Genetic diagnosis of a Chinese multiple endocrine neoplasia type 2A family through whole genome sequencing ZHEN-FANG DU1, , PENG-FEI LI2, , JIAN-QIANG ZHAO3, , ZHI-LIE CAO1, FENG LI1, JU-MING MA1 and XIAO-PING QI1,* 1

Department 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 2 3

XY Biotechnology Co. Ltd, 688 Bin’an Road, Hangzhou 310051, Zhejiang Province, China

Department 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 Multiple endocrine neoplasia type 2 (MEN2, OMIM 171400) is an autosomal-dominant multi-glandular cancer syndrome due to germline mutations of RET (REarranged during Transfection) proto-oncogene (Mulligan et al. 1993; Keywords.

Takahashi et al. 1985). Based on its clinical characteristics, MEN 2 are classified into two subtypes: MEN 2A (OMIM:171400) and MEN 2B (OMIM:162300). MEN2A accounts for 95% of MEN 2 cases and has four variants: classical MEN2A, which is characterized by medullary thyroid carcinoma (MTC, *95%), pheochromocytoma

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 Published online: 11 May 2017

J. Biosci. 42(2), June 2017, 209–218 Ó Indian Academy of Sciences DOI: 10.1007/s12038-017-9686-5

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(PHEO, *50%) and primary hyperparathyroidism (PHPT, *25%); MEN2A with cutaneous lichen amyloidosis (CLA), MEN2A with Hirschsprung’s disease (HSCR), and familial MTC (FMTC, OMIM:155240) (Wells et al. 2015). FMTC is defined as families or individuals with RET germline mutations who have MTC but neither PHEOs nor PHPT (Eng et al. 1996). MEN 2B accounts for \5% of all MEN 2 cases and presents a highly aggressive MTC, PHEO, ganglioneuromatosis, and mucosal neuroma since infancy (Wells et al. 2015). The estimated incidence of the MEN 2 syndromes is *1/30,000 in the general population and 1 to 3 per 100 in all thyroid malignant tumors (American Thyroid Association Guidelines Task et al. 2009). Classical MEN 2A is the most common MEN 2A variant and 95% of patients carry RET germline mutations in codons 609, 611, 618, or 620 of exon 10 or codon 634 of exon 11. Virtually all patients develop MTC, while RET codon-634 mutations are associated with a high penetrance of PHEO or PHPT, but RET mutations in exon 10 with a much lower penetrance (Frank-Raue et al. 2010; Imai et al. 2013). The presence of CLA was initially observed specifically in MEN 2A patients with RET codon-634 mutations, which was approximately 9% (18/199) (Eng et al. 1996). It has been also reported in a patient with a codon-804 mutation (Ceccherini et al. 1994; Rothberg et al. 2009). The RET mutations in patients with MEN 2A and HSCR are point mutations involving codons in exon 10, which occurs in approximately 7% (Verdy et al. 1982; Decker and Peacock 1998). Currently, genetic screening for germline RET mutations is a routine procedure for diagnosis of MEN 2 patients for MTC management (Force et al. 2009). Pre-symptomatic identification of pathogenic RET mutations has a great impact on management including the timing of standard or prophylactic thyroidectomy or dissection of cervical lymph nodes, which significantly improve the prognosis (Force et al. 2009; Qi et al. 2013). Equally important, RET testing spares mutation non-carriers the anxiety of later onset cancer. Recently, the timing and extent of surgery were decided by the type of RET mutation and serum pre-calcitonin (Ct) levels, recommended by both the 2015 American Thyroid 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 survival of MTC (Elisei et al. 2013; Wells et al. 2015). However, current data also indicates that a few families have been detected negative for all known RET mutations, predicting the predisposition that MEN 2 may be caused by mutations at other loci (Sarika et al. 2015). Interestingly, the presence of phenotypic diversity features associated with the specific RET mutation (Eng et al. 1996). The same RET mutation have been found in both MEN 2A or FMTC, or the offspring with RET mutation can appear earlier complex J. Biosci. 42(2), June 2017

