The aim of this study was to observe the outcomes of newborn screening (NBS) in a certain population by using next-generation sequencing (NGS) as a first-tier screening test combined with tandem mass spectrometry (MS/MS). We performed a multicenter study of 29,601 newborns from eight screening centers with NBS via NGS combined with MS/MS. A custom-designed panel targeting the coding region of the 142 genes of 128 inborn errors of metabolism (IEMs) was applied as a first-tier screening test, and expanded NBS using MS/MS was executed simultaneously. In total, 52 genes associated with the 38 IEMs screened by MS/MS were analyzed. The NBS performance of these two methods was analyzed and compared respectively. A total of 23 IEMs were diagnosed via NGS combined with MS/MS. The incidence of IEMs was approximately 1 in 1287. Within separate statistical analyses, the positive predictive value (PPV) for MS/MS was 5.29%, and the sensitivity was 91.3%. However, for genetic screening alone, the PPV for NGS was 70.83%, with 73.91% sensitivity. The three most common IEMs were methylmalonic academia (MMA), primary carnitine deficiency (PCD) and phenylketonuria (PKU). The five genes with the most common carrier frequencies were PAH (1:42), PRODH (1:51), MMACHC (1:52), SLC25A13 (1:55) and SLC22A5 (1:63). Our study showed that NBS combined with NGS and MS/MS improves the performance of screening methods, optimizes the process, and provides accurate diagnoses.

目的：了解广州地区新生儿串联质谱遗传代谢病筛查病种的分布特征、发病率及确诊患者的临床转归，优化筛查指标，提高串联质谱遗传代谢病筛查性能。方法：采用串联质谱技术对2015年1月至2020年12月在广州市出生的272 117名新生儿进行多种遗传代谢病筛查，以主要筛查指标2次超出阳性切值判断为筛查阳性，召回复查，对疑似母源性疾病连同母亲一并召回，复查仍阳性者进行生化及相关基因测序确诊。回顾性分析筛查结果、确诊病例的临床特点及转归，以及优化筛查指标。结果：在272 117名新生儿中，初筛阳性1808例，阳性率为0.66%，召回复查1738例，召回率为96.13%，临床诊断时间中位数为15 d，确诊新生儿遗传代谢病79例，包括氨基酸代谢病23例、有机酸代谢病17例及脂肪酸氧化障碍39例，共计21个病种，常见病种为原发性肉碱缺乏病（26.6%）、甲基丙二酸尿症（12.7%）及苯丙氨酸羟化酶缺乏症（11.4%），发现假阴性病例4例，均为希特林蛋白缺乏症。272 117名新生儿遗传代谢病筛查总体发病率约为1/3444，阳性预测值为4.5%。确诊母亲患者32例，包括原发性肉碱缺乏病30例、异丁酰基辅酶A脱氢酶缺乏症及3-甲基巴豆酰辅酶A羧化酶缺乏症各1例。遗传代谢病筛查总体检出率为1/2451。所有确诊病例随访至今，5例重型患儿（枫糖尿症1例、单纯型甲基丙二酸尿症2例、肉碱-酰基肉碱移位酶缺乏症2例）早期死亡，10例有机酸代谢病患儿出现轻度精神运动发育迟缓。优化各病种筛查指标后，初筛阳性数降至963例，阳性预测值提高至8.6%，未遗漏确诊病例。结论：广州地区新生儿脂肪酸氧化障碍发病率相对较高，串联质谱技术能有效筛查多种遗传代谢病，优化筛查指标可提高筛查性能。

The aim of this study was to improve screening efficiency by establishing reasonable interpretation criteria for the use of flow injection analysis tandem mass spectrometry (FIA-MS/MS) in newborn screening (NBS) for X-linked adrenoleukodystrophy (X-ALD). FIA-MS/MS was employed to analyze very-long-chain acylcarnitines (ACs) and lysophosphatidylcholines (LPCs) and their ratios in dried blood spot (DBS) obtained from five X-ALD patients in the neonatal period (0–7 days old) and 7123 healthy neonate controls. By comparing these results and analyzing receiver operating characteristic (ROC) curves, we identified sensitive indicators for X-ALD screening in newborns. To evaluate the performance of different FIA-MS/MS screening indicators, we simultaneously analyzed 7712 neonatal DBS samples obtained for X-ALD screening using FIA-MS/MS and the established liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantitative detection of C26:0-lysophosphatidylcholine (C26:0-LPC). Furthermore, 84,268 newborn X-ALD screening results were retrospectively analyzed to further evaluate the screening performance of FIA-MS/MS. After the three-step optimization evaluation, the optimized first-tier sensitive screening indicators of FIA-MS/MS were C24:0-AC, C26:0LPC, and C24:0/C22:0-AC. Among the 7712 newborns screened, one case was confirmed to be double-positive. Within separate statistical analyses, based on LC-MS/MS screening alone (positive cutoff > 0.17 µmol/L), only seven cases (0.09%) were initially positive, with a positive predictive value (PPV) of 42.8%, and two additional ABCD1 VUS hemizygous males were detected. Through the retrospective analysis of 84,268 newborns, eight ABCD1 variants (six hemizygous males and two heterozygous females) were ultimately identified. Our study showed that the optimization of first-tier screening performance is particularly important if second-tier screening is not performed. Using LC-MS/MS for second-tier screening for X-ALD can significantly reduce the number of false positives, but the method still misses some false negatives. If it is used as a first-tier assessment, more VUS variant neonates can be detected.