Genetic Diagnosis of a Chinese Multiple Endocrine Neoplasia Type 2A Family Through Whole Genome Sequencing

Genetic diagnosis of a Chinese multiple endocrine neoplasia type 2A family through whole genome sequencing

1, 2, 3, 1 ZHEN-FANG DU ,PENG-FEI LI ,JIAN-QIANG ZHAO ,ZHI-LIE CAO , 1 1 1, FENG LI ,JU-MING MA and XIAO-PING QI * 1Department of Oncologic and Urologic Surgery, Nanjing Military Command Hospital Center for Endocrine and Metabolic Diseases, the 117th PLA Hospital, Wenzhou Medical University, 40 Jichang Road, Hangzhou 310004, Zhejiang Province, China 2XY Biotechnology Co. Ltd, 688 Bin’an Road, Hangzhou 310051, Zhejiang Province, China 3Department of Head and Neck Surgery, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou 310022, Zhejiang Province, China

*Corresponding author (Email, [email protected]) These authors contributed equally to this work.

Approximately 98% of patients with multiple endocrine neoplasia type 2A (MEN 2A) have an identifiable RET mutation. Prophylactic or early total thyroidectomy or pheochromocytoma/parathyroid removal in patients can be preventative or curative and has become standard management. The general strategy for RET screening on family members at risk is to sequence the most commonly affected exons and, if negative, to extend sequencing to additional exons. However, different families with MEN 2A due to the same RET mutation often have significant variability in the clinical exhibition of disease and aggressiveness of the MTC, which implies additional genetic loci exsit beyond RET coding region. Whole genome sequencing (WGS) greatly expands the breadth of screening from genes associated with a particular disease to the whole genome and, potentially, all the information that the genome contains about diseases or traits. This is presumably due to additive effect of disease modifying factors. In this study, we performed WGS on a typical Chinese MEN 2A proband and identified the pathogenic RET p.C634R mutation. We also identified several neutral variants within RET and pheochromocytoma-related genes. Moreover, we found several interesting structural variants including genetic deletions (RSPO1, OVCH2 and AP3S1, etc.) and fusion transcripts (FSIP1-BAZ2A, etc.).

[Du Zhen-Fang, Li Peng-Fei, Zhao Jian-Qiang, Cao Zhi-Lie, Li Feng, Ma Ju-Ming, Qi Xiao-Ping 2017 Genetic diagnosis of a Chinese multiple endocrine neoplasia type 2A family through whole genome sequencing. J. Biosci. 42 209–218]

1. Introduction Takahashi et al. 1985). Based on its clinical characteristics, MEN 2 are classiﬁed into two subtypes: MEN 2A Multiple endocrine neoplasia type 2 (MEN2, OMIM (OMIM:171400) and MEN 2B (OMIM:162300). MEN2A 171400) is an autosomal-dominant multi-glandular cancer accounts for 95% of MEN 2 cases and has four variants: syndrome due to germline mutations of RET (REarranged classical MEN2A, which is characterized by medullary during Transfection) proto-oncogene (Mulligan et al. 1993; thyroid carcinoma (MTC, *95%), pheochromocytoma

Keywords. Multiple endocrine neoplasia type 2A; polymorphisms; RET proto-oncogene; whole genome sequencing

Supplementary materials pertaining to this article are available on the Journal of Biosciences Website. http://www.ias.ac.in/jbiosci J. Biosci. 42(2), June 2017, 209–218 Ó Indian Academy of Sciences 209 DOI: 10.1007/s12038-017-9686-5 Published online: 11 May 2017 210 Zhen-Fang Du et al.

