The development of NGS has revolutionized genetic research and NGS methods have been proven to be effective for detecting genetic disorders (13). Since our previous study demonstrated that DNA extracted from DBS could be used for NGS, we designed a gene panel consisting of 77 genes related to amino acid metabolism, organic acid metabolism, and fatty acid oxidation and compared the specificity and sensitivity of the sequencing data (7). This cohort included 1,173 DBS, among which 1,127 (96.1%) qualified for NGS. Several factors can affect the yield, including individual differences in white blood cells, in addition to DNA degradation rates due to storage of the DBS samples for over a year before retrieval for NGS. The subjects in our study were born in September 2016 and their NBS samples were continuously numbered from ID 83038 to 84210.
Comparisons of biochemical screening and genetic results
Four newborns (all males) had low G6PD enzyme levels with hemizygous variants classified as DM or DM? in the HGMD database. The biochemical results for G6PD were consistent with the genetic variants, indicating the high sensitivity and specificity of G6PD. G6PD deficiency is an X-linked, genetic defect that arises due to a mutation in G6PD and is the most common human enzyme defect. The most frequent clinical manifestations of G6PD deficiency are neonatal jaundice and acute hemolytic anemia, which are usually triggered by an exogenous agent. Heterozygous females generally have less severe clinical manifestations than those in G6PD-deficient males (11). The highest frequencies of G6PD deficiency occur in tropical Africa, tropical/subtropical Asia, the Mediterranean region, and the Middle East. The global distribution of G6PD deficiency is similar to that for epidemic areas of malaria, indicating that G6PD deficiency confers resistance against malaria (14, 15). In this study, the four newborns with low G6PD levels included two males born in Shanghai (ID 83162, 84010) and two males born in Macao (ID 83841, 83847). In this cohort, 557 males were born in Shanghai, two of whom had G6PD deficiency. Among 43 males born in Macao, which is located in the subtropical area of Asia, two males had G6PD deficiency. These findings indicated the higher prevalence of G6PD deficiency in Macao than that in Shanghai.
Three infants had high 17-OHP levels at birth; with normal when they were called back and re-tested again in October 2016. Reexamination for false-positive findings for 17-OHP enzymatic screening at birth could be time-consuming. Thus, a second-tier test may be needed to improve the efficacy of this screening and reduce the number of false-positive findings.
In addition to the cases mentioned above, two children with negative biochemical results harbored DUOX2, and SLC22A5 variants, respectively.
DUOX2 is an autosomal recessive gene related to thyroid hormone synthesis dysfunction (16). One case (ID 84066) harbored two DUOX2 variants, c.3391G>T(p.A1131S) and c.2202G>A(p.W734*). Whether the two DUOX2 variants were located in the same allele could not be distinguished in the integrative genomics viewer, and samples from the parents were required for verification. Our review of the related literature revealed a report of a patient with congenital hypothyroidism harbored compound heterozygous variations p.W734*/p.A1131S (17). The patient (patient 1) in the literature showed a high TSH level. According to the guidelines for variant interpretation from ACMG/AMP, c.2202G>A(p.W734*) can be interpreted as a pathogenic variant (PVS1+PM2+PP4), and c.3391G>T(p.A1131S) can be interpreted as a likely pathogenic variant (PM1+PM2+PM3+PP3+PP4)(18). The newborn (ID 84066) in this study harbored the same variants and a normal TSH level (1.5mU/L; the cut-off value for TSH in our lab is 10). The infant was born at full term (38.5 weeks) with normal weight (3.095 kg); therefore, the possibility of late-onset TSH increase was low. We failed to contact the family of the newborn for further study; however, there are two possible explanations for these findings. First, the two variants may be located at one allele; thus, there would be no impact on her thyroid. Second, this observation could also have occurred due to a false-negative biochemical result. Our study has certain limitations. The proband-only sequencing approach made it hard to distinguish whether the two variants were bi-allelic or located at one allele of the recessive gene, as we did not collect the blood samples of the parents, unless the two variants were not far and could be distinguished using the integrative genomics viewer.
Homozygous or compound heterozygous mutations in SLC22A5 can cause systemic primary carnitine deficiency (SPCD) and lead to impaired fatty acid oxidation in muscles (19). This study identified one case (ID 84123) with compound heterozygous SLC22A5 variants. His C0 (free carnitine) value was 11.6μmol/L (reference: 10-60μmol/L) at birth in September 2016. When he was called back in April 2019 due to SLC22A5 mutations, his C0 value was 4.3μmol/L, which was lower than the normal range and could be classified as SPCD. SPCD encompasses a broad clinical spectrum including metabolic decompensation in infancy, childhood myopathy involving heart and skeletal muscle, pregnancy-related decreased stamina or exacerbation of cardiac arrhythmia, fatigability in adulthood, or absence of symptoms (20). Genomic screening can avoid diagnostic odyssey and allow optimal early treatment strategies in affected newborns. The child is nearly 3 years old, and has not yet shown any symptoms. This child should receive regular physical examination and possible treatment to maintain his plasma carnitine levels and prevent primary manifestations.
Our team focused on IMDs, and we determined the carrier frequencies of 77 genes related to disorders detectable by MS/MS, including amino acid metabolism, organic acid metabolism, and fatty acid oxidation, which are listed in Table S2. A total of 51 genes were identified in 1,127 subjects, the other 26 genes with DM or DM? variants were not detected according to our criteria. The top five genes with the highest carrier frequencies in these newborns were PAH (1.77%), ETFDH (1.24%), MMACHC (1.15%), SLC25A13 (0.98%), and GCDH (0.80%). According to previous studies in our lab, the prevalence of HPA was 1:11,763 and the prevalence of methylmalonic acidemia and hyperhomocysteinemia, cblC type was 1:28,000(21, 22). The carrier frequencies of PAH and MMACHC were consistent with the prevalence, indicating the rationality of our data. To the best of our knowledge, this is the first study to report the carrier frequencies of these many genes in China.
In 2018, a pilot study of expanded carrier screening in China investigated 11 recessive diseases, including PAH deficiency, reporting a PAH carrier frequency of 3.59% among all ethnicities in China. The results varied among different ethnicities; the PAH frequency for Han ethnicity was 3.30% (8), which was higher than that in our study (1.77%). The subjects in our study were mainly born in Shanghai and the population was comprised mostly of individuals of the Han ethnicity. One explanation for the discordance in carrier frequencies between the two studies may lie in the difference in the criteria used to determine variants.
Another study in 2016 applied a molecular approach for NBS for four genetic diseases in Guizhou Province of southern China, including beta-thalassemia, G6PD deficiency, PKU, and non-syndromic hearing loss. The study included 515 newborns and selected 10 common mutations of PAH in the Chinese population, and reported a PAH carrier frequency of 0.78% (23). This frequency was much lower than that in our study (1.77%). Our data indicated 20 newborns carrying variants with mutations in 17 different positions, and all of which were classified as DM or DM? This comparison also suggests that PAH is better screened as whole exons rather than as variants based on mutations in several positions.