phenotypes than their parental, such as incomplete penetrance of PHEO or CLA in p.C634Y/R (Machens and Dralle, 2015). The same RET mutations cause MEN 2A with different penetrance of HSCR (Mulligan et al. 1994; Mulligan and Ponder Mulligan and Ponder 1995). The double mutations seem to be associated with higher risk of MEN 2A progress than mutation alone, indicating the modifying effects of neutral variants within RET (Qi et al. 2011; Toledo et al. 2010). Recent studies indicated that mutations in promoter region were associated with disease development, indicating the necessity to extend the genetic screening to the non-coding regions (Ceccarelli et al. 2016; Weedon et al. 2014). Moreover, it has been demonstrated that germline copy number variants (CNVs) at other loci contribute to a greater predisposition to the development of lymph node metastases in a MEN 2A family with RET p.G533C mutation (Araujo et al. 2014). Therefore, it may occur by the interaction of other modifying factors such as RET polymorphisms or a second mutation which defined different expressions of the same RET mutation (Araujo et al. 2014; Siqueira et al. 2014). Therefore, genetic screening on the whole region of RET and beyond RET proto-oncogene is highly necessary. In the last few years, next-generation sequencing is evolving rapidly, including whole genome sequencing (WGS) and whole exome sequencing and targeted capture combined with next generation sequencing. WGS could potentially provide a rapid and comprehensive diagnostic solution (Pankhurst et al. 2016). WGS greatly expands the breadth of testing from genes associated with a particular disease to the whole genome and, potentially, all the information that the genome contains about diseases or traits (Thomas et al. 2015). In addition, the accuracy has been highly improved, and the cost and time spending on of WGS has been reduced dramatically in recent years (Yu et al. 2012). In this study, we investigated a Chinese MEN 2A pedigree by performing WGS on the proband. We paid our primary attention on RET gene including its coding and noncoding region. Meanwhile, we attempted to find additional germline genetic modifiers which could explain the diversity of MEN 2A phenotype.

2. Subjects and methods 2.1

Subjects

We investigated a Chinese three-generation MEN 2A kindred including 14 members (figure 1A). The total available 11 family members followed the complete clinical and genetic diagnostic testing according to the published criteria of the American Thyroid Association (2009; Ferris et al. 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/normetanephrine, and/or 24-h urinary catecholamine levels neck and abdomen Doppler ultrasound (US) computed tomography (CT) were also performed (Qi et al. 2013). Diagnosis of MTC or C cell hyperplasia was confirmed by histopathology. Tumor staging was performed according to American Joint Committee on Cancer (AJCC) 7th edition tumor-nodemetastasis (TNM) classification system (Edge and Compton, 2010). This study was conducted according to the Helsinki Declaration and approved by the Ethics Committee of the 117th PLA Hospital. After signed informed consent, Genomic DNA was extracted from peripheral blood samples by QIAmp DNA Blood mini kit (Qiagen #51104) and then used for WGS and/or Sanger sequencing. Then, follow-up was carried out. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with 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. Informed consent was obtained from all individual participants included in the study.

2.2

Whole genome sequencing

We performed WGS on the proband. The procedures have been described previously (Jiang et al. 2013). Briefly,

genomic DNA of the proband (II-1; figure 1A) was sequenced on a HiSeq2000 platform (Illumina) with at least 40-fold coverage. Illumina Pipeline v1.6 was used to conduct the raw image analysis and base calling with default parameters. Data Analysis Paired-end reads of sequencing data against the reference genome hg19 (GRCh 37) was aligned using the SOAP2 aligner (Soap2.21) (Li et al. 2009b). The SNPs were filtered out with dbSNP132, 1000 Genome Project and HapMap. Small indels (short insertions and deletions) were detected by SAMtools using its pileup command (http://samtools. sourceforge.net/) (Li 2011; Li et al. 2009a). We used Breakdancer (http://breakdancer.sourceforge.net/) to analyse structural variants (Chen et al. 2009). The functionality of single-nucleotide variants were predicted by PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) (Ramensky et al. 2002) and SIFT (http://sift.jcvi.org/) (Ng and Henikoff 2002).

2.3 Variants confirmed by Sanger sequencing 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 UTRs, as well as selected intronic regions of the RET gene. The primers used for PCR were listed in supplementary table 4. The PCR products were analysed by Sanger sequencing.