(PHEO, *50%) and primary hyperparathyroidism (PHPT, phenotypes than their parental, such as incomplete pene- *25%); MEN2A with cutaneous lichen amyloidosis (CLA), trance of PHEO or CLA in p.C634Y/R (Machens and Dralle, MEN2A with Hirschsprung’s disease (HSCR), and familial 2015). The same RET mutations cause MEN 2A with dif- MTC (FMTC, OMIM:155240) (Wells et al. 2015). FMTC is ferent penetrance of HSCR (Mulligan et al. 1994; Mulligan defined as families or individuals with RET germline and Ponder Mulligan and Ponder 1995). The double muta- mutations who have MTC but neither PHEOs nor PHPT tions seem to be associated with higher risk of MEN 2A (Eng et al. 1996). MEN 2B accounts for\5% of all MEN 2 progress than mutation alone, indicating the modifying cases and presents a highly aggressive MTC, PHEO, gan- effects of neutral variants within RET (Qi et al. 2011; Toledo glioneuromatosis, and mucosal neuroma since infancy et al. 2010). Recent studies indicated that mutations in (Wells et al. 2015). The estimated incidence of the MEN 2 promoter region were associated with disease development, syndromes is *1/30,000 in the general population and 1 to indicating the necessity to extend the genetic screening to the 3 per 100 in all thyroid malignant tumors (American Thyroid non-coding regions (Ceccarelli et al. 2016; Weedon et al. Association Guidelines Task et al. 2009). 2014). Moreover, it has been demonstrated that germline Classical MEN 2A is the most common MEN 2A variant copy number variants (CNVs) at other loci contribute to a and 95% of patients carry RET germline mutations in codons greater predisposition to the development of lymph node 609, 611, 618, or 620 of exon 10 or codon 634 of exon 11. metastases in a MEN 2A family with RET p.G533C muta- Virtually all patients develop MTC, while RET codon-634 tion (Araujo et al. 2014). Therefore, it may occur by the mutations are associated with a high penetrance of PHEO or interaction of other modifying factors such as RET poly- PHPT, but RET mutations in exon 10 with a much lower morphisms or a second mutation which defined different penetrance (Frank-Raue et al. 2010; Imai et al. 2013). The expressions of the same RET mutation (Araujo et al. 2014; presence of CLA was initially observed specifically in MEN Siqueira et al. 2014). Therefore, genetic screening on the 2A patients with RET codon-634 mutations, which was whole region of RET and beyond RET proto-oncogene is approximately 9% (18/199) (Eng et al. 1996). It has been highly necessary. also reported in a patient with a codon-804 mutation (Cec- In the last few years, next-generation sequencing is cherini et al. 1994; Rothberg et al. 2009). The RET muta- evolving rapidly, including whole genome sequencing tions in patients with MEN 2A and HSCR are point (WGS) and whole exome sequencing and targeted capture mutations involving codons in exon 10, which occurs in combined with next generation sequencing. WGS could approximately 7% (Verdy et al. 1982; Decker and Peacock potentially provide a rapid and comprehensive diagnostic 1998). solution (Pankhurst et al. 2016). WGS greatly expands the Currently, genetic screening for germline RET mutations breadth of testing from genes associated with a particular is a routine procedure for diagnosis of MEN 2 patients for disease to the whole genome and, potentially, all the infor- MTC management (Force et al. 2009). Pre-symptomatic mation that the genome contains about diseases or traits identification of pathogenic RET mutations has a great (Thomas et al. 2015). In addition, the accuracy has been impact on management including the timing of standard or highly improved, and the cost and time spending on of WGS prophylactic thyroidectomy or dissection of cervical lymph has been reduced dramatically in recent years (Yu et al. nodes, which significantly improve the prognosis (Force 2012). In this study, we investigated a Chinese MEN 2A et al. 2009;Qiet al. 2013). Equally important, RET testing pedigree by performing WGS on the proband. We paid our spares mutation non-carriers the anxiety of later onset can- primary attention on RET gene including its coding and non- cer. Recently, the timing and extent of surgery were decided coding region. Meanwhile, we attempted to find additional by the type of RET mutation and serum pre-calcitonin (Ct) germline genetic modifiers which could explain the diversity levels, recommended by both the 2015 American Thyroid of MEN 2A phenotype. Association (ATA-MOD, H, HST), the 2012 European (Level-1, 2, 3) recommendations and local practice, which can maximally improve disease-free survival and overall 2. Subjects and methods survival of MTC (Elisei et al. 2013; Wells et al. 2015). However, current data also indicates that a few families have 2.1 Subjects been detected negative for all known RET mutations, pre- dicting the predisposition that MEN 2 may be caused by We investigated a Chinese three-generation MEN 2A kin- mutations at other loci (Sarika et al. 2015). Interestingly, the dred including 14 members (figure 1A). The total available presence of phenotypic diversity features associated with the 11 family members followed the complete clinical and specific RET mutation (Eng et al. 1996). The same RET genetic diagnostic testing according to the published criteria mutation have been found in both MEN 2A or FMTC, or the of the American Thyroid Association (2009; Ferris et al. offspring with RET mutation can appear earlier complex 2015). Basal serum calcitonin (Ct), carcinoembryonic

