1 Introduction
Birth defects (also known as congenital anomalies) are the major causes of neonatal deaths, worldwide. According to the report on prevention and treatment of birth defects-Ministry of Public Health of China in 2012, the incidence of birth defects in China is about 5.6%, with approximately 900000 new cases of birth defects every year1. To reduce the occurrence of birth defects, the World Health Organization (WHO) develops prevention strategies into three levels based on the causes and epidemiology of birth defects: preconception care to increase the likelihood of a healthy infant delivery, pregnancy care to reduce the birth rate of defective infant, newborn infant and child care to decrease disability, mortality, and serious consequences of birth defects2. 3 periods-screening (Preconception screening, Peri-conception screening, Neonatal screening) prevent different diseases of birth defects. Newborn screening (NBS) is the last step against birth defects, which aims to identify seriously harmful diseases in the neonatal period. It is helpful for early diagnosis, early intervention (as reducing exposure to risk factors), and early management of the diseases, thus preventing mortality and morbidity of children3,4.
NBS begins in the 1960s when Professor Guthrie firstly applied the method of bacterial inhibition to screen phenylketonuria (PKU) in the dried blood spots5. Since then, NBS has gradually expanded under the screening guiding principles with the progression of clearly-investigated diseases, the feasibility of screening methods, and the increment of public conscientious 6,7. In 2006, the American college of medical genetics (ACMG) expert group published and continually updated the Recommended Uniform Screening Panel (RUSP) for NBS consensus with 35 primary and 26 secondary conditions8. While in China, NBS started in 1981, mainly focused on the screening of PKU, congenital hypothyroidism (CH) firstly, followed by deafness, glucose-6-phosphate dehydrogenase (G6PD) deficiency, congenital adrenal hyperplasia (CAH), and so on9. With the expansion of screening diseases, NBS methods have developed from the initial low throughput biochemical approach to high throughput molecular biology technology. Tandem mass spectrometry (MS) is the main conventional NBS method and has been widely used in clinical practice for the last 20 years, with the feature of rapid, sensitive, and high throughput10. Although MS accelerated the development of NBS, it is now well established from a variety of studies that MS has a high false-positive/negative ratio4,11,12. Besides, conventional NBS covered a limited range of diseases. New methods are urgently needed for complementing the shortcomings of MS and other conventional NBS.
Recently, high-throughput sequencing technology has been widely used in tumor-targeted genes test13, pathogen detection14, as well as 3 periods-screening of birth defects15. Laboratories have successfully carried out studies on sequencing technology as an NBS method16-18 and indicated sequencing could be used as a second-tier confirmation for conventional NBS of some diseases19,20. Besides, the ability of sequencing for the gene diagnosis of Neonatal Intensive Care Units (NICU) patients has also been uncovered by multiple studies21,22. However, there are several concerns about the use of sequencing in NBS. 1) Interpretation of the gene variant site. Sequencing may identify lots of variants with uncertain significance (VUS), while the relationship between many genes and diseases is not clear. 2) Report of the results. There is no gene-disease association list about conditions that are treatable and preventable so far. 3) Personalized sequencing panel. Sequencing protocols and panels specifically designed for NBS are lacking. 4) Clinical trials of the whole population. Most of the previous studies focused on the use of sequencing in ill infants, or for the screening for a specific disease, studies about multiple monogenic-disease screening in healthy newborns are not fully understood. These big challenges for the application of sequencing in NBS are presented and aroused great interest in this field.
Based on the above-mentioned challenges, researchers devoted to establishing proper criteria of gene-disease association list and reporting strategies for newborn gene sequencing since the year 2012 23,24. However, this gene-disease association list and reporting strategy cannot be fully applied, as the high incidence of monogenic diseases in the Chinese population is quite different from that of other countries, due to different races and geographical areas. Here, we systemically evaluated the NGS methods used in NBS, designed the severe, actionable, and early-onset inherited gene-disease list for the Chinese population, established the featured NGS panel (NeoExome), performed a multi-center study to verify its feasibility with 3423 neonates, and compared the NeoExome panel with other NGS panels of NBS. (Supplementary Fig. 1)