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Subjects

In this family, 11 family members were available (figure 1A). Of them, 3 patients (II-1, II-7, III-4) were diagnosed, with two patients underwent thyroidectomy (II-1, II7) and another underwent prophylactic thyroidectomy (III4). The proband (II-1) was a 54-year-old male referred to us for the presence of a palpable, non-tender distinct thyroid mass lateral to the left lobe of his cervix when he was 23 years old in 1981. Then left thyroidectomy was performed. Histological examination revealed left muti-centric MTC (max size of 2.592.392.0 cm). In 1992, he presented with headache, palpitation, hyperhidrosis, paroxysmal hypertension (180/110 mmHg), and excess serum calcitonin. US and CT scanning confirmed masses in size of 10 cm in the left adrenal glands. After treatment with a-blockers, he underwent left adrenal gland tomorectomy, and left PHEO was confirmed by histopathological examination. In 1997, he underwent right total thyroidectomy with modified bilateral neck dissection based on the evidence of elevated Ct and multi-centric nodules (max size of 2.892.592.0 cm) with calcifications in right thyroid lobe. Histology revealed right multi-centric MTC with bilateral lymph nodes metastatic (T2N1M0). He received L-T4 substitution therapy and was followed up. In 2002, he relapsed after initially developing a contralateral PHEO (right, 3.5 cm) after 10 years, and also accepted tomorectomy on the opposite (right) adrenal gland. Histologically diagnosed as right PHEO. After surgery, hypertensive episodes ceased normalize, the remaining glands was still sufficient and he needn’t steroid replacement therapy. In recent survey of this pedigree in 2012, the Ct 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. In 1998, the proband’s 32-year-old younger brother (II-7) was subjected to a left subtotal thyroidectomy with left level VI lymph node dissection and right total thyroidectomy with

modified right neck dissection after diagnosis of bilateral thyroid hypoechogenic nodules with calcifications (right, 2.391.891.3 cm; left, 0.890.690.6 cm) by US and CT. Histological examination showed bilateral multi-centric MTC with lymph nodes metastatic (T2N1M0). In 2007, He underwent left thyroidectomy with modified left neck dissection based on the evidence of elevated Ct and multi-centric nodules with calcifications in residual left thyroid lobes, which were histologically diagnosed as MTC with lymph node metastases. In 2012, hesitant members agreed to participate in biochemical testing, imaging, and RET screening. Six of the 9 members exhibited normal Ct levels and US images but the other 3 (II-1, II-7, and III-4) had the RET p.C634R mutation. Unfortunately, the proband’s 32-year-old younger brother (II7) yet presented a right adrenal mass (2.9 9 2.3 91.7 cm) by US and CT scanning. Further clinical evaluation including serum catecholamine level, 24 h ambulatory blood pressure and electrocardiogram were all no abnormal. But basal serum calcitonin level still abnormal (104 ng/L). Then laparoscopic right adrenal tumourectomy with CO2 pneumoperitoneum was performed. Histopathological examination revealed right PHEO. The index patient’s 18-year-old niece (III-4), exhibiting slight elevated Ct level (24.3 ng/L) and a hypoechoic nodule (0.4 9 0.3 9 0.3 cm) in left thyroid lobe, was subjected to prophylactic total thyroidectomy. Histopathological examination showed bilateral mulifocal MTC (T1N0M0) and postCt levels was normal. The proband’s mother died of acute cerebral extraction of resistant hypertension at the age of 55 years in 1988, and the proband’s father had no abnormality (negative RET mutation and consistently undetectable Ct), it speculates that the proband’s mother was MEN2A patient with RET p.C634R mutation.