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Figure 1. (A) Genealogy of the Chinese MEN 2A families investigated in this study. (B) Distribution of sequence depth. (C) The accumulative sequencing depth. antigen (CEA), parathyroid hormone, metanephrine/norme- genomic DNA of the proband (II-1; figure 1A) was tanephrine, and/or 24-h urinary catecholamine levels neck sequenced on a HiSeq2000 platform (Illumina) with at and abdomen Doppler ultrasound (US) computed tomogra- least 40-fold coverage. Illumina Pipeline v1.6 was used to phy (CT) were also performed (Qi et al. 2013). Diagnosis of conduct the raw image analysis and base calling with MTC or C cell hyperplasia was confirmed by histopathol- default parameters. Data Analysis Paired-end reads of ogy. Tumor staging was performed according to American sequencing data against the reference genome hg19 Joint Committee on Cancer (AJCC) 7th edition tumor-node- (GRCh 37) was aligned using the SOAP2 aligner metastasis (TNM) classification system (Edge and Compton, (Soap2.21) (Li et al. 2009b). The SNPs were filtered out 2010). This study was conducted according to the Helsinki with dbSNP132, 1000 Genome Project and HapMap. Declaration and approved by the Ethics Committee of the Small indels (short insertions and deletions) were detected 117th PLA Hospital. After signed informed consent, Geno- by SAMtools using its pileup command (http://samtools. mic DNA was extracted from peripheral blood samples by sourceforge.net/)(Li2011;Liet al. 2009a). We used QIAmp DNA Blood mini kit (Qiagen #51104) and then used Breakdancer (http://breakdancer.sourceforge.net/)toanal- for WGS and/or Sanger sequencing. Then, follow-up was yse structural variants (Chen et al. 2009). The function- carried out. ality of single-nucleotide variants were predicted by All procedures performed in studies involving human PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/)(Ra- participants were in accordance with the ethical standards of mensky et al. 2002) and SIFT (http://sift.jcvi.org/)(Ng the institutional and/or national research committee and with and Henikoff 2002). the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. 2.3 Variants confirmed by Sanger sequencing Informed consent was obtained from all individual participants included in the study. The genomic DNA samples from all the available family members were used for amplification of all the 21 exons in conjunction with the flanking splice boundaries, 50 and 30 2.2 Whole genome sequencing UTRs, as well as selected intronic regions of the RET gene. The primers used for PCR were listed in supplementary We performed WGS on the proband. The procedures have table 4. The PCR products were analysed by Sanger been described previously (Jiang et al. 2013). Briefly, sequencing.

