish European population, which is higher than the maximum frequency expected for a highly penetrant variant. Moreover, we identified the p.Leu32Gln variant in 1 control subject who underwent thyroidectomy at 60 years of age and who was free of any PTC, including micro-PTC, on pathological examination. We thus consider the *P2RX5* p.Leu32Gln variant as either a pathogenic variant with low penetrance or as not involved in the pathogenesis of FNMTC. These "negative" results lead us to question the status of the F8 family: should we consider it a 2-case family, since patient I.1 had micro-PTC? The usual definition of FNMTC is at least 2 cases of PTC in first-degree relatives, regardless of size [26]. Therefore, even though one of the patients had micro-PTC, the family can still be considered as having 3 cases. To go further in the genetic analysis, reanalyzing the WGS data to identify variants in the non-coding regions could be useful.

In the F17 family with 4 cases of PTC in 3 generations, the best candidate is the *MST1R* c.345del nucleotide variant identified as heterozygous in all 4 cases and absent in the 3 unaffected controls. *MST1R* encodes a membrane TK receptor, also called RON, for macrophage stimulating factor (MSF). In its mature full-length form (FL-RON), RON is a disulfide-linked α-β heterodimer resulting from cleavage of a 1400 amino-acid precursor at the Lys 305-Arg309 motif (Fig. 2). The α chain is short and extracellular, whereas the β chain is long and transmembrane. The 2 chains are linked by a disulfide bridge between Cys 300 and Cys 367. The SEMA domain (Arg37-Ile522) that forms the ligand-binding domain is shared by the 2 chains (Fig. 2). Alternatively, a short RON isoform, SF-RON, with a constitutively activated TK domain can be expressed under the control of an internal promoter located around *MST1R* intron 10 [27]. Expression of SF-RON was found in a large number of cell lines deriving from breast, pancreatic, lung and colon carcinomas [27].

Moreover, SF-RON expression was associated with faster growth in lung and breast cancer cells [21,27] and promoted invasiveness of breast cancer cells by interacting with PI-3K [28]. Regarding thyroid tissue, RON protein expression is barely detectable by immuno-histochemistry in normal epithelial cells. But Wang et al. [18] reported moderate expression in the majority of thyroid adenomas and frequent over-expression in papillary or follicular carcinomas. Moreover, FL-RON activation by MSF or its over-expression induced Akt, p38 MAPK and Erk1/2 phosphorylation in thyroid cancer cells in vitro [19,29]. These signaling pathways play key roles in thyroid epithelial tumorigenesis [30]. In contrast, *MST1R* is rarely mutated at the somatic level in thyroid cancer: only 1 out of 492 PTCs (0.2%) in the TCGA database (Fig. 2).

Interestingly, *MST1R* was suggested to be a susceptibility gene in nasopharyngeal carcinoma (NPC) in the Asian population [20]. The authors identified 11 germline variants in NPC patients with early onset or familial history of the disease: 10 missense variants distributed along the entire length of the receptor and 1 frame shift variant in exon 1: c.834_835del, p.Arg279Profs*5. This variant, classified as pathogenic by the authors, was identified in a 55-year-old male patient with familial history of NPC. The variant is predicted to lead to the synthesis of a truncated RON isoform devoid of the end of the α chain and of the totality of the β chain. The *MST1R* c.345 del variant, identified here in all F17 patients, is also located on *MST1R* exon 1 and leads to synthesis of a truncated protein lacking part of the α chain and the entirety of the β chain. In the present case, the α chain was even shorter since it ended after Thr147. Thus, the c.345del variant could have the same carcinogenesis mechanisms as the NPC c.834_835 del variant.

One hypothesis is that this variant is associated with a switch from long to short pro-oncogenic RON isoform expression. Such a switch was previously reported at transcript level in NPC tumor tissue [20]. However, we did not find loss of the *MST1R* wild type allele in thyroid tumor tissue; and there was no hypermethylation of 2 CpG sites in the CGI1 island in any of the 3 thyroid tissues examined (2 PTCs and 1 vesicular adenoma). There was thus no evidence for decreased expression of the FL-RON isoform in PTC tissues in F17 patients. In parallel, there was no alteration of the *MST1R* genomic region surrounding intron 10, which is likely to activate the internal promoter controlling SF-RON expression [27], in the germline DNA of 3 F17 patients with *MST1R* mutation, 2 with PTC and 1 with vesicular thyroid adenoma. However, in tissue, part of the proximal region covering end-intron 10 to mid-exon 11 (chr3: 49,933,560 to 49,933,470) was not PCR-amplified and not analyzable for the 3 PTCs, unlike for the vesicular adenoma. Whether this was linked to low quality DNA extracted from the FFPE-PTCs or to a PTC-specific somatic rearrangement involving this region of *MST1R* was undetermined. Unfortunately, no RNA samples were available from the PTC tissues to check SF-RON transcript expression by RNASeq or Q-PCR.

## 5. Conclusion

Implementation of WGS plus exome analysis in 2 families with a high probability of familial forms of PTC did not lead to identification of new, clearly pathogenic variants. In the first family, we found the *P2RX5* p.Leu32Gln variant previously described in a Brazilian family. However, its frequency in the general population – especially in Europe – is much higher than expected for a highly penetrant variant. Thus, we should consider it either as a low-penetrant pathogenic variant predisposing to familial PTC or as a variant not associated with familial PTC. In the second family, a variant that modifies the reading frame of *MST1R*, encoding the receptor tyrosine kinase RON which is frequently overexpressed in PTC and FTC, co-segregated with the familial PTC. The nature of the mutated protein (receptor) as well as the signaling pathways
it controls are relevant to thyroid carcinogenesis. However, further studies are needed to determine how precisely this RON variant alters thyroid epithelial growth.

## Informed consent and patient details

The authors declare that this report does not contain any personal information that could lead to the identification of the patient(s).

## Funding

This work has been supported by: UF 7045, BPR Oncologie endocrinienne (grant to Dr Do Cao and Dr Cardot Bauters) and in part by the Institut de biochimie, CHU de Lille (grant UF 8860 to Pr Pigny).

## Author contributions

Christine Do Cao: resources, validation and writing original draft. Rachel Desailloud: resources, validation and writing original draft. Hélène Topolinski: resources, validation and writing original draft. Michel Crepin: investigation, resources, data curation. Cathy Flamant: investigation, resources, data curation. Emmanuelle Leteurtre: validation and writing original draft. Catherine Cardot Bauters: resources and writing original draft. Frederic Frenois: Investigation and resources. Emilie Ait-Yahya: resources, formal analysis and data curation. Fabrice Bonte: resources, formal analysis and data curation. Julie Leclerc: methodology, validation, resources and writing original draft. Pascal Pigny: design, validation, supervision, project administration and writing original draft.

## Disclosure of interest

The authors declare that they have no competing interest.

## Acknowledgements

The authors would like to thank the PBC endocrine tumor team for their expert technical assistance. Ms. Dondeyne for her help with the patient records, and all the colleagues who have referred their family cases to us.

## Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ando.2025.102456.

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