4 Discussion
NGS plays an important role in the whole 3-step prevention to control birth defects. For the primary prevention of birth defects, Carrier screening using the NGS approach should be carried out for couples who were willing to give birth, and genetic counseling and birth guidance should be given to couples who were positive for genetic screening. Genetic screening for Carrier focuses on diseases that were leading to severe genetic defects in children37. For the second-step prevention of birth defects, Peri-conception screening using the NGS approach could detect chromosomal abnormalities of fetal free DNA in maternal peripheral blood, mainly involved in diseases caused by chromosomal abnormalities, such as Trisomy 21, 18, 13 syndromes38. For the third-step prevention of birth defects, NBS using the NGS method was suggested to conduct in neonates to find out the diseases seriously harmful, mainly focused on serious and actionable diseases3. In our study, we aimed to determine the suitable technology, rational diseases and gene-disease association list for newborn sequencing screening. Firstly, we systemically evaluated the NGS methods to select the targeted sequencing for NBS. We then designed a NeoExome panel covering diseases that are severe, actionable, and early-onset for the Chinese population, and verified it in multiple NBS centers. We also compared our NeoExome panel with other NBS NGS panels to illustrate the respective characteristics. Our study designed a targeted-sequencing NeoExome panel for Chinese in NBS with applicable performance.
The gene-disease list is critical in NGS panel design. The study of BabySeq project curated a catalog of gene-disease pairs based on ACMG, the ClinGen clinical validity classification framework criteria, penetrance, and age of onset. They finally screened out 954 genes that are met the criteria reported in their project23. The NC NEXUS project, designed an age-based framework to assess the gene-disease list. They assessed 822 gene-disease pairs and divided them into 4 different categories 24. In China, the NGS screening for NBS is also conducted recently. Dr. Yu has designed a panel of 573 genes for the screening of severe inherited disorders36. While Dr. Li explored the application of their 465-genes panel in clinical practise34. Dr. Zhao designed an NBS genetic sequencing panel including 134 genes of 74 inborn disorders using multiplex PCR39, and Dr. Xu investigated 164 pathogenic genes with 94 common genetic diseases40,41. However, all studies did not elaborate in detail on why their study included these genes. In our study, we comprehensively integrated the diseases catalog of Rare Diseases in China, routine NBS diseases in China, ACMG and mainland experts’ recommendation, and database of genetic diseases in 40000 ill-children, to generate the severe, actionable, and early onset monogenic inherited diseases list of NeoExome panel with 601 genes for NBS.
The positive rate of our NGS panel in “1000 Genomes Project” is 7.6%, higher than other previous studies42. We found thatFLG and GJB2 are the most frequently mutated genes in our analysis. FLG is a pathogenic gene for ichthyosis vulgaris, which encodes filaggrin and plays a key role in epidermal terminal differentiation and skin barrier formation. The proportion of FLGgene variation in ichthyosis vulgaris was reported to be 55.6%, and the ichthyosis vulgaris subjects with FLG gene variant suffered more severe diseases43. However, FLG also has higher mutations in the normal population, Palmer and colleagues carried out FLG analysis with 1008 people of European origin and found functional deletion mutations of theFLG gene were approximately 9%44. Besides,GJB2 gene variation is generally considered to be a common cause of non-syndromic deafness45, while GJB2 c.109G>A mutation was considered as a pathogenic variant with incomplete penetrance and high carrier rate in Asian46. Our analysis identified 5 GJB2variants, 4 of them was c.109G>A mutation (Supplementary Table2). Our study further demonstrated the importance of genetic counseling for such genetic variations.
It was reported that the healthcare cost of NBS using WGS is high and increased the financial burden47,48.Subsequently, researchers implied target sequencing might highly decrease the cost as well as TAT 33,35. In our study, the targeted sequencing of NeoExome approach is designed, carried out, and the TAT is 14 days, which is much shorter than that of WGS/WES. Moreover, the positive rate of our NeoExome panel for NBS (-) neonates was 8.9% (271/3049) in our pilot study, which is consistent with the results of BabySeq project31. Despite the advantages, the use of targeted sequencing in NBS increased the risk of missed detection as exome sequencing only covers approximately 1%-2% of the entire genome. Recently, one study conducted transcriptome sequencing (RNA-seq), WES, and WGS in 115 undiagnosed patients with diverse phenotypes, and the results found RNA-seq could help diagnose 17% patients that had negative results of WES/WGS49. This suggests that it is necessary to simultaneously conduct WGS, RNA-seq, and other methods for symptomatic children with negative results of WES/targeted NGS approach. In addition, the targeted panel should be updated with the increased evidence-based studies of some new gene-disease associations.
