RET Germline Mutations Identified by Exome Sequencing in a Chinese Multiple Endocrine Neoplasia Type 2A/Familial Medullary Thyroid Carcinoma Family

Background Whole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC (multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma) using this approach. Methodology/Principal Findings We sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband's daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband's husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years). Conclusions/Significance The results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.


Introduction
The REarranged during Transfection (RET) protooncogene containing 21 exons is mapped on chromosome 10q11.2 and encodes a tyrosine kinase receptor that plays a crucial role in regulating cell proliferation, migration, differentiation, and survival through embryogenesis [1,2]. Germline mutations of RET are found in multiple endocrine neoplasia type 2 (MEN 2) and Hirschsprung disease (HSCR) in an autosomal dominant pattern [3,4]. MEN 2 is subtyped into MEN 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC) based on the different tissues involved [5]. MEN 2A, the most common clinical subtype, is characterized by the MTC triad in nearly 95%, pheochromocytoma (PHEO) in 50-57%, and hyperparathyroidism (HPT) in 15-30% of cases [5][6][7]. MEN 2B is similar to MEN 2A except that HPT is rare, and characteristic developmental abnormalities, such as marfanoid habitus and ganglioneuromatosis of the intestinal tract, are present [5]. FMTC is defined by the sole presence of MTC as the disease phenotype [5]. MEN 2 and HSCR are usually distinct but occasional families have been reported with both diseases [4]. In HSCR, RET mutations are dispersed throughout the gene with poor genotype-phenotype correlation [4], while all the MEN 2 mutations target within exons 5,8,10,11, and 13 to 16 with strong genotype-phenotype correlations [5][6][7]. More than 95% of MEN2A and FMTC patients have a RET mutation typically occurring at one of the five cysteine residues (codons 609, 611, 618, and 620 within exon 10, and codon 634 within exon 11). Biochemical and biological analyses revealed that these cysteine mutations induce disulfide-linked dimerization of the RET protein, leading to constitutive activation of its intrinsic tyrosine kinase activity [8]. Other mutations within exons 8, 10, 11, and 13 to 16 appear to account for a small percentage of FMTC families [7,9]. MEN 2B is mainly associated with mutation of codon 918 within exon 16 [5]. A second mutation at codon 883 within exon 15 and tandem RET mutations of codons 805, 806, and 904 in cis configuration with the p.V804M mutation have also been reported in individuals with MEN 2B [4,8,10,11]. Recently, Castellone et al found a p.V292M mutation within exon 5 in an Italian man with MTC and PHEO and suggested that the mutant had low-grade transforming potential [12]. Emerging reports of compound RET mutations appear to show modulation of the degree of aggressiveness and the development of unusual features of MTC [13]. p.R982C, a rare polymorphism within exon 18 with a frequency of about 2%, was first reported in HSCR [14]. p.R67H combined with p.R982C was reported to be associated with congenital central hypoventilation syndrome [15].
Protein-coding genes constitute only approximately 1% of the human genome but harbor nearly 85% of the disease-causing mutations at individual Mendelian loci [16][17][18]. Therefore, efficient strategies for selectively sequencing complete coding regions can serve as a genome-wide scan for the pathogenic gene [17][18][19]. Whole exome capture and massively parallel DNA sequencing has provided a laborsaving, reproducible and robust method for disease gene discovery and clinical diagnosis [18,20]. Here, we used whole exome sequencing (WES), coupling the Agilent whole exome capture system to the Illumina HiSeq 2000 DNA sequencing platform, to identify a Chinese female with MEN 2A-related MTC carrying the p.C634Y/V292M/ R67H/R982C mutation of RET, in whom p.C634Y was maternal with 5 carriers with MEN2A, and p.V292M/R67H/R982C was paternal with 6 carriers.

Subjects
A 22-year-old woman with MEN 2A-related MTC and her brother, and 10 paternal and 12 maternal individuals from a four-generation southern Chinese family (Fig. 1), and 100 unrelated healthy matched controls were studied.
This study was conducted in conformity with the Helsinki Declaration and approved by the Ethics Committee of the 117 th PLA Hospital. Written informed consent was given by all subjects.

Clinical Investigation
Clinical evaluation of MEN 2A/FMTC was performed according to the published criteria [6,7,21]. The following parameters were studied: clinical and diagnostic data (age, gender and clinical features), biochemical markers (serum calcitonin (Ct), carcinoembryonic antigen (CEA), parathyroid hormone, catecholamines), Doppler ultrasound (US) and computerized tomography (CT) scans. Prophylactic or surgical thyroidectomy with neck dissection and adrenalectomy was performed after confirmation of RET mutations for the diagnosis. Then follow-up was carried out.

