Application of Next Generation Sequencing Approach to Molecular Diagnosis Of Hereditary Colorectal Cancer: Identification Of A Novel Heterozygous Single Nucleotide Germline Deletion In Msh2 Gene Cause Lynch Syndrome

mutations of MSH2 and MLH1 are together accounting for 90% cases of LS Peltomäki et al. [7]. During DNA replication, to repair the mismatched bases, the MSH2 bind with MSH6 or MSH3 to form the MutSα/β complexes and translocate into nucleus to bind to the DNA and start the mismatched base repair Boland et al. [8]. Germline mutations in the Abstract Germline mutations in DNA mismatch repair (MMR) genes including MSH2, MLH1, MSH6 and PMS2 causes Lynch syndrome (LS). Lynch syndrome (LS) is the autosomal dominantly inherited familial form of colorectal cancer (CRC) predisposing syndrome. In our present study, we identified and screen a five generation Chinese family with LS. According to the Amsterdam II criteria, clinical diagnosis has been done for the affected members of this family. In this study, we performed genetic molecular study for 79 members (11 patients and 68 unaffected members) of this five generation Chinese family and 100 normal healthy control individuals of same ethnic background. Targeted next generation sequencing and Sanger sequencing identified a novel heterozygous single nucleotide germline deletion (c.1427delC) in MSH2 gene in the proband, which is co-segregated well with the disease phenotype among all the affected family members. This novel heterozygous deletion (c.1427delC) in MSH2 gene leads to the formation of a premature stop codon (p.Pro476Leufs*6) which finally results into the formation of a truncated MSH2 protein. Our present finding expands the mutation spectrum for the MSH2 gene as well as establishes the significance of targeted next generation sequencing for identifying novel candidate mutations for patients with Lynch syndrome. The novel heterozygous deletion identified in MSH2 gene through targeted next generation sequencing were verified through Sanger sequencing using the primers: F-5’- AAGGAGTTGTTCGTTTTCCACTT -3’, R-5’- TTACCAAAAGCCAGGTGACATTC -3’. The reference sequence NM_000251 of MSH2 was used.


Introduction
LS is a colorectal cancer (CRC) predisposing syndrome with an autosomal dominant mode of inheritance Lynch et al. [1] approximately, 1-13% among all the colorectal cancer patients has been suffering from LS primarily Vasen et al. [2]. The Clinical symptoms or diagnostic features of LS are early onset CRC, along with extracolonic manifestations; i.e. endometrial, pancreatic or gastrointestinal cancers Canard et al. [3]. Clinical diagnosis for the patients with LS has been done according to the Amsterdam II criteria Canard et al. [3]. Identification of germline mutations of DNA mismatch repair (MMR) genes and positive family history are also significant factor for clinical diagnosis Chung et al. [4]; Goecke 302 DNA MMR genes cause formation of non-functional MMR protein which in turn increases the rate of spontaneous somatic mutation.
Non-functional MMR protein associated somatic mutations are majorly occurring in microsatellite sequences Woods et al. [9]. In addition, till now, there were 500 germline mutations of DNA MMR genes have been reported and 39% of these are MSH2 mutations Vasen et al. [10].
Takeda et al [11] reported that germline loss-of-function mutation in MSH2 gene also causes synchronous endometrial and ovarian cancer (SEOC) in a 41-year-old Japanese patient with a positive family history of colorectal and gastric cancers Takeda et al. [11]. This patient was diagnosed with Lynch syndrome and it is a rare example of a patient with genetically diagnosed  [13].

Clinical Description
Family recruitment and clinical examination. We identified a five generation Chinese pedigree of 89 members ( Figure 1) with LS.
In (Table 1), we described the detailed clinical information for all the affected and unaffected members in this family.

Sanger Sequencing And Confirmation
This novel heterozygous deletion; c.1427delC, p.Pro476Leufs*6 in MSH2 gene was confirmed by Sanger sequencing in proband and among all the affected family members in this family ( Figure 2).

Discussion
Here, we identified a five generation Chinese family with LS.

Volume 3 -Issue 5 Copyrights @ Yan Zhang, et al. Open Acc J Oncol Med
According to recent reports it has been showed that LS patients with germline MSH2 mutations used to develop extracolonic cancers while LS patients with MLH1 mutations never develop extracolonic manifestations Lucía Pérez et al. [15]. In addition, LS patients with MSH6 mutations have the highest risk of developing endometrial cancer Lucía Pérez et al. [15]. In South-East Asia, LS patients are usually developed endometrial and stomach cancer Li [17]. In LS patients with MSH2 germline mutations, the lifetime risk of developing colorectal cancer is ranging between 57 % -80 % Bonadona et al. [18]; Vasen et al. [10].

Ethical statement
Family members of this five generation Chinese family have given written informed consent as they are participating in this study. The Ethical Committee of the Maternal and Child Health Hospital of Hainan Province, Haikou, China, reviewed and approved our study protocol in compliance with the Helsinki declaration.
Diagnosis of the patients for Lynch syndrome was made by oncologists, on the basis of Amsterdam II criteria.

Open Acc J Oncol Med
The novel heterozygous deletion identified in MSH2 gene through targeted next generation sequencing were verified through Sanger sequencing using the primers: F-5'-AAGGAGTTGTTCGTTTTCCACTT -3', R-5'-TTACCAAAAGCCAGGTGACATTC -3'. The reference sequence NM_000251 of MSH2 was used.

Acknowledgments
We thankful to the proband and all the family members for their participation in this study.

Funding
There is no specific funding.

Availability Of Data And Materials
The data used to support the findings of this study are included within the article.

Authors'contributions
MX, YZ and SB designed the study. ZL performed the molecular diagnosis by Sanger sequencing. LY carried out the clinical analysis.
YZ and MX contributed to the molecular diagnosis analysis based on NGS. ZL and LY wrote the paper. This project has been done under the guidance of ZL, MX, YZ and SB. All authors read and approved the final manuscript.

Ethics Approval and Consent to Participate
The present study was approved by the Ethics Committee of Maternal and Child Health Hospital of Hainan Province, Haikou, China, and written informed consent was obtained from all participants.

Consent for publication
Informed consent was obtained from all individual participants included in the study.