3.2

Variants identified by WGS

Our data showed that a total of approximate 1.8 billion clean 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 RET RET RET RET RET RET NF1 SDHB SDHA

synonymous SNV synonymous SNV nonsynonymous SNV synonymous SNV nonsynonymous SNV synonymous SNV synonymous SNV synonymous SNV synonymous SNV synonymous SNV

c.135A[G c.1296A[G c.1900T[C c.2037C[T c.2071G[A c.2307G[T c.2712C[G c.2034G[A c.18C[A c.891T[C

p.A45A p.A432A p.C634R p.P679P p.G691S p.L769L p.S904S p.P678P p.A6A p.P297P

0.74 0.81 NA 0.003 0.16 0.72 0.14 0.48 0.93 0.59

rs1800858 rs1800860 rs75076352 rs55862116 rs1799939 rs1800861 rs1800863 rs2285892 rs2746462 rs1126417

NA NA 0 NA 0.62 NA NA NA NA NA

NA NA 0.999 NA 0.373 NA NA NA NA NA

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Table 2. Genetic deletions identified by WGS Gene

Function

Chr.

Begin position

End position

Type

RSPO1 OVCH2 AP3S1

splicing exonic exonic; splicing

1 11 5

38077350 7716916 115238688

38077420 7716916 115249056

deletion deletion deletion

Table 3. Fusion genes identified by WGS Begin position

Gene

End position

Gene

Function

chr21: 11022748 chr21: 11097543 chr15: 40854194 chr15: 39994624 chr15: 39994637 chr15: 68687991 chr15: 22373078 chr6: 43655533 chr1: 145092947 chr1: 220436373 chr1: 121138760 chr1: 121138286 chr19: 4962159 chr19: 21268377

BAGE5 BAGE C15orf57 FSIP1 FSIP1 ITGA11 LOC101927079 MRPS18A NBPF9 RAB3GAP2 SRGAP2-AS1 SRGAP2-AS1 UHRF1 ZNF714

chr22: 45991517 chr7: 152099216 chr7: 26252971 chr12: 56990100 chr12: 56989716 chrX: 13744217 chr15: 22383653 chr9: 33130549 chr1: 145097083 chr1: 220440886 chr1: 148855317 chr1: 148855838 chr8: 52703374 chr19: 21327838

FBLN1 KMT2C CBX3 BAZ2A BAZ2A TRAPPC2 OR4N4 B4GALT1 SEC22B AURKAPS1 NBPF25P NBPF25P PXDNL ZNF431

Translocation Translocation Translocation Insertion Insertion Translocation Deleltion Translocation Deletion Deletion/insertion Inversion Inversion Translocation Deletion

approximate 160 billion bases of sequence were generated with a 47-fold average coverage sequencing depth on whole genome. More than 99% of target region in simulation results was supported by at least four reads sufficiently for variant calling (supplementary table 1). Sequencing revealed a highly sequencing distribution and depth consisting of single nucleotide polymorphisms (SNPs), copy number variants (CNVs) and structure variants (SVs) (figure 1B and 1C, supplementary table 2). In WGS data from the proband, we firstly focused on variants in RET gene. We found RET p.C634R (c.1900T[C) mutation in the proband (table 1). We also found 106 variants within intronic region of RET gene (supplementary table 2). Secondly, previous studies indicate that more than 30% of PHEO cases carry germline mutations in a growing list of susceptibility genes, which are involved in diverse but interconnecting pathways (Dahia 2014). Recently, rare germline mutations have been identified by targeted nextgeneration sequencing of susceptibility genes in PHEO (Dahia 2014). Considering the presence of PHEO in this 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 supplementary table 2). Thirdly, we identified three interesting genetic deletion in RSPO1, OVCH2 and AP3S1 (table 2 and supplementary table 3). Fourthly, we also identified several chromosome rearrangements, including FSIP1-BAZ2A and BAGE5-FBLN1 (table 3).

3.3 Variants confirmed by Sanger sequencing Firstly, all variants within coding-region of RET identified by WGS were verified by Sanger sequencing. The most common mutation p.C634R and other SNPs within coding region of RET were validated by Sanger sequencing. RET p.C634R (c.1900T[C) also appeared in II-7 and III-4, with other members absent of p.C634R (figure 2). We also focused on the polymorphisms in coding-region of RET gene, and confirmed SNPs including c.A135G (p.A45A) in exon2, c.1296A[G (p.A432A) in exon7, c.2037C[T (p.P679P) in exon11, c.2071G[A (p.G691S) in exon11, c.2307G[T (p.L739L) in exon 13, c.2712C[G (p.S904S) in exon15 (table 4). Secondly, we confirmed 4 intronic variants of RET gene which were not annotated by 1000 Genome Project (table 5). Sanger sequencing in additional family members indicated that these variants did not co-segregated with any clinical characteristics of MEN 2A in this family (Data not shown). The primers used for Sanger sequencing were listed in supplementary table 4.