Exome Capture and Massively Parallel DNA Sequencing
The peripheral blood genomic DNA samples from each of six family members (II-5, III-10, III-11, III-12, IV-10 and IV-11) were randomly fragmented by Covaris to provide DNA fragments with a base pair peak at 150 to 200 bp, then adapters were ligated to both ends of the resulting fragments. The adapter-ligated templates were purified by Agencourt AMPure SPRI beads, and fragments with an insert size of ,200 bp were excised. Extracted DNA was amplified by ligation-mediated PCR (LM-PCR), purified, and hybridized to the SureSelect Biotinylated RNA Library for enrichment; hybridized fragments were bound to strepavidin beads whereas non-hybridized fragments were washed out. Captured LM-PCR products were subjected to an Agilent 2100 Bioanalyzer to estimate the magnitude of enrichment. Each captured library was then loaded onto the Hiseq2000 platform, and we performed GA sequencing for each captured library independently to ensure each sample had at least 50-fold coverage. Raw image files were processed by Illumina Pipeline v1.6 for basecalling with default parameters and the sequences of each individual were generated as 90 bp paired-end reads.
SOAPaligner (soap2.21) was used to align the clean reads to the human reference genome. Based on the SOAP alignment results, the software SOAPsnp was used to assemble the consensus sequence and call genotypes in target regions [22]. We filtered the SOAPsnp results as follows: base quality more than 20, depth between 4 and 200, estimate copy number equal to or less than 2 and the distance between two SNPs no less than 5. Detailed information of each parameter for the two algorithms is available at http://soap.genomics.org.cn/.

RET Mutation Confirmation
To further confirm the RET substitution mutations and the pattern of the compound mutation in the proband's daughter, we isolated DNA and total RNA from peripheral blood and tumor tissues (MTC from III-11 and III-20, PHEO from III-12) respectively, and performed polymerase chain reaction (PCR) amplification of all RET exons followed by Sanger sequencing, restriction endonuclease digestion, reverse transcription PCR (RT-PCR)/sequencing, and haplotype analysis of four RET-flanking microsatellite markers (D10S197, D10S196, D10S1652 and D10S537).
All new data have been deposited in GenBank.

Clinical Features and Phenotypic Data
The proband (III-12) was a 43-year-old woman who had been diagnosed as having MTC in 1995 with a 4-year history of a palpable neck mass in the right thyroid. Biochemical examination disclosed increased CEA (17.7 ng/mL, normal ,5 ng/mL) and Ct (408.32 ng/L, normal ,100 ng/L), while US scanning disclosed multi-centric hypoechoic nodules with calcifications in both thyroid lobes. Then a total bilateral thyroidectomy with modified neck dissection was performed and histopathological examination showed bilateral multi-centric MTC with lymph node metastases (T2N1aM0, Table 1). Since surgery, she has been receiving L-T4 substitution therapy. Her father died of thyroid cancer in 1992. In 2001, the proband's 13-year-old daughter (IV-10) was diagnosed as having MTC with a 1-year history (first diagnosed as multi-nodular goiter rather than MTC). Surveillance found that the palpable neck mass in the left thyroid lobe increased in size and reached a maximum diameter of 3.9 cm. Ct was elevated to 343.92 ng/L and CEA was 14.7 ng/mL. US and CT scanning disclosed multi-centric nodules with calcifications in both lobes. Then, IV-10 received a total thyroidectomy with modified bilateral neck dissection. Histopathology revealed multi-centric MTC with lymph node metastases (T2N1aM0, Table 1). In 2005, the proband's 45-year-old male cousin (III-23) presented with headache, paroxysmal hypertension, dizzy spells, and excess serum catecholamine (Tables 1 and 2). US and CT scanning confirmed bilateral masses in the adrenal glands. After treatment with ablockers, he underwent a total bilateral laparoscopic adrenalectomy with CO 2 pneumoperitoneum; bilateral PHEO was confirmed by histopathological examination. In 2006, the proband's 52-yearold female cousin (III-20) underwent a total thyroidectomy with modified bilateral neck dissection based on the evidence of elevated Ct and CEA and multi-centric nodules with calcifications in both thyroid lobes but without adrenal abnormalities. In 2010, some previously hesitant members of the family (III-13, III-15, III-18, III-26, IV-11, IV-13, IV-16, IV-17, IV-18 and IV-21) agreed to participate in biochemical testing, image examinations and RET screening (Tables 1 and 2). All had normal Ct, CEA and US images except III-15, III-23, IV-21 and III-12 with p.C634Y. Then a total thyroidectomy with modified bilateral (III-15 and III-23) or bilateral level VI lymph node dissection (IV-21) and a total bilateral laparoscopic adrenalectomy with CO 2 pneumoperitoneum (III-12) were performed. Histopathological examination revealed bilateral PHEO in III-12 and bilateral multi-focal MTC with lymph node metastases in III-15, III-20 and III-23, while IV-21 showed no evidence of lymph node metastases. All six patients (III-12, III-15, III-20, III-23, IV-10 and IV-21) were followed up. Biochemical and imaging examination did not reveal any evidence of recurrence of MTC or PHEO, and showed no HPT during this period except for III-12. Biochemical examination of III-12 showed Ct was 1360 ng/L and CEA was 13.2 ng/ mL. US-guided fine needle aspiration and cytology was suggestive of MTC with lymph node metastases. The proband's husband (III-11) and his family members have not felt any special abnormality to date. As p.V292M/R67H/R982C and p.C634Y/V292M/R67H/R982C mutations of RET were found by WES in him and his daughter (IV-10) respectively in 2010, neck US screening was performed on all 6 carriers and the results disclosed multi-focal hypoechoic nodules (range 0.4-0.8 cm) in the upper third of left/right or both thyroid lobes in II-5, III-3, III-8 and III-11 but not in IV-3 and IV-7. Ct was normal while the pentagastrin-stimulated Ct was slightly elevated in II-5 and III-11. In 2010, a total thyroidectomy with modified bilateral level VI lymph node dissection was performed in II-5, then histopathological examination revealed bilateral MTC without lymph node metastases (T1N0M0, Table 1). III-11 underwent a prophylactic total thyroidectomy in 2010 at the age of 44, and histopathology revealed bilateral multi-focal C-cell hyperplasia (CCH) without evidence of MTC. III-3 and III-8 await thyroidectomy, while IV-3 and IV-7 are being followed up ( Table 1). None of the 6 carriers showed abnormalities in the parathyroid or adrenal glands.
We excluded the possibility of HSCR in this family based on the fact that none of the affected individuals presented evidence of HCSR involving severe constipation, vomiting, abdominal distension, or enterocolitis. Meanwhile, cutaneous lichen amyloidosis was also excluded because there was no evidence of pruritic lichenoid skin lesion located on the upper back.