Moreover, it is also necessary to pay attention to whether the genes associated with these adult-onset diseases need to be detected at neonatal stages. In the previous study of the BabySeq project, aBRCA2 pathogenic variant, which is related to a 45% risk of breast cancer and 11% risk of ovarian cancer in women, was found in one infant31. Because of moral distress, the researchers proposed to return these adult-onset genetic variants50. But it raised a strong ethical debate, Lainie FR and colleagues then published an article and argued that researchers should avoid identifying adult-onset genetic variants, as this may cause psychological impact to the child and his/her parents, and deprive the child of the right to an open future51. Our current panel included 12 adult-onset genes and 12 genes with unknown onset age, which we are planning to excluded in the next round. Probably, “Age-based genetic screening strategies” should be used for reference and applied in China3.
Our study has several limitations. Firstly, lacking whole follow-up data of some recruited subjects in our validation cohort is the main disadvantage. In our study, low concordance between NGS and conventional NBS (Table 2) was indicated, which was also reported in the BabySeq project 52. One of the reasons is that the conventional NBS data collected in our study are incomplete due to the great difference of detection indexes in different regions. On the other hand, sequencing could not fully represent phenotypes, there were always some subjects with gene-positive, but clinical phenotype-negative34. This may be caused by two reasons: one is that some diseases are later onset due to the differences in the time of diseases onset, while our study period is limited and these disease-related symptoms have not shown. The other is due to the low association of genes and disease. Once the penetrance of a gene variant is low, the individual with this variant has a low chance to be symptomatic53. This is also one limitation in our study, we did not include penetrance in our report interpretation category. Our report category needs to be modified and the follow-up needs to be strengthened in our next study.
Although studies have shown that NGS may play a role as second-line screening in NBS and cannot replace MS, we believe that sequencing screening can be performed simultaneously with conventional NBS if the cost is reasonable. For the diseases that have no biochemical markers, such as spinal muscular atrophy (SMA), duchenne muscular dystrophy (DMD), and cardiovascular disease, sequencing could predict disease at early stage. Therefore, we proposed this NeoExome panel may have the following applications in the clinical practice: 1) As a first-tier NBS for genetic diseases with no biochemical detections; 2) As an adjunct diagnostic tool for monogenic inherited diseases with obvious abnormal phenotype; 3) As a second-tier NBS. A combination of NeoExome with conventional NBS can enhance the clinical utility of NBS. For example, sequencing screening can be further carried out in children with ambiguous-positive results in conventional NBS to improve diagnostic accuracy. In addition, the application of sequencing in healthy infants with negative conventional NBS results can help to find out the gene variants in the early stage of their lifetime to prevent mortality. 4) As an exclusion (quasi-first-line) screening for infants with low phenotypic specificity (jaundice, etc.), to assist the excluding the common genetic diseases. More specific clinical trials are needed in the future to validate the use of NeoExome in clinical practice.
In all, we designed a personal targeted-sequencing panel for Chinese NBS, evaluated this approach in a multi-center pilot study. Our study indicated our panel needs to be further optimized for the whole population screening. Besides, more specific clinical trials need to be carried out to verify the applicability of the panel in the next step.
Acknowledgments: We thank all the participants and their guardians in the research.
Conflict of Interest: The authors declare no conflicts of interest.
Funding : This work is partially supported by the Chongqing Science and Technology Bureau (cstc2019jscx-msxm0189), Shanghai Hospital Development Center (SHDC2020CR1047B) and the Natural Science Foundation of China (82070167, 81870126).
Authorship : Conceptualization, LZ, GJY, and JW.; Investigation, ZYC, XYH, DJW, MSG, FS, RQ, RXZ, CRS, XHW BZ, DHC, HHY, YPQ, GSS, JW, XBW, ZYJ, and PPW; Validation, ZYC, JXW, HYZ, ZJY, and LZ; Writing – Original Draft Preparation, ZYC and LZ; Writing – Review & Editing, ZYC and LZ; Visualization, ZYC and PPW; Supervision, LZ, and GJY; Funding acquisition, LZ, and GJY; All authors have read and agreed to the published version of the manuscript.
Data sharing Statement: All data are available in the main text or the supplementary materials.