4. Discussion In this study, through WGS, we identified a common RET p.C634R (c.1900T[C) mutation in a Chinese MEN 2A family. We also found several exonic polymorphisms and intronic alterations within RET proto-oncogene. Moreover, J. Biosci. 42(2), June 2017

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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

Exon 2 Exon 7

p.A45A p.A432A

p.A45A –

p.A45A –

– –

Exon 11

p.C634R; p.P679P; p.G691S – p.L769L p.S904S

–

p.P679P; p.G691S

–

–

– p.L769L –

– p.L769L p.S904S

– – –

– p.L769L –

Exon 12 Exon 13 Exon 15

II-6

II-8

III-1

III-2

p.A45A –

p.A45A –

– –

p.C634R

–

p.P679P; p.G691S

–

– – –

– p.L769L –

– p.L769L p.S904S

– – –

p.A45A p.A45A p.D489N p.A432A

we found several structural variants including RSPO1 deletion and FSIP1-BAZ2A fusion gene. There is a strong correlation between RET mutations and specific phenotypes of MEN 2 (Eng et al. 1996). In 98% of families with MEN 2A, molecular genetic testing of the RET gene reveals that disease-causative germline mutations cluster to the extracellular cysteine-rich domain at codons 609, 611, 618, 620, 630, and 634. Approximately 63% carried an abnormal RET 634-codon mutations while 9.2%, 6.8%, and 2.9% for codons 618, 620 and 790, respectively (Machens and Dralle, 2015). The majority of PHEO cases cluster with exon 11 mutations, specifically with the 634-codon mutations. RET p.C634R

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II-7

III-3

III-4

p.A45A p.A45A p.A432A; – p.D489N – p.C634R – – –

– –

mutation account for the majority of codon-634 mutations associated with MEN 2A (Zhou et al. 2007). Activating mutations also in codon 634 are rarely involved in MEN2A families with CLA (Force et al. 2009; Qi et al. 2015). In this study, RET p.C634R (c.1900T[C) mutation exhibited a typical MEN2A phenotype consistent with previously reported Chinese families (Qi et al. 2012; Zhou et al. 2007). In the last decade, several studies have focused on the potential role of neutral RET sequence variants in modifying the clinical course of MEN 2-related MTC. Although controversial, an increased prevalence of the RET polymorphisms p.G691S, p.L769L, p.S836S, and p.S904S has

Genetic diagnosis of MEN 2A by WGS Table 5. Variants within RET non-coding regions identified by WGS and verified by Sanger sequencing No.