Exome Sequencing Identified Four RET Missense Variants
We generated an average of approximately 4.0 billion bases of sequence per affected individual as paired-end, 88-bp reads, and the fraction of effective bases on target was about 64% with a

Discussion
We used WES here and successfully confirmed several pathogenic variants in a Chinese family with MEN 2A/FMTC. WES can serve as a genome-wide scan for the causative gene [20]. Moving along the spectrum from rare monogenic disorders to complex common diseases, it is likely that the increasing extent of genetic heterogeneity will need to be matched by increasingly large sample sizes [23]. Meanwhile, although this approach could be underemployed for mutation analysis of known genes, especially those with hot-spot mutations, our case serves as a proper example of genetic diagnosis of a pathogenic gene that harbors a large myriad of exons, particularly those without hot-spot mutational sites, such as Cornelia de Lange syndrome with causative gene NIPBL embracing 47 exons without hot-spot mutational sites [24].
WES can reduce the missed detection of some rare mutations in cases with hot-spot mutations and serve as a more direct method than cumbersome Sanger sequencing of a myriad of PCR fragments [19]. Moreover, WES could be a better choice for some diseases with overlapping symptoms that might be ambiguously associated with many pathogenic genes, in which event the detection of mutation by Sanger sequencing could be time-consuming and labor-intensive [18,19]. The standard sequencing of a single PCR fragment in molecular diagnosis costs 600 Yuan (,$91) for Zhejiang University-Adinovo Center for Genetic and Genomic Medicine. This price includes consumables, equipment amortization, personnel salary and corporate profit. Therefore, the total cost of sequencing the entire RET coding sequence, which requires 21 PCR fragments, is 12600 Yuan (,$1909). In comparison, the all-inclusive cost of WES per sample is 25000 Yuan (,$3788), about twice as much as Sanger sequencing. However, WES would be a more cost-effective and robust method for a causative gene embracing more than 42 exons (25200 Yuan, ,$3819) such as NIPBL. Additionally, with rapid commercialization, the cost of WES for gene testing will be considerably reduced in the coming years [19].
The most frequent RET mutations responsible for MEN 2A are p.C634Y/R/W/F/S/G/L, which target to one of five cysteine residues in the extracellular domain of RET [5,25]. It seems that different amino acid substitutions within codon 634 present different prevalence in different populations but cause indistinguishable phenotypes, although one study suggested that individuals with p.C634R have significantly more distant metastases at diagnosis than those with p.C634Y/W [25,26]. In this study, the clinical features seemed similar to those reported by Zhou et al Previous studies provided evidence that some variants play roles in modifying other variants in the genesis of MEN 2A or FMTC. The p.V292M mutation maps to the third cadherin-like domain of RET and is a gain-of-function alteration with weaker pathogenicity [12]. An Italian MEN 2A patient with p.V292M presented at the age of 44 with classic PHEO and a 4-mm unifocal MTC in the right thyroid lobe [12]. Dvorakova et al reported another mutation, p.G321R, within exon 5 in a threegeneration Czech kindred with FMTC [27]. Among 4 affected paternal members, one (aged 70) had MTC, one (aged 42) had CCH, and two (aged 27 and 52) had no evidence of either [27].