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3. Results modified right neck dissection after diagnosis of bilateral thyroid hypoechogenic nodules with calcifications (right, 3.1 Subjects 2.391.891.3 cm; left, 0.890.690.6 cm) by US and CT. Histological examination showed bilateral multi-centric MTC In this family, 11 family members were available (fig- with lymph nodes metastatic (T2N1M0). In 2007, He under- ure 1A). Of them, 3 patients (II-1, II-7, III-4) were diag- went left thyroidectomy with modified left neck dissection nosed, with two patients underwent thyroidectomy (II-1, II- based on the evidence of elevated Ct and multi-centric nodules 7) and another underwent prophylactic thyroidectomy (III- with calcifications in residual left thyroid lobes, which were 4). The proband (II-1) was a 54-year-old male referred to us histologically diagnosed as MTC with lymph node metas- for the presence of a palpable, non-tender distinct thyroid tases. In 2012, hesitant members agreed to participate in bio- mass lateral to the left lobe of his cervix when he was 23 chemical testing, imaging, and RET screening. Six of the 9 years old in 1981. Then left thyroidectomy was performed. members exhibited normal Ct levels and US images but the Histological examination revealed left muti-centric MTC other 3 (II-1, II-7, and III-4) had the RET p.C634R mutation. (max size of 2.592.392.0 cm). In 1992, he presented with Unfortunately, the proband’s 32-year-old younger brother (II- headache, palpitation, hyperhidrosis, paroxysmal hyperten- 7) yet presented a right adrenal mass (2.9 9 2.3 91.7 cm) by sion (180/110 mmHg), and excess serum calcitonin. US and US and CT scanning. Further clinical evaluation including CT scanning confirmed masses in size of 10 cm in the left serum catecholamine level, 24 h ambulatory blood pressure adrenal glands. After treatment with a-blockers, he under- and electrocardiogram were all no abnormal. But basal serum went left adrenal gland tomorectomy, and left PHEO was calcitonin level still abnormal (104 ng/L). Then laparoscopic confirmed by histopathological examination. In 1997, he right adrenal tumourectomy with CO2 pneumoperitoneum underwent right total thyroidectomy with modified bilateral was performed. Histopathological examination revealed right neck dissection based on the evidence of elevated Ct and PHEO. multi-centric nodules (max size of 2.892.592.0 cm) with The index patient’s 18-year-old niece (III-4), exhibiting calcifications in right thyroid lobe. Histology revealed right slight elevated Ct level (24.3 ng/L) and a hypoechoic nodule multi-centric MTC with bilateral lymph nodes metastatic (0.4 9 0.3 9 0.3 cm) in left thyroid lobe, was subjected to (T2N1M0). He received L-T4 substitution therapy and was prophylactic total thyroidectomy. Histopathological exami- followed up. In 2002, he relapsed after initially developing a nation showed bilateral mulifocal MTC (T1N0M0) and post- contralateral PHEO (right, 3.5 cm) after 10 years, and also Ct levels was normal. The proband’s mother died of acute accepted tomorectomy on the opposite (right) adrenal gland. cerebral extraction of resistant hypertension at the age of 55 Histologically diagnosed as right PHEO. After surgery, years in 1988, and the proband’s father had no abnormality hypertensive episodes ceased normalize, the remaining (negative RET mutation and consistently undetectable Ct), it glands was still sufficient and he needn’t steroid replacement speculates that the proband’s mother was MEN2A patient therapy. In recent survey of this pedigree in 2012, the Ct with RET p.C634R mutation. level was 32.4 ng/L (normal for male: 8.4, female: 5.0) on the day after the surgery, and he presented with neck abnormalities by imaging. 3.2 Variants identified by WGS In 1998, the proband’s 32-year-old younger brother (II-7) was subjected to a left subtotal thyroidectomy with left level Our data showed that a total of approximate 1.8 billion clean VI lymph node dissection and right total thyroidectomy with reads with an average length of 90 bp comprising

Table 1. Variants in coding region of MEN2A-related genes identiﬁed by WGS

Gene Effect cDNA alteration AA alteration 1000 db dbSNP SIFT PolyPhen2

RET synonymous SNV c.135A[G p.A45A 0.74 rs1800858 NA NA RET synonymous SNV c.1296A[G p.A432A 0.81 rs1800860 NA NA RET nonsynonymous SNV c.1900T[C p.C634R NA rs75076352 0 0.999 RET synonymous SNV c.2037C[T p.P679P 0.003 rs55862116 NA NA RET nonsynonymous SNV c.2071G[A p.G691S 0.16 rs1799939 0.62 0.373 RET synonymous SNV c.2307G[T p.L769L 0.72 rs1800861 NA NA RET synonymous SNV c.2712C[G p.S904S 0.14 rs1800863 NA NA NF1 synonymous SNV c.2034G[A p.P678P 0.48 rs2285892 NA NA SDHB synonymous SNV c.18C[A p.A6A 0.93 rs2746462 NA NA SDHA synonymous SNV c.891T[C p.P297P 0.59 rs1126417 NA NA