Region

Chr_start

Chr_end

Alteration

1 2 3 4

Intron 1 Intron 1 Intron 5 Intron 15

43587077 43595836 43603007 43616380

43587077 43595836 43603007 43616382

–[A –[T –[CA AAG–[

been described in individuals with MTC (Gimm et al. 1999; Robledo et al. 2003; Elisei et al. 2004; Rocha et al. 2007; Siqueira et al. 2010). Other studies have shown that these variants could interfere in disease presentation (Robledo et al. 2003; Elisei et al. 2004; Magalhaes et al. 2004; Siqueira et al. 2010; Ceolin et al. 2012b). Interestingly, recent findings suggest a potential additive effect of the different RET variants on the susceptibility and clinical course of sporadic MTC. Individuals harboring haplotypes with three or more RET polymorphic alleles have higher risks for MTC development and lymph node and distant metastases (Ceolin et al. 2012a). Also, individuals harbored two RET genetic variants presented with an age-related increased risk for developing PHEO. RET p.C634Y/Y791F double mutation carries a codon 634-like pattern of MTC development, while it is associated with increased susceptibility to unusually large bilateral PHEO (Toledo et al. 2015; Toledo et al. 2010). There is also report indicating that p.G691S variant allele does contribute to the MTC occurrence (Lantieri et al. 2013). Meanwhile, it has been demonstrated that germline intronic variants are associated with different types of cancers (Liu et al. 2004; Pagenstecher et al. 2006). Recently, polymorphisms within noncoding region of RET have been reported to associate with Hirschsprung disease (Gunadi et al. 2014). Meanwhile, two intronic RET variants were associated with the risk of developing sporadic MTC and its aggressiveness (Fugazzola et al. 2008). In this study, we found many polymorphisms both in coding and non-coding regions of RET. The neutral variants did not co-segregate with the clinical characteristics of MEN 2A in this family, so we excluded their causative effects in the pathogenesis of MEN 2A. The association between these neutral variants and clinical features of MEN 2A remains elusive as the sample size was limited. Together with previous studies, our report highlight the importance of further investigation in large cohort on the identification of variants located in the non-coding regions of RET and also their association with the development of MEN 2A. Non-coding regulatory mutations have been reported to associate with a variety of cancers including thyroid cancer (Horn et al. 2013; Chen et al. 2014; Weinhold et al. 2014; de Biase et al. 2015; Diederichs et al. 2016; Yin et al. 2016;

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Kim et al. 2016; Chang et al. 2017). In this study, we found a 71-bp deletion within RSPO1 promoter region. RSPO1 was demonstrated to be a strong potentiator of Wnt signaling (Hao et al. 2012). RSPO2, another family member of RSPO family, was reported to be a tumor suppressor gene inactivated by promoter CpG methylation in colorectal cancer (Wu et al. 2014). RSPO proteins regulate Wnt signaling by a common mechanism (Kim et al. 2008). Thus, this 71-bp deletion possibly impaired the transcription regulatory machine and subsequently implicated in the development of MEN 2A. Although RET mutations are important to determine cancer risk, additional germline CNVs in MEN 2Aaffected individuals may predispose to MTC aggressiveness (Araujo et al. 2014). Meanwhile, germline fusion genes and chromosomal rearrangements have been reported to involve in the pathogenesis of monogenic and complex diseases (Trevino et al. 2009; van Heesch et al. 2014; Grunewald et al. 2015). Somatically, it has been confirmed that RET fusion is a novel driver of sporadic MTC (Grubbs et al. 2015). EML4-ALK fusion possibly drives the development of sporadic MTC (Ji et al. 2015). In this study, we identified several fusion genes including FSIP1-BAZ2A. Recently, it is reported that FSIP1 may be involved in the initiation and invasion of breast cancer (Zhang et al. 2015). BAZ2A was involved in epigenetic alterations and its overexpression served as a useful marker for metastatic potential in prostate cancer (Gu et al. 2015). Our study highlights the importance of using WGS to identify additional genetic modifiers associated with MEN 2A. As the cost of sequencing the human genome falls, medical use of whole-genome sequencing will rapidly advance (Ashley et al. 2010). WGS greatly expands the breadth of testing from genes associated with a particular disease to the whole genome and, potentially, all the information that the genome contains about diseases or traits (Ormond et al. 2010). Recently, WGS has been successful applied in the genetic diagnosis of monogenic diseases (Ashley et al. 2010; Talkowski et al. 2012). In this study, by using WGS, we identified the pathogenic RET p.C634R (c.1900T[C) mutation in the proband of a Chinese MEN2A family. We also found variants within the non-coding region of RET and PHEO-related genes. Besides, we identified three interesting genetic deletions which were possibly involved in the pathogenesis of MEN 2A. Although challenges exist, WGS could be an instructive, robust and time-effective tool in the genetic diagnosis of monogenic diseases.

Acknowledgements The authors thank all the patients and their families who agreed to participate in this study. This work was supported J. Biosci. 42(2), June 2017

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by the National Natural Science Foundation of China (81472861), the Key Project of Zhejiang Province Science and Technology Plan, China (2014C03048-1), the Key Scientific Research Project of Nanjing Military Command, China (09Z038, 10Z036), and the Medical Science and Technology Project of Zhejiang Province, China (2014KYB219).

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MS received 12 January 2017; accepted 15 March 2017 Corresponding editor: SEYED E HASNAIN

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