Long-term follow-up and novel splice donor mutation in MEN1 in a Chinese family

Heterozygous germline mutation of the MEN1 tumor suppressor gene is responsible for multiple endocrine neoplasia type 1. Parathyroid and thoracic neuroendocrine tumor specimens and DNA from two Han Chinese MEN1 family patients were analyzed using whole exome and Sanger sequencing. The proband (II-3) was sequentially diagnosed with pituitary adenoma, pancreatic tumor, adrenal cortical tumor, abdominal lipoma, and parathyroid adenoma during the 6-year follow-up. The son of the proband (III-6) was also diagnosed with a thoracic neuroendocrine tumor and a parathyroid adenoma during this period. Splice alterations were studied by RT-PCR and sequencing. The mutation impact was evaluated using bioinformatics. Sequence analysis revealed a novel splice donor mutation, MEN1 IVS9 + 1G > C, that changed the splicing mode of MEN1 to halt translation before two nuclear localization signals in the menin protein. Novel somatic mutations, MEN1 c.1402_1405delGAGG and c.286 C > T, were identified in the parathyroid adenoma of II-3 and thoracic neuroendocrine tumor of III-6, respectively, indicating a two-hit etiology of MEN1 syndrome. Our study revealed the clinical and genetic basis of MEN1 in this Han Chinese family and provides insight into MEN1 mechanisms, diagnosis, and management.


INTRODUCTION
Multiple endocrine neoplasia type 1 (MEN1) syndrome is an autosomal dominant disease characterized by the occurrence of tumors involving two or more endocrine glands within a single patient [1]. The estimated worldwide prevalence of MEN1 is between 1/30000 and 1/500000 [1,2]. More than 1000 families presenting with MEN1 syndrome have been described since the cloning of the MEN1 gene in 1997 [3]. However, clinical treatment and long-term follow-up are rarely reported in the literature. MEN1-associated tumors usually occur in multiples, readily metastasize, involve multiple organs, and are larger than sporadic endocrine tumors. The treatments for each type of MEN1-associated endocrine tumor are similar to those of non-MEN1 patients, but the outcomes of MEN1-associated tumors are less successful [1]. Therefore, a thorough understanding of clinical features with long-term follow-up data is essential for the management of MEN1.
Inactivation of the MEN1 tumor suppressor gene causes MEN1 [4]. MEN1 is localized to chromosome 11q13 and encodes the 610 or 615 amino acid nuclear protein, menin. Menin is a widely expressed protein that consists of three nuclear localization signals (NLSs) and five putative GTPase sites. Menin interacts with

Research Paper: Chromosome
Oncotarget 1578 www.impactjournals.com/oncotarget proteins involved in transcriptional regulation, genome stability, cell division, and proliferation, but its function is still poorly understood [5]. More than 600 different germline mutations and 160 different somatic mutations have been identified in MEN1 patients [3,6]. The splicesite mutation rate was 9% among these mutations. The significance of splicing donor mutations is insufficiently reported in the literature.
A novel germline-splicing donor mutation, MEN1 IVS9 + 1G > C, was identified in a Han Chinese MEN1 family with a 6-year follow-up period and its functions were investigated. Two novel somatic mutations, MEN1 c.1402_1405delGAGG and c.286 C > T, and loss of IVS9 + 1G > C heterozygosity (LOH) were found in MEN1 tumor tissues. Here, we elucidated the relationship between clinical features and MEN1 mutations in a Han Chinese MEN1 family and enriched the theoretical basis of MEN1 clinical diagnosis and therapy.