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Table 2. Genetic deletions identiﬁed by WGS

Gene Function Chr. Begin position End position Type

RSPO1 splicing 1 38077350 38077420 deletion OVCH2 exonic 11 7716916 7716916 deletion AP3S1 exonic; splicing 5 115238688 115249056 deletion

Table 3. Fusion genes identiﬁed by WGS

Begin position Gene End position Gene Function chr21: 11022748 BAGE5 chr22: 45991517 FBLN1 Translocation chr21: 11097543 BAGE chr7: 152099216 KMT2C Translocation chr15: 40854194 C15orf57 chr7: 26252971 CBX3 Translocation chr15: 39994624 FSIP1 chr12: 56990100 BAZ2A Insertion chr15: 39994637 FSIP1 chr12: 56989716 BAZ2A Insertion chr15: 68687991 ITGA11 chrX: 13744217 TRAPPC2 Translocation chr15: 22373078 LOC101927079 chr15: 22383653 OR4N4 Deleltion chr6: 43655533 MRPS18A chr9: 33130549 B4GALT1 Translocation chr1: 145092947 NBPF9 chr1: 145097083 SEC22B Deletion chr1: 220436373 RAB3GAP2 chr1: 220440886 AURKAPS1 Deletion/insertion chr1: 121138760 SRGAP2-AS1 chr1: 148855317 NBPF25P Inversion chr1: 121138286 SRGAP2-AS1 chr1: 148855838 NBPF25P Inversion chr19: 4962159 UHRF1 chr8: 52703374 PXDNL Translocation chr19: 21268377 ZNF714 chr19: 21327838 ZNF431 Deletion approximate 160 billion bases of sequence were generated 3.3 Variants confirmed by Sanger sequencing with a 47-fold average coverage sequencing depth on whole genome. More than 99% of target region in simulation Firstly, all variants within coding-region of RET identified by results was supported by at least four reads sufficiently for WGS were verified by Sanger sequencing. The most com- variant calling (supplementary table 1). Sequencing revealed mon mutation p.C634R and other SNPs within coding a highly sequencing distribution and depth consisting of region of RET were validated by Sanger sequencing. RET single nucleotide polymorphisms (SNPs), copy number p.C634R (c.1900T[C) also appeared in II-7 and III-4, with variants (CNVs) and structure variants (SVs) (figure 1B and other members absent of p.C634R (figure 2). We also 1C, supplementary table 2). focused on the polymorphisms in coding-region of RET In WGS data from the proband, we firstly focused on gene, and confirmed SNPs including c.A135G (p.A45A) in variants in RET gene. We found RET p.C634R (c.1900T[C) exon2, c.1296A[G (p.A432A) in exon7, c.2037C[T mutation in the proband (table 1). We also found 106 vari- (p.P679P) in exon11, c.2071G[A (p.G691S) in exon11, ants within intronic region of RET gene (supplementary c.2307G[T (p.L739L) in exon 13, c.2712C[G (p.S904S) in table 2). Secondly, previous studies indicate that more than exon15 (table 4). Secondly, we confirmed 4 intronic variants 30% of PHEO cases carry germline mutations in a growing of RET gene which were not annotated by 1000 Genome list of susceptibility genes, which are involved in diverse but Project (table 5). Sanger sequencing in additional family interconnecting pathways (Dahia 2014). Recently, rare members indicated that these variants did not co-segregated germline mutations have been identified by targeted next- with any clinical characteristics of MEN 2A in this family generation sequencing of susceptibility genes in PHEO (Data not shown). The primers used for Sanger sequencing (Dahia 2014). Considering the presence of PHEO in this were listed in supplementary table 4. pedigree, we then analysed additional 9 PHEO-related genes. Within the 10 PHEO-related genes, non-synonymus SNP were only found in RET gene (table 1 and supple- 4. Discussion mentary table 2). Thirdly, we identified three interesting genetic deletion in RSPO1, OVCH2 and AP3S1 (table 2 and In this study, through WGS, we identified a common RET supplementary table 3). Fourthly, we also identified several p.C634R (c.1900T[C) mutation in a Chinese MEN 2A chromosome rearrangements, including FSIP1-BAZ2A and family. We also found several exonic polymorphisms and BAGE5-FBLN1 (table 3). intronic alterations within RET proto-oncogene. Moreover,

