**3. Germline risk factor of the familial FCC**

Cancer is a disease caused by one or multiple mutation(s) in the genome of cells. A familial cancer has one or multiple germline causal mutations that are inherited from parents of the individual. Identification of germline causal variants has important value for both cancer prevention factors of human thyroid cancer. GWAS is a statistical method to identify genomic regions that are associated with targeted traits/disease taking advantage of linkage disequilibrium between genomic variants. Linkage disequilibrium is the non-random association of alleles at different genomic loci in a given population. It makes it possible to identify the genomic region associated with the investigated trait/disease even when the causal loci are not genotyped. After a GWAS analysis, fine-mapping can be performed to identify the causal variants in the targeted genomic region. Whole-genome sequencing data are usually used in fine-mapping, which can identify all the variants when compared with the reference genome. In order to confirm the causal variants and underlying molecular mechanism, further *in vitro* or *in vivo* experiments are needed., diagnosis, and novel drug development. Next-generation sequencing technologies make it easy to obtain wholegenome sequencing data of an individual. However, identification of germline causal mutations for a disease/cancer is still challenging. In the past decade, many genomewide association studies (GWASs) have been performed to identify germline risk.

Identification of germline risk factor of a hereditary form of thyroid cancer is still challenging. In humans, only approximately 5% of a form of familial non-medullary thyroid cancers have well-documented germline risk factors [13]. We performed a series of analyses based on a combination of SNP array genotype data and wholegenome sequencing data to identify the germline risk mutation that confers a higher risk for thyroid carcinoma in Dutch GLPs [14]. We combined a GWAS analysis and a homozygosity mapping to identify the genomic region that is associated with the FCC. This combined strategy was used because clear population stratification was observed between the genotyped affected and unaffected dogs. In the GWAS analysis, to correct over false-positive discoveries, genomic relationship matrix estimated based on genotype data was incorporated as a random effect. Homozygosity mapping was also used because the FCC in these dogs has very likely an autosomal recessive inheritance pattern according to pedigree. Homozygosity mapping used in that study is based on runs of homozygosity (ROH)-based approach, which is a powerful method to identify genomic region that is associated with a recessive disease. A common genomic region was identified by both the GWAS and homozygosity mapping analyses. Next, we performed fine-mapping using WGS data of 11 affected and 11 unaffected GLPs to identify the germline mutations that are in the targeted region. A series of stringent filtering was performed on the variants identified in the targeted

#### **Figure 2.**

*Graphic abstract of identification of the germline risk mutations in the familial FCC [14]. 170K SNP array genotype data were obtained from 36 healthy German longhaired pointer (GLP) dogs and 28 GLPs affected by the familial thyroid follicular cell carcinoma. The genotype data were used in the genome-wide association analysis and homozygosity mapping to identify genomic region associated with the familial thyroid follicular cell carcinoma.*

genomic region based on WGS data: 1) must be deleterious predicted by pathogenicity prediction tools; 2) must fit an autosomal recessive inheritance pattern; 3) must be rare in general dogs; 4) must be conserved across species. At the end, we identified two deleterious mutations, chr17:800788G>A (p.686F>V) and chr17:805276C>T (p.845T>M), in the *TPO* gene. We further genotyped these two variants in 186 GLPs (59 affected and 127 unaffected) using PCR-RFLP experiment and revealed 16.94 and 16.64 of the relative risk of homozygous recessive genotypes compared with homozygous genotypes for the reference allele (**Figure 2**).

The genetic cause of general canine thyroid cancer is still poorly studied. There is no study that investigated the genetic causes of canine thyroid cancer at a genomewide scale, except for our study. Genetic causes of canine thyroid cancer need to be revealed since thyroid tumor is the most frequent endocrine neoplasm in dogs.