Extended family presentation of MEN1 syndrome
The proband (II-3), a male born in December 1945, visited Xiangya Hospital in August 2010 for dizziness. Magnetic resonance imaging (MRI) revealed a 2.0 × 2.5 × 2.5 cm pituitary tumor ( Figure 1A) that was presumed to be a functional pituitary adenoma due to the high serum prolactin level (Table 1). The patient refused surgical treatment. Therefore, 10 mg/day of bromocriptine was administered, and regular follow-up visits were instituted. Subsequent examinations indicated that the pituitary adenoma had been controlled. In March 2014, the patient revisited Xiangya Hospital for epigastric pain, sicchasia, and vomiting. Abdominal computed tomography (CT) revealed a 4 × 3 cm solid mass localized in the pancreatic head, bilateral renal stones, bilateral adrenal nodules, and abdominal lipoma ( Figure 1). Cervical ultrasonography revealed a left 8 × 5 mm and a right 9 × 5 mm auxetic parathyroid nodule. Barium meal examination revealed chronic non-atrophic gastritis and multiple polyps of the duodenal bulb (data not shown). The pancreatic tumor was surgically resected and histologically diagnosed as a pancreatic neuroendocrine tumor (data not shown). The blood biochemistry prolactin and parathyroid levels were outside the normal range (Table 1). A patient with 2 or more MEN1-associated tumors should be diagnosed with MEN1 according to the clinical diagnostic criteria [7]. In August 2016, the patient returned to Xiangya Hospital as the parathyroid nodule was enlarging progressively. Single-Photon Emission Computed Tomography (SPECT) revealed multiple nodules behind the thyroid (Figure 1). The parathyroid tumors were resected and diagnosis of adenoma with lymphatic metastasis was confirmed by pathology (data not shown).
Patient III-6, the proband's elder son, was diagnosed with a parathyroid tumor and a thoracic tumor at the age of 37. The parathyroid tumor and thoracic tumor were surgically resected and were histologically diagnosed as parathyroid adenoma and thoracic neuroendocrine tumor, respectively (data not shown).
Novel germline MEN1 splicing donor mutation IVS9 + 1 G > C Whole exome sequencing (WES) of peripheral blood lymphocyte DNA from both patients revealed 7 single nucleotide variants in the MEN1 gene. The novel variant NM_130799.2: c.1350 + 1 G > C, or IVS9 + 1 G > C, was not found in the HGMD, dbSNP, 1000G_ ALL, and ClinVar databases. The other 6 variants were known benign SNPs in 1000G_ALL. Sanger sequencing confirmed the presence of heterozygous IVS9 + 1 G > C in II-3 and III-6 ( Figure 2A). IVS9 + 1 G > C was inherited from father to son and co-segregated with MEN1 syndrome. Furthermore, IVS9 + 1 G > C was not found in other members of this family or 50 HCs. Restriction fragment length polymorphism (RFLP) analysis with HphI verified that II-3 and III-6 had the germline heterozygous IVS9 + 1 G > C mutation ( Figure 2C, 2D).

Functional analysis of MEN1 IVS9 + 1 G > C
The significance of nucleotide alterations in MEN1 IVS9 + 1 G > C was determined by RT-PCR. The total RNA was extracted from proband tumor tissues and peripheral blood lymphocytes of III-6 and HC to identify the impact of splicing donor mutation on MEN1 transcription. Agarose gel electrophoresis of the proband and III-6 RT-PCR products showed an additional larger band migrating slower than the normal band Figure 4A.
MEN1 RT-PCR products were cloned into pGEM-T for Sanger sequencing to obtain the precise sequence. The larger band contained the MEN1 intron 9 sequence (217 bp), but 202 bp of the MEN1 exon 10 sequence were www.impactjournals.com/oncotarget sheared ( Figure 4B). As a result, the additional band was 15 bp larger than the normal band. The splicing mode of MEN1 was changed due to the IVS9 + 1 G > C mutation. A transcription variant splicing donor mutation (SDM) was identified and abnormal mRNA was produced in MEN1 patients. We identified a new stop codon TGA in the undeleted MEN1 intron 9 sequence of the transcription variant SDM open reading frame (ORF), which prevented translation of the MEN1 exon 10 sequence ( Figure 4B). This mutation resulted in the loss of the two NLSs in exon 10 ( Figure 4C), which suggested that the mutant menin protein product cannot enter the nucleus for proper functioning [8]. We concluded that this was the mechanistic cause of this MEN1 family.