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Figure 2. RET p.C634R (c.1900T[C) was conﬁrmed by Sanger sequencing in the proband. (A) Wild-type. (B) RET c.1900T[C (p.C634R).

Table 4. Variants within coding region of RET gene conﬁrmed in this study

No. II-1 II-2 II-3 II-5 II-6 II-7 II-8 III-1 III-2 III-3 III-4

Exon 2 p.A45A p.A45A p.A45A – p.A45A p.A45A p.A45A p.A45A – p.A45A p.A45A Exon 7 p.A432A – – – p.D489N p.A432A – – – p.A432A; – p.D489N Exon 11 p.C634R; – p.P679P; – – p.C634R – p.P679P; – – p.C634R p.P679P; p.G691S p.G691S p.G691S Exon 12 – – – – – – – – – – – Exon 13 p.L769L p.L769L p.L769L – p.L769L – p.L769L p.L769L – – – Exon 15 p.S904S – p.S904S – – – – p.S904S – –

we found several structural variants including RSPO1 dele- mutation account for the majority of codon-634 mutations tion and FSIP1-BAZ2A fusion gene. associated with MEN 2A (Zhou et al. 2007). Activating There is a strong correlation between RET mutations mutations also in codon 634 are rarely involved in and speciﬁc phenotypes of MEN 2 (Eng et al. 1996). In MEN2A families with CLA (Force et al. 2009;Qiet al. 98% of families with MEN 2A, molecular genetic testing 2015). In this study, RET p.C634R (c.1900T[C) mutation of the RET gene reveals that disease-causative germline exhibited a typical MEN2A phenotype consistent with mutations cluster to the extracellular cysteine-rich domain previously reported Chinese families (Qi et al. 2012; at codons 609, 611, 618, 620, 630, and 634. Approxi- Zhou et al. 2007). mately 63% carried an abnormal RET 634-codon muta- In the last decade, several studies have focused on the tions while 9.2%, 6.8%, and 2.9% for codons 618, 620 potential role of neutral RET sequence variants in modify- and 790, respectively (Machens and Dralle, 2015). The ing the clinical course of MEN 2-related MTC. Although majority of PHEO cases cluster with exon 11 mutations, controversial, an increased prevalence of the RET poly- speciﬁcally with the 634-codon mutations. RET p.C634R morphisms p.G691S, p.L769L, p.S836S, and p.S904S has

