**7. The usage of identified germline and somatic mutations**

Identification of germline risk mutations can be valuable for cancer prevention. For hereditary cancer with a simple inheritance pattern, the identified germline risk mutation can be used to identify those individuals that have a higher risk for the cancer through a genetic test. Those animals then can be removed from the breeding program to eradicate the inherited cancer from the population. Without a genetic test, it is hard to completely remove a disease with an autosomal recessive inheritance pattern from the population because of difficulty in identifying those heterozygous animals. For a cancer with a complex inheritance pattern, a polygenic risk score can be calculated based on all identified germline risk mutations to predict the risk of the cancer development for an individual. For those people with a high risk score for certain disease, early prevention, such as healthy dietaries and life styles, can be taken to diminish the risk. Frequent examination can also be arranged to detect the disease earlier and cure easier.

We developed a genetic test based on one of the germline risk mutation (chr17:800788G>A) in the *TPO* gene to identify GLPs that have a higher risk for the FCC. This genetic test is now commercially available for GLP breeders and owners after testing 142 GLPs at the Animal Breeding and Genomics laboratory. To date, this genetic test has been performed on more than 150 GLPs from a few countries. The frequency of germline risk allele is 25.4% in those tested GLPs. This frequency is rather high and indicates that the risk allele is hard to be completely eradicated from the population by conventional breeding strategy. The genetic test can be especially valuable for GLP breeders to breed healthy dogs. It enables breeders to find those dogs at a high risk for the FCC before any signs of the disease and then those dogs can be excluded from the breeding program.

### **8. Germline and somatic mutation interaction**

*TPO* gene encodes an enzyme, thyroid peroxidase, which plays an important role in production of thyroid hormones. There are seven key steps in the thyroid hormone synthesis: 1) iodine uptake into thyroid follicular cells by the sodium/iodide symporter (NIS); 2) synthesis of two key proteins, thyroid peroxidase (TPO) and thyroglobulin (TG), and secretion of TG into the follicular lumen; 3) iodide transport into the follicular lumen; 4) iodide oxidation to form iodine by TPO; 5) iodination of TG tyrosine residues to generate monoiodotyrosine (MIT) and diiodotyrosine (DIT) by TPO; 6) coupling of iodotyrosines to form thyroxine (T4) and triiodothyronine (T3) by TPO; 7) endocytosis of TG-thyroid hormone complex and T3 and T4 cleaved from it by proteases in the lysosomes [26, 27]. TPO is involved in steps 4, 5, and 6. Meanwhile, hydrogen peroxide (H2O2) is needed in those reactions catalyzed by the TPO. We suspect that germline mutations identified in the *TPO* gene may impair the activity of the TPO enzyme and result in less consumption of H2O2, therefore increased level of H2O2. This assumption needs to be validated in future using, for instance, cell experiments. However, hydrogen peroxide is a type of reactive oxygen species that can induce DNA damages. Elevated H2O2 probably induces many somatic mutations occurring in the thyroid follicular cells and finally a cancerous cell form when a driver mutation occurs. In those familial FCCs, one of driver mutations is the recurrent somatic mutation identified in the *GNAS* gene.

### **9. Medullary thyroid cancer in dogs**

Regarding MTC, both spontaneous and hereditary forms have been reported in dogs [7, 11, 28, 29]. Up to 20%–30% of human hereditary MTC is caused by activating mutations in the *RET* proto-oncogene [30]. In the hereditary MTCs that were studied by Lee et al., the authors sequenced the *RET* gene but identified no mutation in that gene [11]. The germline genetic causes of canine MTC including the somatic driver mutations are still not clear. Canine familial MTC is similar to human familial MTC in clinical symptoms and morphology of histology, suggesting their value to be used as a disease model. However, unraveling the genetic basis of canine MTC is needed for that purpose.

### **10. Thyroid cancer in other species**

Besides the occurrence in dogs, thyroid tumor has also been reported in many other species, such as guinea pig [31], cat [32], horse [33], cattle [34, 35], barred owl [36], rat [37], and ferret [38, 39]. Thyroid hyperplasia was seen in fish over one hundred years ago, and thyroid neoplasms were also reported in fish [40]. Thyroid carcinoma has been induced in mice using transgenic technology for the study of

pathogenesis [41]. Genetic causes of thyroid carcinoma in these species (except for mice) are generally not known. Mapping of the genetic causes of thyroid carcinoma in these species is needed to elucidate the molecular mechanism of tumorigenesis. However, it is challenging due to the difficulty in identifying sufficient amount of cases for causal mutation mapping in those species.
