Novel Germline Mutation in BCAR1 in Two Siblings With Lung Cancer: a Possible Susceptibility Gene


 Background: Lung cancer is the most prevalent malignancy worldwide. Most patients were sporadic and carried somatic mutations in hotspot genes. At present, accumulating researches had identified several germline mutations that predispose patients to lung cancer. In this report, two siblings were diagnosed as lung squamous cell carcinoma and lung adenocarcinoma, respectively. Results: The two siblings shared similar features of a germline insertion of 11 bp (GCCCTGGCATT) in BCAR1 producing a frameshift at codon 314 which is located at the substrate domain. The BCAR1 was previously demonstrated to be associated with lung cancer. The variant detected in this report would impair the regulation and functions of BRCA1 in some extent, thus may promote lung cancer tumorigenesis. Our findings suggest that BCAR1 is a possible susceptibility gene for lung cancer, and its functional analyses in lung cancer need further investigation.Conclusions: In this study, we first reported a novel causative mechanism of Lung cancer: an insertion of 11 bp in BCAR1 gene, which can be helpful in the genetic diagnosis of this disease.


Background
At present, lung cancer still ranks top one in cancer incidence and mortality [1], in which, non-small cell lung cancer is the predominant histologic type, mainly including adenocarcinoma and squamous cell carcinoma. The primary non-inherited risk factor for lung cancer is smoking, however, approximately 25% of lung cancer cases worldwide are not attributable to tobacco use [2]. Multiple risk factors, including environment, hormone and genetic predisposition, have been associated with the pathogenesis of lung cancer in never smokers. Among these factors, familial lung cancer had been reported [3], and persons with family history of lung cancer had a double-risk of developing the disease [4,5]. Although familial forms of lung cancer is rarely described, several susceptibility genes have been identi ed, such as EGFR [3,6], HER2 [7], BAP1 [8], and BRCA2 [9], among which EGFR germline mutations were the most reported. In the current report, we presented two siblings with lung cancer harboring germline mutation in breast cancer anti-estrogen resistance protein 1 (BCAR1). BCAR1, encoding a scaffold protein, has been shown to be overexpressed in lung cancer and linked to adverse features in lung cancer [10][11][12]. Therefore, we suspected that BCAR1 germline mutation potentially predisposes patients to lung cancer.

Subjects
Peripheral blood samples were obtained from the proband and his family after informed consent was signed respectively. Experiment on human subjects was approved by Institutional Review Board of Huazhong University of Science and Technology, Wuhan, China. The two affected siblings' brain MRI results were evaluated by two pediatric neuroradiologists.   Figure 1C). However, the patient was readmitted for cough, chest distress and asthma in January, 2019. The repeated CT scan revealed tumor recurrence ( Figure 1D). Subsequent treatment was not administrated because of the patient's willingness, and he died on July, 2019.

Patient 2
Patient 2, a 51-year-old female, is the younger sister of patient 1. She complained of intermittent coughing for 1 month and sough medical consultation at a local hospital. Auxiliary examination revealed the presence of nodule in the right parahilar region, as well as the increased number and enlarged size of mediastinal lymph nodes. She transferred to our department in June, 2018. Subsequent PET-CT and enhanced CT showed a 2.7×2.2 cm mass in the right lower lung (Figure 2A). Multiple enlarged lymph nodes were also presented in the right parahilar region and mediastinum. CT-guided lung biopsy was performed on June 12, 2018. Pathological examination indicated lung adenocarcinoma ( Figure 2B Figure 2C). On August 28, 2019, lung lesions were stable, however, brain MRI revealed potential metastasis ( Figure 2D), and the patient is under treatment with alectinib at present.

Follow-up and outcomes
Two siblings were diagnosed with lung cancer, leading to the request of an evaluation of possible hereditary predisposition. Whole-exome sequencing was performed, and two shared deleterious germline mutations (BCAR1 and FAM20C) were identi ed. Of these, BCAR1 was considered as the best candidate, because germline mutation in FAM20C was also found in spouses of their relatives, and because BCAR1 have previously been associated with lung cancer. Both patients harbored a germline insertion of 11 bp (5'-GCCCTGGCATT-3') producing a frameshift at codon 314 ( Figure 3 and Figure 4A). The BCAR1 variant detected in this report could not be found in the ExAC database, indicating it as a novel variant. Among the sixteen family members tested, three were found to carry the BCAR1 variant ( Figure 4B). To date, the three mutation carriers did not reveal any features of lung cancer.

