Clinical and molecular findings
Of 45 referred MTC patients, 7 cases had FMTC or MEN syndrome, with more than one affected members. Thirty-eight patients seemed to be apparently sporadic MTC. All six RET exons (10, 11, 13, 14, 15 and 16 exons) were sequenced in 45 index patients. Eight RET known distinct mutations, one unclassified variant, and one novel variant were detected (Table 1).
Table 1
RET mutations and number of affected subjects, carriers and healthy individuals.
ID | Family | Type | Mutation | Polymorphism | Variant | Number of affected | Number of carriers | Number of healthy | ATA risk |
| | | | Familial Cases | | | | |
1 | IR-F4 | FMTC | L790F | L769L & S836S | | 4 | 5 | 11 | A (MOD) |
2 | IR-F15 | MTC | C634R | | | 2 | | | C (H) |
3 | IR-F18 | FMTC | C634R | G691S & S904S | | 8 | | | C (H) |
4 | IR-F25 | MEN2A | C630R | G691S, L769L & S904S | | 3 | 6 | 4 | B (MOD) |
5 | IR-F31 | MEN2A | C634R | | | 4 | 2 | | C (H) |
6 | IR-F33 | MEN2A | C618R | G691S | | 2 | 3 | 2 | B (MOD) |
7 | IR-F36 | MEN2A | C634R | | | 2 | 2 | | C (H) |
Sporadic Cases |
8 | IR-F11 | Metastatic MTC | M918T | | | 1 | | | D (HST) |
9 | IR-F14 | MTC | C611Y | G691S & L769L | | 1 | 1 | 2 | B (MOD) |
10 | IR-F17 | MEN2A | C634R | | | 1 | 1 | | C (H) |
11 | IR-F19 | MTC | | | V648I (uncertain) | 1 | | | |
12 | IR-F22 | MTC | C634F | G691s, L769L, S836S & S904S + A > T IVS16 | | 1 | 1 | 1 | C (H) |
13 | IR-F23 | MEN2B (metastatic MTC + ambigous genitalia) | M918T | | | 1 | | 4 | D (HST) |
14 | IR-F29 | MEN2A | C634R | | | 1 | 1 | | C (H) |
15 | IR-F37 | MTC | M918T | | | 1 | | | D (HST) |
16 | IR-F38 | MTC | C634Y | | H658R (novel) | 1 | | | C (H) |
18 | IR-F39 | MTC | C634Y | G691S | | 1 | | | C (H) |
The family IR-F4 was an extended kindred with two affected individuals in the second generation (II2 and II3), two affected (III3 and III10) and three carriers in the third generation (III5 − 7), and two carriers in the fourth generation (IV2 and IV8) (Fig. 1-a). The 44-year-old proband III3 and other affected individuals of the pedigree had a known heterozygous mutation (c.2370G > T) in exon 13 (p.L790F). According to the ATA risk classification, p.L790F mutation is stratified as level A, indicating moderate risk of aggressive MTC with ~ 10% incidence of PHEO. Carriers of this mutation are advised to undergo prophylactic thyroidectomy only if it occurs as FMTC or they have a high level of calcitonin. Therefore, these carriers were referred to the endocrinologist for more clinical follow-up.
The proband of the family IR-F15 (II2) also had a known mutation (c.1900T > C) in exon 11 (p.C634R). Her mother (I2) died because of MTC and was not available but two other family members did not have any RET mutations in the six tested exons (Fig. 1-b).
The family IR-F18 was another family with three affected daughters (II2, II6 and II7), one affected son (II4), three affected grandchildren (III3, III4 and III8), and one carrier granddaughter (III6) (Fig. 1-c). A known heterozygous mutation in exon 11 [c.1900T > C (p.C634R)] was found in all affected individuals (II2, II4, II6, II7, III3, III4 and III8) and one carrier granddaughter (III6). Interestingly, individual III6 had two genetically normal triplets. According to the ATA risk classification, she was advised to undergo prophylactic thyroidectomy before 5 years of age.The family IR-F25 was an extended kindred consisting of a healthy father (II3) and an affected mother (II4) with one affected son (III1), one affected daughter (III2), two carrier daughters (III4 and III6), one phenotypically healthy son (III3) and one genetically healthy daughter (III5), three carrier grandsons (IV1, IV4 and IV9), one carrier granddaughter (IV2), and three genetically healthy grandchildren (IV3, IV7 and IV8) (Fig. 1-d). The 63-year-old proband (II4) was diagnosed with MEN2A (MTC, pheochromocytoma (PHEO), and hyperparathyroidism) and underwent thyroidectomy, parathyroidectomy and adrenalectomy. Sequencing of six RET exons of the proband and her affected children revealed a heterozygous mutation (c. 1888T > C) in exon 11 (p.C630R) classified as a pathogenic variant. All carrier relatives of c. 1888T > C mutation with elevated calcitonin levels, III4, III6, IV1, IV4 and IV9, underwent prophylactic thyroidectomy at the time of diagnosis (age: 36, 24, 13, 12, and 5 years old, respectively). The 19-year-old carrier granddaughter (IV2), who had a normal calcitonin level, did not undergo prophylactic thyroidectomy. However, according to the ATA risk level, p.C630R RET mutation is stratified as level B with a moderate risk of aggressive MTC (MOD) and the patients are recommended to receive prophylactic thyroidectomy before the age of 5 years old.
