Newborn Screening for Inborn Errors of Metabolism by Next-Generation Sequencing Combined with Tandem Mass Spectrometry.

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.

Genomic Sequencing as a First-Tier Screening Test and Outcomes of Newborn Screening.

Importance: Newborn screening via biochemical tests is in use worldwide. The availability of genetic sequencing has allowed rapid screening for a substantial number of monogenic disorders. However, the outcomes of this strategy have not been evaluated in a general newborn population. Objective: To evaluate the outcomes of applying gene panel sequencing as a first-tier newborn screening test. Design, Setting, and Participants: This cohort study included newborns who were prospectively recruited from 8 screening centers in China between February 21 and December 31, 2021. Neonates with positive results were followed up before July 5, 2022. Exposures: All participants were concurrently screened using dried blood spots. The screen consisted of biochemical screening tests and a targeted gene panel sequencing test for 128 conditions. The biochemical and genomic tests could both detect 43 of the conditions, whereas the other 85 conditions were screened solely by the gene panel. Main Outcomes and Measures: The primary outcomes were the number of patients detected by gene panel sequencing but undetected by the biochemical test. Results: This study prospectively recruited 29 601 newborns (15 357 [51.2%] male). The mean (SD) gestational age was 39.0 (1.5) weeks, and the mean (SD) birth weight was 3273 (457) g. The gene panel sequencing screened 813 infants (2.7%; 95% CI, 2.6%-2.9%) as positive. By the date of follow-up, 402 infants (1.4%; 95% CI, 1.2%-1.5%) had been diagnosed, indicating the positive predictive value was 50.4% (95% CI, 50.0%-53.9%). The gene panel sequencing identified 59 patients undetected by biochemical tests, including 20 patients affected by biochemically and genetically screened disorders and 39 patients affected by solely genetically screened disorders, which translates into 1 out of every 500 newborns (95% CI, 1/385-1/625) benefiting from the implementation of gene panels as a first-tier screening test. Conclusions and Relevance: In this cohort study, the use of gene panel sequencing in a general newborn population as a first-tier screening test improved the detection capability of traditional screening, providing an evidence-based suggestion that it could be considered as a crucial method for first-tier screening.

Application of an optimized interpretation model in capillary hemoglobin electrophoresis for newborn thalassemia screening.

INTRODUCTION: Newborn screening is an important supplement to thalassemia control and prevention. Capillary electrophoresis (CE) technology has several advantages for thalassemia screening but with low sensitivity, especially for thalassemia carriers. This study aims to illustrate the application of an optimized interpretation model in newborn thalassemia screening by capillary hemoglobin electrophoresis. METHODS: Two thousand, two hundred fifty-eight neonates selected from four regions in China were enrolled and were screened for α-thalassemia and ß-thalassemia by capillary electrophoresis. Results were interpreted based on an optimized model integrated with multiple parameters. Molecular analysis was carried out in synchrony and used as the gold standard for the screening performance assessment. The consistency among different regions and thalassemia genotypes were also investigated. RESULTS: Among the 2258 neonates, 485 were identified to have a likely diagnosis of thalassemia, and 422 α-thalassemia, 80 ß-thalassemia, and 21 α/ß-thalassemia cases were confirmed by molecular analysis, including 277 α-thalassemia silent carriers, 135 α-thalassemia trait carriers, 10 Hemoglobin H disease, and 80 ß-thalassemia trait carriers. The screening sensitivity, specificity, positive, and negative predictive value for α-thalassemia and ß-thalassemia were 84.83%, 99.14%, 95.98%, 96.41%, and 88.75%, 98.73%, 76.34%, and 99.48%, respectively. The optimized interpretation model showed higher performance for thalassemia carriers, though some neonates with silent α-thalassemia genotypes (-α3.7 /αα, -α4.2 /αα, and αWS α/αα) and ß-28 /ßN genotype were still missed. The screening performance among different regions was comparable. CONCLUSIONS: Capillary hemoglobin electrophoresis with the optimized interpretation model shows reliable performance for newborn thalassemia screening. It is applicable to large-scale population screening.

Variants of the phenylalanine hydroxylase gene in neonates with phenylketonuria in Hainan, China.

OBJECTIVE: To investigate the incidence of phenylalanine hydroxylase (PAH) deficiency and PAH genotypes in neonates in Hainan, China. Methods: We performed heal stick to collect blood and obtain dry blood spot specimens from newborns in Hainan from January 2007 to December 2016. Phenylalanine (Phe) concentration in these dry blood spots was measured by the fluorescence method to screen phenylketonuria (PKU). For suspicious samples, the genotypes of the PAH gene were amplified by biotin labeled oligonucleotide primers. Polymerase chain reaction (PCR) products were then analyzed by flow-through hybridization to detect genotypes. At the same time, peripheral blood samples of children suspicious of PKU and their parents were used to perform gene sequencing. Results: Of the 914,520 newborns screened, 29 of them had PAH deficiency. The incidence of PAH deficiency in Hainan was 3.17/100,000. A total of 58 mutant alleles belonging to 15 different types were identified in the 29 patients. In terms of genotypes frequency, the top 4 were: c.611A > G 20.7% (12/58) , c.728G > A 17.2%, c.158G > A 15.2% (9/58) and c.721C > T 13.8% (8/58). The frequencies of other genotypes were all below 10%. Conclusion: The incidence of PAH deficiency in Hainan is relatively high among all provinces in southern China. With a total frequency of 67.2%, c.611A > G, c.728G > A, c.158G > A and c.721C > T, and are the most common PAH gene genotypes.

[Analysis of Genetic Screening in Couples of Reproductive Age for Thalassemia in Lingshui Li Autonomous County of Hainan Province].

OBJECTIVE: To investigate the genetic carrier rate of thalassemia and its gene mutation types as well as the distribution characteristics among the people in Lingshui Li autonomous county of Hainan province, so as to provide the basis for making the prevention programs of thalassemia in administrative departments. METHODS: Samples were collected from couples undergoing premarital and pregestational screenings, in which the positive ones in preliminary screening were further tested by genetic diagnoses and the genotypes were analyzed. RESULTS: The rate of thalassemia gene carriers was 19.41% （274/1412） of the couples of childbearing age in Lingshui Li autonomous County of Hainan Province. In these carriers,α-thalassemia accounted for 83.21%（228/274）, ß-thalassemia for 8.03%（22/274）, and both α-and ß-thalassemia gene accounted for 8.76% （28/274）. CONCLUSION: The carrying rate of thalassemia gene in population Lingshui Li autonomous county of Hainan province is high, and its distribution has geographical characteristics,the major type is α-thalassemia. Blood screening and genetic diagnosis of thalassemia should be strengthened, and corresponding measures should be taken to reduce its gene frequency.