Discussion
Lung cancer remains the most prevalent malignancy worldwide. In the past decades, numerous somatic mutations have been identi ed in lung cancer tissues and further established an oncogene-centric molecular classi cation paradigm in this disease. In contrast, limited germline mutations have been identi ed. Here, we encountered a family in which two siblings developed lung cancer, leading to the evaluation of possible hereditary predisposition. Using whole-exome sequencing, two shared germline mutation in BCAR1 and FAM20C were identi ed. Germline testing was further extended to their family members. Eight family members and two spouses were found to carried FAM20C variant, and BCAR1 variant was identi ed in three family members, indicating BCAR1 variant as the potential candidate. In theory, the allele frequency of BCAR1 variant should be approximately either 50% or 100% (heterozygote or homozygote), however, the allele frequency of both patients revealed by whole-exome sequencing were less than 1/3. This result was caused by the low detection rate of inserted mutation. Therefore, the veri cation of sanger sequencing was further performed, and both patients were identi ed as heterozygote.
BCAR1 encodes p130cas protein which possesses an amino-terminal Src-homology 3 domain, a substrate binding domain, proline-rich and serine-rich regions, and a bipartite Src-binding domain in carboxy-terminal sequence [10,13]. p130Cas itself has no intrinsic enzymatic or transcriptional activity, but they could control signaling events via phosphorylation and dephosphorylation and their association with effectors proteins in multimolecular complexes. Tyrosine phosphorylation is the major posttranslational modi cation of p130Cas, mainly occurring in the substrate binding domain, which help to open binding sites for a variety of effector proteins [11]. p130Cas has been demonstrated in many studies to play a vital role in cell migration [14,15], apoptosis [16,17] and cell transformation [18]. p130Cas is also reported to be involved in the development and progression of several human cancers, including breast cancer [19,20], prostate cancer [21,22], as well as lung cancer [23]. In lung cancer, BCAR1 was usually overexpressed, and increased BCAR1 predicts poorer prognosis in non-small lung cancer [23,24]. In this report, the detected BCAR1 variant occurred an 11 bp insertion at codon 314 which locates at the substrate binding domain, indicating that its association with effectors proteins would be impaired in some extent, thus leading to a wide-range downstream consequence. This in uence cannot be underestimated, and may predispose patients to lung cancer. Therefore, BCAR1 is a possible susceptibility gene for lung cancer. Although the biological function of BCAR1 in lung cancer remains unclear, we believed that BCAR1 plays an important role in the development and progression of lung cancer. It is worth noting that the two siblings developed two different histologic type of lung cancer, revealing that BCAR1 may undergo different mechanisms to induced different histologic type of lung cancer. To further prove our hypothesis, investigation concerning the biological function of BCAR1 in lung cancer is urgent and necessary. In addition, there are another three carriers in good health condition, and a long-term follow-up is of great necessity.

Conclusion
Above all, our report revealed a possible susceptibility gene, BCAR1, and further investigation of biological function of BCAR1 in lung cancer is need. These ndings may help to conclude and reveal speci c variation types in BCAR1 , which is bene t for the diagnosis and then for the future precision treatment of Lung Cancer.

Declarations
Kuikui Zhu designed the study experiments. Yingchao Zhao collected the data and conducted the clinical evaluations. Lu Wu, Sijia Zhang, Yan Zong and Zhenyu Li performed wholeexome sequencing and sanger sequencing. Qianwen Li and Fang Cheng wrote the article. Rui Meng supervised the study experiments. All authors revised and approved the article.

Funding
The author(s) received no nancial support for the research, authorship, and/or publication of this article.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Consent for publication
Publication of data was informed consent to all individuals involved in this study.