Two hit of MEN1 in parathyroid carcinoma
DNA of the peripheral blood lymphocytes and tumor specimens were sequenced by WES, and the results were compared to identify somatic mutations in MEN1 tumors. A novel MEN1 mutation, C.1402_1405del GAGG, was found in the parathyroid carcinoma tissue of II-3 and verified by RT-PCR and clone sequencing of MEN1 mRNA in tumor tissue ( Figure 2B). The in silico analysis of this mutation indicated it was a diseasecausing frameshift variant (Table 2). In addition, Sanger sequencing revealed the LOH of MEN1 IVS9 + 1 G > C in the parathyroid carcinoma tissue of II-3 ( Figure 2A). The novel MEN1 nonsense mutation, MEN1 c.286 C > T, was found in the formalin fixed paraffin-embedded (FFPE) tissue of thoracic neuroendocrine tumor resected from III-6 by WES. Two additional FFPE DNA samples were poor quality and unsuitable for WES. The in silico analysis of MEN1 c.286 C > T indicated it was a disease-causing nonsense variant ( Table 2). The two-hit pathogenic mechanism of MEN1 was revealed by two different somatic mutations in the parathyroid carcinoma tissue of II-3 and a novel somatic mutation in the thoracic neuroendocrine tumor tissue of III-6.

DISCUSSION
A total of 29 MEN1 mutations have been found in Chinese populations. Long-term follow-up analysis is rarely reported [9][10][11][12][13][14]. Here, we identified a novel germline-splicing donor mutation and two novel somatic mutations using WES in a Han Chinese MEN1 family with a 6-year follow-up period. A two-hit mechanism was determined to cause MEN1. Our study elucidated the clinical and genetic features of MEN1 in Han Chinese.
The proband was diagnosed with functional pituitary adenoma with high serum prolactin in 2010, but the patient did not elect for surgical treatment and was treated with bromocriptine instead. During the 6-year followup he underwent two surgical procedures. The pituitary adenoma was well-controlled and decreased in size. Therefore, MEN1 tumors should be considered curable if the patient is adequately treated by multidisciplinary cooperation. This conclusion was consistent with the results of Ning [15].
Positional cloning of the MEN1 gene in 1997 enabled the accurate genetic diagnosis of MEN1 and early or presymptomatic diagnosis of at-risk patient relatives, which led to long-term outcome improvement  Oncotarget 1581 www.impactjournals.com/oncotarget [4,16]. We conducted genetic screening among a Han Chinese MEN1 family, and a novel splicing donor germline mutation (IVS9 + 1 G > C) was discovered in the proband and one of his sons. This novel mutation caused the retention of intron 9 and deletion of exon 10 during splicing that resulted in the early stop of MEN1 translation with subsequent truncation of the normal menin protein. MEN1 intron 9 had additional splicing mutations, MEN1 IVS9 + 1 G > A [17] and IVS9-1 G > T [19], that caused the retention of intron 9 and resulted in an ORF frameshift of MEN1 similar to IVS9 + 1 G > C. However, the clinical manifestation of IVS9 + 1 G > C varied from that of the other two mutations. IVS9 + 1 G > A patients manifested familial isolated primary hyperparathyroidism and tested negative for multiple endocrine neoplasia. IVS9-1 G > T patients manifested familial isolated primary hyperparathyroidism. IVS9 + 1 G > C patients presented pituitary adenoma, pancreatic tumor, adrenal cortical tumor, abdominal lipoma, thoracic neuroendocrine tumor, and parathyroid adenoma. In addition, MEN1 IVS9 + 1 G > C and G > A had the same mutation site with dissimilar clinical features. In contrast to the studies of Ning and Raghavan, this result indicated that a different missense mutation at the same site could lead to a different phenotype [15,18].
MEN1 occurrence could be explained by Knudson's two-hit theory. In the dominantly inherited form, one mutation is inherited via the germinal cells and the second via somatic cells [20]. According to Thakker et al., the sequential inactivation of the MEN1 gene is an essential step in the development of MEN1 tumors [21]. The novel somatic mutation, MEN1 c.1402_1405del GAGG, and  (C, D) Restriction fragment length polymorphism confirmed the MEN1 IVS9 + 1 G > C heterozygous germline mutation in MEN1 patients. MEN1 gene fragments including IVS9 + 1 were amplified by PCR with primers, E9-3F and E10-3R. The wild-type PCR product had three HphI sites, and HphI digestion generated four fragments with lengths of 128, 39, 133, and 188 bp. However, the mutation IVS9 + 1 G > C was located in an HphI site. The mutant PCR product had only two HphI sites and was cut to three fragments with lengths of 167, 133, and 188 bp. In RFLP analysis, II-3 and III-6 had the additional 167 bp fragments compared with healthy controls (HCs).
Oncotarget 1582 www.impactjournals.com/oncotarget LOH was identified in the parathyroid carcinoma tissue of II-3, and a novel somatic mutation, MEN1 c.286 C > T, was found in the thoracic neuroendocrine tumor of III-6. This result supported the two-hit mechanism of MEN1 development.
We identified the clinical features of a Han Chinese MEN1 family within a 6-year follow-up period and the novel splicing donor and somatic mutations by WES. Our study illuminated the genetic mechanism, clinical features, and management of Chinese MEN1 patients.