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Table 5. Variants within RET non-coding regions identified by Kim et al. 2016; Chang et al. 2017). In this study, we found WGS and verified by Sanger sequencing a 71-bp deletion within RSPO1 promoter region. RSPO1 was demonstrated to be a strong potentiator of Wnt signaling No. Region Chr_start Chr_end Alteration (Hao et al. 2012). RSPO2, another family member of RSPO 1 Intron 1 43587077 43587077 –[A family, was reported to be a tumor suppressor gene inacti- 2 Intron 1 43595836 43595836 –[T vated by promoter CpG methylation in colorectal cancer 3 Intron 5 43603007 43603007 –[CA (Wu et al. 2014). RSPO proteins regulate Wnt signaling by a [ 4 Intron 15 43616380 43616382 AAG– common mechanism (Kim et al. 2008). Thus, this 71-bp deletion possibly impaired the transcription regulatory machine and subsequently implicated in the development of been described in individuals with MTC (Gimm et al. MEN 2A. Although RET mutations are important to deter- 1999; Robledo et al. 2003;Eliseiet al. 2004;Rochaet al. mine cancer risk, additional germline CNVs in MEN 2A- 2007;Siqueiraet al. 2010). Other studies have shown that affected individuals may predispose to MTC aggressiveness these variants could interfere in disease presentation (Araujo et al. 2014). Meanwhile, germline fusion genes and (Robledo et al. 2003;Eliseiet al. 2004; Magalhaes et al. chromosomal rearrangements have been reported to involve 2004; Siqueira et al. 2010;Ceolinet al. 2012b). Interest- in the pathogenesis of monogenic and complex diseases ingly, recent findings suggest a potential additive effect of (Trevino et al. 2009; van Heesch et al. 2014; Grunewald the different RET variants on the susceptibility and clinical et al. 2015). Somatically, it has been confirmed that RET course of sporadic MTC. Individuals harboring haplotypes fusion is a novel driver of sporadic MTC (Grubbs et al. with three or more RET polymorphic alleles have higher 2015). EML4-ALK fusion possibly drives the development risks for MTC development and lymph node and distant of sporadic MTC (Ji et al. 2015). In this study, we identified metastases (Ceolin et al. 2012a). Also, individuals harbored several fusion genes including FSIP1-BAZ2A. Recently, it is two RET genetic variants presented with an age-related reported that FSIP1 may be involved in the initiation and increased risk for developing PHEO. RET p.C634Y/Y791F invasion of breast cancer (Zhang et al. 2015). BAZ2A was double mutation carries a codon 634-like pattern of MTC involved in epigenetic alterations and its overexpression development, while it is associated with increased suscep- served as a useful marker for metastatic potential in prostate tibility to unusually large bilateral PHEO (Toledo et al. cancer (Gu et al. 2015). Our study highlights the importance 2015;Toledoet al. 2010). There is also report indicating of using WGS to identify additional genetic modifiers that p.G691S variant allele does contribute to the MTC associated with MEN 2A. occurrence (Lantieri et al. 2013). Meanwhile, it has been As the cost of sequencing the human genome falls, demonstrated that germline intronic variants are associated medical use of whole-genome sequencing will rapidly with different types of cancers (Liu et al. 2004;Pagen- advance (Ashley et al. 2010). WGS greatly expands the stecher et al. 2006). Recently, polymorphisms within non- breadth of testing from genes associated with a particular coding region of RET have been reported to associate with disease to the whole genome and, potentially, all the Hirschsprung disease (Gunadi et al. 2014). Meanwhile, two information that the genome contains about diseases or intronic RET variants were associated with the risk of traits (Ormond et al. 2010). Recently, WGS has been suc- developing sporadic MTC and its aggressiveness (Fugaz- cessful applied in the genetic diagnosis of monogenic dis- zola et al. 2008). In this study, we found many polymor- eases (Ashley et al. 2010; Talkowski et al. 2012). In this phisms both in coding and non-coding regions of RET.The study, by using WGS, we identified the pathogenic RET neutral variants did not co-segregate with the clinical p.C634R (c.1900T[C) mutation in the proband of a Chi- characteristics of MEN 2A in this family, so we excluded nese MEN2A family. We also found variants within the their causative effects in the pathogenesis of MEN 2A. The non-coding region of RET and PHEO-related genes. association between these neutral variants and clinical Besides, we identified three interesting genetic deletions features of MEN 2A remains elusive as the sample size was which were possibly involved in the pathogenesis of MEN limited. Together with previous studies, our report high- 2A. Although challenges exist, WGS could be an instruc- light the importance of further investigation in large cohort tive, robust and time-effective tool in the genetic diagnosis on the identification of variants located in the non-coding of monogenic diseases. regions of RET and also their association with the development of MEN 2A. Non-coding regulatory mutations have been reported to Acknowledgements associate with a variety of cancers including thyroid cancer (Horn et al. 2013; Chen et al. 2014; Weinhold et al. 2014;de The authors thank all the patients and their families who Biase et al. 2015; Diederichs et al. 2016;Yinet al. 2016; agreed to participate in this study. This work was supported

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