Here, we report a new method of genomic sequencing for newborn screening, called “NeoSeq”, which is based on multiplex PCR amplicon sequencing. As seen from preliminary clinical applications, it has the following advantages: (1) Similar to few nGS studies, NeoSeq can not only detect most of the diseases included in traditional TMS-NGS screening but can also reduce the false-positive rate considerably. (2) It can screen greater number of diseases and provide more genetic health-related information. (3) Current studies usually use the WGS and WES technologies, which are technically difficult to perform and have more complicated experimental procedures than NeoSEq. In addition, WGS and WES have longer reporting cycles and cost significantly more than NeoSeq.
It is well known that TMS has helped to expand the NBS program. NGS technology can be used as the second key step for diagnosis after TMS screening. The combination of both is an enhanced plan for NBS. Currently, more and more countries have adopted TMS-NGS as the primary method for NBS, with good results4,17,18. With the rapid advancement of sequencing and the subsequent decrease in costs, new-born genomic sequencing (nGS) may become a new milestone in the field of NBS, after TMS, and shows the prospect of broad applications. For example, Bodian’s group presented a comparison of the results of WGS and blood-based NBS from a cohort of 1,696 new-borns. There was generally good agreement between both techniques, with 88.6% true positives and 98.9% true negatives being identified. In addition, the false-positive rate of WGS was far lower than that of the traditional NBS8. BabySeq and NC NEXUS projects also reported 60.0–88.0% sensitivity and 100% specificity12,15. Our results showed similar specificity (99.4%), but the sensitivity was relatively lower (55.6%). This may be because we used MTA-Seq as the screening technology rather than WES. Meanwhile, when we designed the NeoSeq panel, we selected criteria of variants in 135 genes as: (1) high frequency in Chinese, Eastern Asian or Asian population; (2) common pathogenic variants in databases, including ClinVar and ClinGen; (3) LOF (Loss of function) variants in Asian population (≥ 10 allele count in Gnomad); and (5) high frequency in local databases. This indicates that NeoSeq precisely reported the definite pathogenic variation; its clinical application is more reliable and genetic consultation is more certain.
Newborn genomic screening is a new research area. To explore the effects of its clinical applications, we conducted a meta-analysis and comparison from a search of the literature (Table 3). Four groups have conducted studies since 2016, which include Newbie Seq19, NC NEXUS15, BabySeq12 and Dale L’ group8. All the studies were designed by combining a methodological comparison with the findings from a retrospective cohort. With the exception of Dale L’ group (WGS)8, the studies used the WES or ES technology. The sensitivity and specificity were 60.0–88.6% and 93.7–100%, respectively. Most studies have reported additional findings beyond the traditional newborn screening. Instead of using WES, we adopted multiplex PCR amplicon sequencing (MTA-Seq) for a number of reasons such as ease of use, simple and standardized procedure, and cost effectiveness. Compared with that of the traditional TMS-NGS screening, the sensitivity of NeoSeq was 55.6% and specificity was 99.4%. Furthermore, the results revealed some interesting additional findings. Importantly, this method can significantly reduce the false-positive rate and the duration of reporting cycle (7–10 days). It is yet unclear whether nGS can completely replace TMS-NGS. Current studies agreed that nGS could be used as an important supplement to common blood-based NBS while not completely replacing it. Based on a population screening of 4.5 million infants, the NBSeq project19 suggested that exome sequencing was not recommended as a first-line method for NBS of IEM. However, it could be used as a secondary test after TMS screens. We support this suggestion. All the studies have shown that the sensitivity of nGS was approximately 55.6–88.6%, which means that some infant diseases diagnosed with traditional NBS may not be detected using nGS. Both complement each other in order to achieve the best effect. Notably, in the present study, five out of eight ill infants missed by NeoSeq only had one pathogenic variant, while the other variant was of uncertain significance. We are following these children closely. On the other hand, is it necessary to screen for these diseases in NBS? It is a scientific problem worthy of discussion. Many countries are constantly optimizing the disease spectrum detected by NBS4.
Table 3
Comparison with other similar studies
Study | Year | Samples | Project | Method | Panel | Sensitivity | Specificity | Additional discovery | Carrier rate |
Dale L1 | 2016 | 1696 infants | - | WGS | 163 genes | 88.6% | 98.9% | G6PD etc | - |
Aashish N2 | 2020 | 1190 805 with IEM 385 with TMS false positives | Newbie Seq | WES | 78 genes associated with the 48 IEMs | 88.0% | 93.7% | - | 34.0% |
Tamara S3 | 2020 | 106 17 with IEM 28 with hearing loss cases 61 healthy newborns | NC NEXUS | ES | 466 genes | 88.0% for IEM 18.0% for hearing loss | 100% | OTC deficiency Amilial hypercholesterolemia Actor XI deficiency Arrhythmogenic right ventricular dysplasia | - |
Monica H4 | 2021 | 316 12 with NBS Positive 147 with NBS Negative 127 healthy newborns | BabySeq | ES | 954 genes | 60.0% | 100% | Cardiomyopathy Hereditary breast and ovarian cancer Supravalvular aortic stenosis KBG syndrome Atypical hemolytic-uremic syndrome Glomuvenous malformation Cystinuria Non-syndromic hearing loss Lynch syndrome | - |
Our | 2021 | 196 36 with IEM 60 with TMS false positives 100 with TMS negative | Neoseq | MTA-Seq | 135 genes related to 75 diseases | 55.6% (20/36) | 99.4% (159/160) | Hear loss G6PD MELAS CH | 26.3% (42/160) |
It is certain that the advantages of nGS primarily include fewer false-positive results, accurate diagnosis and distinction of disorders, and more useful information for newborn life. Due to the high sensitivity of TMS, the high false-positive rate and low positive predictive value of TMS are always a problem, particularly for some special populations such as premature infants20. Additionally, the technique is easily affected by external interference21. The high false-positive rate will bring a large number of healthy infants to be excessively recalled, which will not only burden the medical service but also result in psychological burden to the parents22. Some researchers even began to question whether TMS should be used to expand NBS23. In the present study, three of 60 infants with TMS positive results might have the risk of disease. Therefore, as a supplementary method of TMS screening, nGS can effectively reduce the false-positive rate.
Currently, three methods are primarily used in new-born genomic sequencing: WGS, ES, or gene panel sequencing. However, there are still some doubts whether new-born WGS/WES should be used routinely in clinical applications24. They are difficult to popularize due to the complexity of project technology and cost involved. Therefore, they may be not suitable for screening projects. At the same time, they can provide a plethora of genetic information, which could bring great challenges to clinical genetic counselling and also involve a lot of ethical problems. NeoSeq, reported here, is based on multiplex PCR amplicon sequencing (MTA-Seq). This technology is considered a simple, customizable, and targeted sequencing method, which is conducive to the wide application of high-throughput sequencing, such as genome diagnosis, population genetic analysis and so on25. Recently, the Yang group26 applied it to screen genetic hearing loss variants in newborns, demonstrating 100% sensitivity and specificity. We tried to use it to screen 75 kinds of inborn disorders. The results were quite satisfactory. At the same time, most of the experimental detection could be completed in one week, and the cost was only one fifth of that of WES.
In conclusion, NeoSeq is an effective method of genomic sequencing for newborn screening. It can detect most inborn errors of metabolism, reduce the rate of false positive results, shorten the porting cycles, and reduce the screening cost.