WES and bioinformatics analysis
WES was performed using DNA isolated from leukocytes of II-3 and III-6 and the parathyroid carcinoma specimen of II-3 with the Dzup kit (Dzup, Sangon Biotech Co. Ltd, Shanghai, China), and the other three paraffin-embedded tumor tissues with the QIAGEN kit (QIAGEN, Sample & Assay Technologies, Germany). Sequencing libraries were constructed using a modified Illumina HiSeq X-Ten DNA sample preparation kit (BGI, Shenzhen, China). Qualified genomic DNA sample was randomly fragmented by the Covaris Acoustic System, and adapters were ligated to both ends of the resulting fragments. Extracted DNA was amplified by ligationmediated PCR (LM-PCR), purified, and hybridized to the Nimblegen SeqCap EZ Library v3.0 (Roche/NimbleGen, Madison, WI) for enrichment. Both non-captured and captured LM-PCR products were subjected to quantitative PCR to estimate the magnitude of enrichment. Each captured library was loaded on the Hiseq2500 platform (Illumina, San Diego, CA). We performed high-throughput sequencing for each captured library to ensure that each sample met the desired average sequencing depth (903). Raw image files were processed by Illumina base-calling Software 1.7 for base-calling with default parameters, and the sequences were generated as 90 bp pair-end reads. Single nucleotide polymorphisms (SNPs) and insertion/ deletions were called using the Genome Analysis Toolkit Oncotarget 1583 www.impactjournals.com/oncotarget (GATK v1.5.11). Novel mutations were determined by searching the Human Gene Mutation Database (HGMD), dbSNP, 1000 Genomes Project data (1000G_ALL) and ClinVar database. The functions of MEN1 mutations were analyzed using predictive algorithms in silico, such as MutationTaster (http://www.mutationtaster.org) and Fathmm (http://fathmm.biocompute.org.uk) [22,23].

Total RNA extraction, RT-PCR, and cloning
Total RNA was extracted from frozen tumor tissues and fresh peripheral blood cells using TriZol Reagent ; values above 0.5 are predicted to be deleterious, while those below 0.5 are predicted to be neutral or benign. h: MutationTaster is an in silico predictive algorithm that predicts an alteration as one of four possible types: disease-causing, disease-causing automatic, polymorphism, or polymorphism automatic; i, j: In MutationTaster, PhastCons (range [0, 1]) reflects the probability that each nucleotide belongs to a conserved element, and PhyloP (range [-14, + 6]) measures conservation at individual columns and ignores the effects of their neighbors. The higher the values of PhastCons and PhyloP, the more probable the nucleotide is conserved. www.impactjournals.com/oncotarget (Ambion, USA). Frozen tumor tissues were pulverized with liquid nitrogen before TriZol treatment. Fresh peripheral blood cells were separated by lymphocyte separation medium (TBD, Tianjin, China) to obtain lymphocytes. Reverse transcription (RT) was performed with total RNA using the Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific, USA). MEN1 cDNA fragments were then amplified by PCR with primers E8-1F (5′-ATGTCATCCCCAACCTGCTG-3′) and E10-1R' (5′-CAGCTCCTTCATGCCCTTCA-3′), which were designed based on the sequences spanning from exon 8 to exon 10. Finally, RT-PCR fragments were cloned into 1R', II-3 and III-6 had two bands, one normal band and a larger abnormal band, but HC had only one normal band. (B) Sanger sequencing of the MEN1 abnormal mRNA, transcript variant SDM, using RT-PCR and cloning. In the transcript variant SDM, the intron 9 (217 bp) was not deleted, but 202 bp of exon 10 was sheared. In the ORF of transcript variant SDM, the stop codon TGA of intron 9 resulted in the untranslated exon 10 sequence. (C) Nuclear localization signals were not translated in transcript variant SDM. IVS9 + 1 G > C resulted in the loss of two nuclear localization signals in menin, presumably disrupting nuclear translocation and functionality.