The synergy of germline C634Y and V292M RET mutations in a northern Chinese family with multiple endocrine neoplasia type 2A

Abstract Genetic analysis for germline mutations of RET proto‐oncogene has provided a basis for individual management of medullary thyroid carcinoma (MTC) and pheochromocytoma. Most of compound mutations have more aggressive phenotypes than single point mutations, but the compound C634Y/V292M variant in MTC has never been reported. Thus, we retrospectively investigated synergistic effect of C634Y and V292M RET germline mutations in family members with multiple endocrine neoplasia type 2A. Nine of 14 family members in a northern Chinese family underwent RET mutation screening using next‐generation sequencing and PCR followed by direct bidirectional DNA sequencing. Clinical features of nine individuals were retrospectively carefully reviewed. In vitro, the scratch‐wound assay was used to investigate the difference between the cells carrying different mutations. We find no patients died of MTC. All 3 carriers of the V292M variant were asymptomatic and did not have biochemical or structural evidence of disease (age: 82, 62 and 58). Among 4 C634Y mutation carriers, 2 patients had elevated calcitonin with the highest (156 pg/mL) in an 87‐year‐old male. Two carriers of compound C634Y/V292M trans variant had bilateral MTC with pheochromocytoma or lymph node metastasis (age: 54 and 41 years, respectively). Further, the compound C634Y/V292M variant had a faster migration rate than either single point mutation in vitro (P < .05). In conclusion, the V292M RET variant could be classified as ‘likely benign’ according to ACMG (2015). The compound variant V292M/C634Y was associated with both more aggressive clinical phenotype and faster cell growth in vitro than was either single mutation.

V292M RET germline mutations in family members with multiple endocrine neoplasia type 2A. Nine of 14 family members in a northern Chinese family underwent RET mutation screening using next-generation sequencing and PCR followed by direct bidirectional DNA sequencing. Clinical features of nine individuals were retrospectively carefully reviewed. In vitro, the scratch-wound assay was used to investigate the difference between the cells carrying different mutations. We find no patients died of MTC. All 3 carriers of the V292M variant were asymptomatic and did not have biochemical or structural evidence of disease (age: 82, 62 and 58). Among 4 C634Y mutation carriers, 2 patients had elevated calcitonin with the highest (156 pg/ mL) in an 87-year-old male. Two carriers of compound C634Y/V292M trans variant had bilateral MTC with pheochromocytoma or lymph node metastasis (age: 54 and 41 years, respectively). Further, the compound C634Y/V292M variant had a faster migration rate than either single point mutation in vitro (P < .05). In conclusion, the V292M RET variant could be classified as 'likely benign' according to ACMG (2015). Their results suggested that the p.C634Y/V292M/R67H/R982C transmutation of RET exhibited a more aggressive clinical phenotype than did the p.C634Y or p.V292M/R67H/R982C cismutation. However, they could not confirm the isolated effect of the V292M mutation since it was accompanied by the mutation of R67H/R982C.

| INTRODUC TI ON
Mutations for cysteine in extracellular domain 634 have been reported to occur in MEN2A. 15,16 The most common mutation in codon 634 is the amino acid Cys-Arg(C634R), while the Cys-Tyr(C634Y) mutation being less common and less aggressive than the former one. 17 Along with all the mutations emerged in codon 634, the C634Y RET mutation is classified as 'high risk' according to the 2015 ATA guideline. Sanchez et al (1999)  have been reported to be associated with MEN2A kindreds. 19,20 Herein, we are the first to describe a compound C634Y/V292M transmutation of RET, occurring in a northern Chinese family. The compound mutation was associated with a more aggressive clinical phenotype than was either the C634Y or V292M single point mutation, demonstrating the synergistic effect of compound non-synonymous germline RET mutations.

| Study patient description
The pedigree of the 14 members of the family is shown in Figure 1.
Three of the participants (designated II-3, II-5 and III-3) had histopathologically confirmed MTC. Nine family members underwent The compound variant V292M/C634Y was associated with both more aggressive clinical phenotype and faster cell growth in vitro than was either single mutation.

K E Y W O R D S
compound mutation, hereditary medullary thyroid carcinoma, MEN2, RET proto-oncogene F I G U R E 1 Features of the C634Y and V292M RET mutations among the 14 family members. Squares and circles represent the male and female family members, respectively. and indicate positive for MTC and RET mutation; and indicate negative for MTC; and indicate not tested testing using RET full-exon next-generation sequencing and polymerase chain reaction followed by direct bidirectional DNA sequencing (MyGenostics, Beijing). The following were studied for these nine individuals: clinical and diagnostic data (age, sex and clinical features), serum calcitonin (Ct) level (normal range: 0-11 pg/mL in females, 0-18 pg/mL in males), carcinoembryonic antigen (CEA) level (normal range: 0-5 ng/mL), parathyroid hormone, and Doppler ultrasound and computed tomography images including abdominal region.