This indicated that mutations in the extracellular cadherin-like domain cause a comparatively mild clinical phenotype. Clinical investigation in our patients revealed that only one (II-5) had bilateral MTC at the age of 70, one (III-10) had bilateral CCH at age 44, and two (III-8 and III-3) had bilateral multiple nodules in both thyroid lobes at ages 46 and 48, which may be caused by the RET p.V292M/R67H/R982C mutation. In contrast to the previous literature, our patients presented with a later onset of MTC or CCH and showed no evidence of PHEO even in the 70year-old male (II-5), while the previously reported case with p.V292M had PHEO before MTC [12]. The p.V292M/R67H/ R982C compound mutation may predispose to the FMTC phenotype. Previously-reported RET double mutations associated  with MEN 2 are summarized in Table 4. Susceptibility to MEN 2A seems to be impaired by certain RET polymorphisms [28][29][30], while polymorphisms involving genes whose products interact with RET or components of the RET signaling pathway have also been reported to modify the risk of MTC [31,32]. Iwashita [14,37], and p.R67H/R982C was reported to be associated with congenital central hypoventilation syndrome [15]. Previous studies have shown that double RET mutations may be associated with unusual MEN 2 phenotypes (Table 4). The case with p.C634Y/V292M/R67H/R982C (IV-10) had a more advanced and large bilateral MTC with lymph node metastases (Table 1), in contrast to p.C634Y (including contemporary IV-21) or p.V292M/R67H/R982C (including contemporaries IV-3 and IV-7) carriers, but lacked PHEO and HPT. Nunes et al believed that the RET trans p.C634R/V648I double mutation may have modified and contributed to the rare MEN 2A phenotype in a patient with bilateral adrenocorticotropin-producing PHEO and MTC [38]. A previous study showed that the potential physiochemical effects of p.V804M and p.E805K substitutions in silico are moderately deleterious in isolation but severely deleterious in cis combination due to their distinctly synergistic effect [39].
These two cases provided the evidence that these two pathogenic mutations together amount to a more deleterious double mutation than that in isolation, which might explain the higher MTC pathogenicity of p.C634Y/V292M/R67H/R982C due to the additive effect of p.V292M/R67H/R982C and p.C634Y in trans. A ''second hit'' mechanism advocates that allelic imbalance between mutant and wild-type RET is a possible mechanism of tumorigenesis in some patients with MEN 2A-related MTC [40]. Our data illustrated that the consistency of sequencing of genomic DNA from blood and tumor tissues and the cDNA from tumor tissues refuted the loss of heterozygosity and are inconsistent with the ''second hit'' mechanism [40]. However, the wild-type RET might play a neutralizing role and thereby partly compensate for the activating effects of the mutant allele, but this protective effect of wild-type RET may disappear in the trans double mutant, which, from another perspective, could explain the more aggressive pathogenicity of the p.C634Y/V292M/R67H/R982C compound mutation [40]. Due to the young age of 22 years in IV-10, the combination of p.C634Y and p.V292M/R67H/R982C, and the reduced penetrance of PHEO and HPT, it is too early to ascertain whether she will develop PHEO and HPT.
Briefly, WES provides an instructive, comprehensive, fast and accurate method for genetic diagnosis of a disease whose pathogenic genes embrace a large cohort of exons. Our case suggests the importance of extending RET sequencing studies to the gene's entire coding region, even though initial testing of a patient with clinical signs of MEN-2 reveals RET hot-spot mutations, or when genotype-phenotype discrepancies emerge. Meanwhile, in the present study, the p.R67H/R982C polymorphism had a modifying effect on the disease and may inhibit the genesis of PHEO, and p.C634Y/V292M/R67H/R982C may lead to higher MTC pathogenicity due to the additive effect of p.V292M/R67H/R982C and p.C634Y in trans combination.