A pilot study of assessing whole genome sequencing in newborn screening in unselected children in China

We investigated whether screening by whole genome sequencing (WGS) in unselected newborns provides more information of potentially curable or treatable medical conditions than routine newborn screening (NBS). We demonstrated that compared with routine NBS, WGS pro-duced fewer false positive results and identified more actionable pathogenic or likely pathogenic variants in the selective 246 genes. 3–5 Sequencing one actionable PGx variant. The CYP2C19 second-highest carrier rate, where 209 (65.11%) carried at least one clinically relevant variant. In addition, and 122 infants carried actionable PGx variants at the gene UGT1A1 and NUDT15 , respectively. No actionable variant was identified in the DPYD gene in the 321 children. Among the selected gene–drug pairs, irinotecan, azathioprine, mercaptopurine and tioguanine to paediatric patients, while codeine and clopidogrel are restricted for use of children under 18 years old. Our findings suggest that participants may obtain benefit from PGx profiling already in early childhood

For the 164 PIDs, 9 heterozygous P/LP variants in 6 genes were identified in 9 newborns (2.80%), all in a heterozygous state ( Table 2). Four newborns were shown to carry heterozygous variant of unknown significance (VUS) in the gene SLC25A13 (c.2T>C, p.M1T), which was predicted as start loss and likely affecting the initiator methionine of the SLC25A13 mRNA. Two newborns carried a VUS in ASS1(c.-4C>T, p.?). Although these VUSs were not included in the final report to the participants, follow-up of the children with VUSs will be conducted till 3 years of age.
Sanger sequencing confirmed 143 out of 145 mutations identified by WGS, resulting in an accuracy of 98.62%. Carriers of SMN1 mutations were validated by multiplex ligation-dependent probe amplification and real-time quantitative PCR, showing that five out of the six predicted carriers were true.
Of the 321 newborns, 312 (97.20%) had the results of 48 IMDs screening and genetic hearing loss screening on 20 loci, which identified one newborn with PKU and one infant with increased blood level of isovalerylcarnitine  (Table 1). In addition, 18 carriers harbouring 20 pathogenic mutations causing hearing impairment were detected by genetic hearting loss screening, albeit all 321 children passed the physical hearing screening at hospital (Supporting Information). The newborn WGS also identified the PKU case and 18 hearing loss carriers ( Figure 2A). However, the child with increased level of isovalerylcarnitine was confirmed to be a carrier of 3-methylcrotonyl-CoA carboxylase deficiency by WGS. In addition, WGS identified two infants carrying compound heterozygous P/LP variants in GJB2 ( Figure 3) and four children carrying pathogenic mutations in MT-RNR1 (c1095T > C), suggesting an increased risk of late-onset deafness or drug-induced hearing loss, respectively. Although currently non-symptomatic, the two newborns with GJB2 variants were scheduled to undergo hearing tests every 6 months, and the four newborns with the m.1095T mutation in MT-RNR1 were advised to avoid using aminoglycosides.
Interestingly, we observed that 313 newborns (97.51%) carried at least one actionable PGx variant ( Figure 1C). This result is in line with a European 44 000 biobank participants study, where 99.8% of the participants had a genotype associated with increased risks to at least one medication. 8 Furthermore, we found three common PGx variants in the Qingdao cohort, CYP2D6*10 (48.60%), NUDT15*3 (13.08%) and UGT1A1*6 (21.18%) ( Figure 2B and Table 3), that showed significant frequency differences as compared to East Asian populations (p < 0.05). An impor-tant aspect when screening for disorders in a given population is the use of a matched control database as variants can be highly specific for a given ethnic group. 9 Most databases published to date are based on individuals of European descent and many populations have limited or poor representation.
Limitations of the current study are the small sample size and restricted metabolic tests. A large-scale NBS effort is needed to validate our findings and fully investigate the treatable or curable medical conditions in newborns. The technical challenge of newborn WGS is to screen genes with high homology due to the misalignment of short-read sequencing. Therefore, the customized pipeline is needed to improve the accuracy and sensitivity of SNVs at genes with high-level homology. Albeit the present cost and turnaround time of WGS is several times more than the present NBS methods, in the forseeable future the pitfalls of WGS cost and turnaround time will likely facilitate the application of newborn WGS in NBS programs.
In our study, selective identification of genomic data, where therapeutic options are available, did not violate the Wilson-Jungner criteria 10 . Our work provides a basis for future research on expanding screening genes and diseases in newborn screening program. Given adequate cost-effectiveness, WGS should be considered in future newborn screening programs. Further discussion of the interpretation accuracy and ethical use of genomic information needs to take place on a global scale.

F I G U R E 2 (A)
Comparison between the findings of newborn WGS and routine NBS in the Qingdao cohort (n = 321). The findings of routine NBS tests are summarized on the left side of (A). In total, 18 carriers of hearing loss associated genes, one patient with PKU and one false positive result of C5-OH were detected by existing routine methods. The findings of WGS are shown on the right side of (A). The WGS results confirmed the positive routine NBS findings of 18 carriers of hearing loss and the case with increased level of Phe. However, the infant with a routine NBS showing an increased level of C5-OH (sample ID 18110806) was found to carry one pathogenic mutation in MCCC1, corresponding to being a carrier of 3-methylcrotonyl-CoA carboxylase deficiency (3MCCD). The newborn WGS also identified more infants carrying extra hearing loss mutations that were not identified by the routine NBS method, including 2 newborns carrying compound heterozygous P/LP variants in GJB2, 4 newborns harbouring a pathogenic mutation in MT-RNR and 17 additional carriers harboured altogether 19 variants. Moreover, newborn WGS identified 59 extra carriers carrying 66 P/LP variants corresponding to 18 inherited metabolic diseases that could not be identified by the routine NBS tests. The abbreviations of diseases and the summary of are listed in Table S1. (B) Comparison of allele frequency of actionable PGx variants between the Qingdao cohort and five subpopulations of the 1000 Genome Project dataset, including East Asians (EAS), South Asians (SAS), Africans (AFR), Europeans (EUR) and Americans (AMR). In most cases, the allele frequency of the Qingdao cohort is consistent with the EAS, but differed significantly with the SAS, the AFR, the EUR and the AMR, such as CYP2C19*2, CYP2C19*3 and NUDT15*6. It should be noted, however, that three common PGx variants in the Qingdao cohort, CYP2D6*10 (48.60%), NUDT15*3 (13.08%) and UGT1A1*6 (21.18%), showed significant frequency differences with the EAS (p < 0.05), indicating population diversity within the East Asians. Notably, two rare variants, CYP2D6*8 (1.87%, n = 11) and CYP2C19*6 (0.16%, n = 1) which have not been reported in any subpopulation in the 1000 Genome phase 3 dataset and were first detected in our Qingdao 321 newborns

A C K N O W L E D G E M E N T S
We appreciate the participation of the volunteers and their families. Without their support, this work would not have been possible. This work was also supported by the China National GeneBank (CNGB). This study was funded by the National Natural Science Foundation of China (No.31800765), the Shenzhen Municipal Government of China (JCY20170817145047361) and the Guangdong Provincial Key Laboratory of Genome Read and Write (No. 2017B030301011).