NIH3T3 cells were cultured in high-glucose Dulbecco's modified
Eagle's medium containing 10% foetal bovine serum (HyClone Laboratories). Cells were changed to a fresh medium containing 5 μg/mL hexadimethrine bromide (polybrene) and infected with a lentivirus (multiplicity of infection, 50). Cells were then switched to a fresh culture medium. Puromycin (2 μg/mL) was added to the medium 48 hours after infection. Next, the cells were cultured in medium with 2 μg/mL puromycin and regularly changed medium. After several passages, the stable cells were used for subsequent studies.

| Scratch-wound assay
Obtained stable cell lines were seeded in a 24-well plate (~30 000 cells/well). Twenty-four hours later, the cell layer was scratched with a 200-μL pipette tip. The width of the wound was about 200 μm.
Cells were then washed once with phosphate-buffered saline and changed to a fresh culture medium. Three spots on the bottom of the plate were labelled and then the wound area was photographed and calculated at the time of 0, 12 and 24 hours after the scratch. The cell growth rate was calculated as the percentage of newly extended area to the initial wound area.

| Statistical analyses
Data presented as mean ± SD were analysed using SPSS version 19.0 (SPSS Inc). The differences between groups were performed with t test or one-way ANOVA. A value of P < .05 was defined as statistically significant Results. All in vitro assays were triplicated.

| Characterization and distributions of RET mutations among 14 family members
We purified genomic DNA from participant II-5's peripheral blood and tested it using RET full-exon next-generation sequencing. In total, we identified two RET missense mutations in participant II-5.  Figure 2). We tested the genomic DNA from participant II-5's parents using the same method and found the p.C634Y and p. V292M mutations in her father and mother, respectively. We then performed polymerase chain reaction followed by direct bidirectional DNA sequencing for other available family members to examine for C634Y and V292M mutations. We found the compound C634Y/V292M mutation in participant II-3 and II-5; V292M mutation in participants I-2, II-1 and II-2; and the single mutation of C634Y in participants I-1, III-3, IV-2 and IV-3. Table 1

| Analysis of our in vitro study
We used the scratch-wound assay to investigate the growth of cells harbouring the RET mutations described above, which are We performed Western blotting of RET protein expression in these cells using an anti-FLAG antibody. The results showed that RET protein could be expressed stably in each transfected cell line ( Figure 3D). Composed of 21 exons, the proto-oncogene RET is located on chromosome 10 (10q11.2) and encodes for a transmembrane receptor tyrosine kinase for members of a glial cell line. 21,22 The RET protein is composed of an extracellular ligand-binding domain containing a cysteine-rich region, a series of cadherin homology domains, a transmembrane domain and a cytoplasmic tyrosine kinase domain. The highly conserved cysteine-rich region is important for disulphide bond formation, which is required for maintaining the native tertiary structure, allowing for receptor dimerization. 22 In the majority of MEN2A families (more than 90%), germline muta- There are six different amino acid substitutions for the same cysteine on codon 634 (F/G/R/S/W/Y), and all of them display comparable transforming activity. The C634Y mutation is believed to confer lower penetrance of the MEN2A phenotype and less aggressive behaviour of MTC than does the C634R mutation. [23][24][25] There is higher penetrance of MTC, phaeochromocytoma and hyperparathyroidism in patients harbouring C634R mutation. The age-related penetrance of bilateral phaeochromocytoma in C634R is significantly higher than in other types of codon 634 mutations. Lymph node and distant metastases occurred earlier in C634R carriers than in the C634Y carriers. The C634R mutation is reported to be an independent factor for recurrent or persistent disease. In Chinese population the most frequent RET proto-oncogene mutation was localized at codon 634 of exon 11, with the C634Y mutation as the most common, followed by C634R, C634W and the rarer mutations C634F, C634S and C634G. 26,27 In the present study, the 87-year-old C634Y carrier (I-1) had unexpected and slightly high levels of Ct (156 pg/ mL), suggesting that his MTC would be associated with relatively moderate biological behaviour and lesser aggressiveness of disease.

| CON CLUS ION
We are the first to describe the compound C634Y/V292M RET trans association. Additionally, the compound C634Y/V292M mutation resulted in an apparently more aggressive phenotype than did either the C634Y or V292M single point variant. Treatment of MEN2A should be individualized based on particular associated mutations and any available family history about the behaviour of family members with the same mutations, while research should continue to elucidate the optimal therapeutic schedule for carriers of each mutation.

ACK N OWLED G EM ENTS
We thank all of the participants in this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Zheng Yang: Data analysis and manuscript preparation. Xinmeng Qi: Performing of the assays and manuscript preparation. Neil Gross: Review of the manuscript. Huang: Conception of the study.

I N FO R M E D CO N S E NT
This study was approved by the Ethics Committee (TRECKY2014-011) of Beijing Tong Ren Hospital. All participants and/or their legal guardians provided written informed consent for participation in the present study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.