The family IR-F31 was another kindred comprising an affected father (I1), a healthy mother (I2), two affected daughters (II2 and II4), one affected son (II5), one genetically healthy son (II7), and two carrier grandsons (III3 and III4) (Fig. 1-e). MEN2A was diagnosed in the 45-year-old grandfather as the proband with clinical manifestations of MTC and pheochromocytoma. He underwent thyroidectomy and adrenalectomy. His affected children also had MEN2A, including MTC and pheochromocytoma. We identified a heterozygous known mutation in exon 11 (c.1900T > C, p.C634R) in this family which has been classified as a pathogenic variant with ATA risk level C. Prophylactic thyroidectomy is recommended for this mutation before 5 years of age because of high risk of aggressive MTC (H). To conduct a follow-up study, two carrier grandsons (III3, III4) were referred to the endocrinologist and underwent prophylactic thyroidectomy at age 5.
The family IR-F33 had a known RET mutation (c.1852T > C) in exon 10 (p.C618R) (Fig. 1-f). The proband (II5) was first referred for RET genetic testing because of a high calcitonin level (above 1500) and histopathological results showed MTC. Initially, a heterozygous known mutation (c.1852T > C) was seen in exon 10 (p.C618R) in the proband (II5) at age 27 and subsequently, this variant was found in his affected father I1 and three carrier siblings (II1, II2 and II3). According to the ATA risk classification, p.C618R mutation is stratified as level B (MOD) and carriers are recommended to undergo prophylactic thyroidectomy before 5 years of age. Follow-up visits showed that the calcitonin level was above the normal range (300 pg/ml) in II5 even after prophylactic thyroidectomy.
Thirty-eight index patients who seemed to be apparently sporadic MTC were tested for RET mutation at six mentioned exons. The results showed that 10 out of 38 patients had germline RET mutations. In addition, in four families, we found four carriers of a causative mutation in presymptomatic children who had affected parents, including family IR-F14 with c.1832G > A mutation in exon 10 (p.C611Y), families IR-F17 and IR-F29 with c.1900T > C mutation in exon 11 (p.C634R) and family IR-F22 with c.1901G > T mutation in exon 11 (p.C634F).
In the family IR-F17, a 32-year-old affected mother (II8) with a p.C634R mutation had a -year-old daughter with elevated calcitonin who was found to have the same mutation as observed in her mother. The proband in the family IR-F29 (III16) had an asymptomatic 10-year-old boy who carried the same mutation (p.C634R). Moreover, a 15-year-old carrier of p.C611Y in the family IR-F14 with an ATA risk level B (MOD) and a 12-year-old carrier of p.C634F in the family IR-F22 were found. All of the carriers in these four families were referred to the endocrinologist for further clinical follow-up and prophylactic thyroidectomy. Furthermore, three index patients in families IR-11, IR-F23 and IR-F37 had a known RET germline mutation (c.2753T > C) in exon 16 (p.M918T) which is a MEN2B specific mutation with ATA risk level D (highest risk of aggressive MTC-HST). However, no available persons in their families had this mutation. The apparent sporadic patient IR-F39 had a known RET mutation (c.1901G > A) in exon 11 (p.C634Y). Likewise, another apparently sporadic patient (IR-F38) had a RET germline mutation (p.C634Y) and a novel uncertain variant (c.1973A > G) causing p.H658R, both in exon 11. RET novel variant p.H658R was predicted to be of uncertain significance by InterVar, benign by polyphen2, disease causing by mutation taster and tolerated with SIFT score 0.5 by SIFT web server (Table 2). RET sequencing results of another sporadic patient (IR-F19-II7) showed an uncertain variant, c.1942G > A, in exon 11 (p.V648I). This variant was predicted to be likely benign by InterVar, benign by polyphen2, disease causing by mutation taster and tolerated with SIFT score 0.43 by SIFT web server (Table 2).
Table 2
Variants according to in silico analysis
| Variant | InterVar | Polyphen2 | Mutation Taster | SIFT | MEN2 database | Report |
1 | p.H658R | Uncertain significance | Benign with 0.006 score: Sensitivity 0.97 Specificity 0.75 | Disease causing | Tolerated (SIFT score 0.5) | | Novel |
2 | p.V648I | Likely benign | Benign with 0.006 score: Sensitivity 0.97 Specificity 0.75 | Disease causing (NMD, amino acid sequence change, protein features might be affected, splice site changes) | Tolerated (SIFT score 0.43) | Uncertain | [24] (2002) |