Development of multiple-PCR and next-generation sequencing techniques to establish a breast cancer gene panel and its clinical applications

Abstract

Breast cancer is a malignant tumor caused by a variety of gene mutations. Early diagnosis and treatment are likely to be the most effective means of reducing human cancer mortality. Molybdenum targets and breast color ultrasound are commonly used in clinical screening for breast cancer. However, it is sometimes difficult to identify the tumor only depending on imaging examination. In clinical practice, invasive puncture pathology or surgical pathology should be used to identify the tumor. At the same time, it should be noted that a large number of unpalpable breast malignancies are missed clinically, which delays the diagnosis and treatment of breast cancer and leads to a poor prognosis. Precision medicine is a very effective method based on scientific cognition of individual differences from genome variation, environment, and lifestyle, then through precise gene diagnosis to achieve individualized therapy, and early prevention. Advances in DNA sequencing techniques, such as next-generation sequencing (NGS) which have been applied to identify breast cancer susceptibility genes. High-throughput sequencing technology, also known as second-generation sequencing technology and NGS technology, greatly improves sequencing efficiency. Its application and derived various relevant technology are mature, such as microarray comparative genomic hybridization, and target gene sequence, which can achieve large pieces of samples at the same time sequencing. The clinical application uses all kinds of gene detection methods, and usually use the detection of whole-exon sequencing (WES). However, the WES cost is very high, and cannot be widely applied in patients with demand, gene diagnosis technology remains to be further improved. It is necessary to develop a low-cost, and effective gene panel which is suitable for early prevention, early detection, and early diagnosis. In this study, first, the tissues for 52 patients with breast cancer and the blood samples from 18 normal donors were collected, and then gDNA was extracted. Additionally, the variants in the AKT1 (12%), ERBB2 (10%), ESR1 (8%), TWIST1 (8%), and PIK3R1 (4%) gene were found in less than 15% in all patients. The number of pathogenic variants was 68 (68/358) through the Polyphen2 and the Sorting Intolerant from Tolerant (SIFT) analysis, which likely damage protein functions. In conclusion, a suitable, low-cost, high-risk gene panel has been successfully established for early detection/diagnosis. It will help for early detection/diagnosis, earlier prevention, and early treatment for people with breast cancer, especially those from developing or undeveloped countries. The new variants found in our cohort not only expand the knowledge and information for clinical application gene diagnosis in breast cancer patients but also provide genetic counseling related to breast cancer patients and family pedigree.