Genotypic and Phenotypic Spectrum of maple syrup urine disease in Zhejiang of China.

BACKGROUND: Maple Syrup Urine Disease (MSUD) is an autosomal recessive metabolic disorder originating from defects in the branched-chain α-ketoacid dehydrogenase (BCKDH) complex encoded by BCKDHA, BCKDHB, and DBT. This condition presents a spectrum of symptoms and potentially fatal outcomes. Although numerous mutations in the BCKDH complex genes associated with MSUD have been identified, the relationship between specific genotypes remains to be fully elucidated. AIM: Our objective was to predict the pathogenicity of these genetic mutations and establish potential links between genotypic alterations and the clinical phenotypes of MSUD. DESIGN: Retrospective population-based cohort. METHODS: We analyzed 20 MSUD patients from the Children's Hospital at Zhejiang University School of Medicine (Hangzhou, China), recorded from January 2010 to May 2023. Patients' blood samples were collected by heel-stick through neonatal screening, and amino acid profiles were measured by tandem mass spectrometry. In silico methods were employed to assess the pathogenicity, stability, and biophysical properties. Various computation tools were utilized for assessment, namely PredictSNP, MAGPIE, iStable, Align GVGD, ConSurf and SNP effect. RESULTS: We detected 25 distinct mutations, including 12 novel mutations. The BCKDHB gene was the most commonly affected (53.3%) compared to the BCKDHA gene (20.0%) and DBT gene (26.7%). In silico webservers predicted all novel mutations were disease-causing. CONCLUSIONS: This study highlights the genetic complexity of MSUD and underscores the importance of early detection and intervention. Integrating neonatal screening with advanced sequencing methodologies is pivotal in ensuring precise diagnosis and effective management of MSUD, thereby significantly improving the prognosis for individuals afflicted with this condition.

Progress of newborn screening in China. / 中国新生儿筛查进展.

Newborn screening (NBS) plays a significant role in reducing the risk of birth defects. NBS in China began in the early 1980s. Under the protection of laws and regulations and the leadership of the national health administration, approved screening centers in public hospitals took the responsibility for publicity, screening, diagnosis, treatment, follow-up and management of birth defects. As of 2022, 31 provinces (autonomous regions and municipalities directly under the central government) have carried out NBS for phenylketonuria, congenital hypothyroidism, and hearing loss, 23 provinces have carried out screening for glucose-6-phosphate dehydrogenase (with a screening rate of 89.24%), and 24 provinces have carried out screening for congenital adrenal cortical hyperplasia (91.45% screening rate). Over the past four decades, screening techniques have evolved from bacterial inhibition, fluorescence analysis, and tandem mass spectrometry for the detection of biochemical markers to genetic testing, which has greatly contributed to the expansion of the types of diseases screened for. The combined use of metabolomics and genomics is currently being explored. Effective management and rigorous quality control of NBS are prerequisites for improving the quality and ensuring the accuracy of screening. The Quality Management System for Newborn Screening System Network (QMS-NBS), established by the National Center for Clinical Laboratories, covers all screening centers and related blood collection agencies. The operation of the QMS-NBS allows the quality and performance of screening to be transparent and measurable, ensuring the quality and efficiency of screening. This article provides an overview of the history of NBS, especially the evolution of policies for the NBS in China, the construction of screening institutions, the number of newborns screened, the incidence rates of screened diseases, the changes in screening technology, the expansion of new diseases screened for, and the quality control of NBS. Overall, the progress in NBS in China has not only benefited from the development and standardization at the technological level, but also benefited from the construction of policies, regulations and ethics.

Results of neonatal screening for congenital hypothyroidism and hyperphenylalaninemia in Zhejiang province from 1999 to 2022. / 浙江省1200ä¸‡ä¾‹æ–°ç”Ÿå„¿å ˆå¤©æ€§ç”²çŠ¶è ºåŠŸèƒ½å‡é€€ç—‡ã€é«˜è‹¯ä¸™æ°¨é ¸è¡€ç—‡ç­›æŸ¥ç»“æžœåˆ†æž.

OBJECTIVES: To analyze the results of neonatal screening for congenital hypothyroidism (CH) and hyperphenylalaninemia (HPA) in Zhejiang province from 1999 to 2022. METHODS: A total of 11 922 318 newborns were screened from September 1999 and December 2022 in Zhejiang province. The blood thyroid stimulating hormone (TSH) levels were measured by a fluorescence method and blood phenylalanine (Phe) levels were measured by fluorescence method or tandem mass spectrometry. TSH≥9 µIU/mL was considered positive for CH, while Phe>120 µmol/L and/or Phe/Tyr ratio>2.0 were considered positive for HPA. The positive newborns in screening were recalled, and the gene variations were detected by high-throughput sequencing and MassARRAY tests. RESULTS: The overall neonatal screening rate during 1999-2022 was 89.41% (11 922 318/13 333 929) and the screening rate was increased from 6.46% in 1999 to 100.0% in 2022. A total of 8924 cases of CH were diagnosed among screened newborns with an incidence rate of 1/1336. A total of 563 cases of HPA were diagnosed, including 508 cases of classic phenylketonuria (cPKU) and 55 cases of tetrahydrobiopterin deficiency (BH4D), with an incidence rate of 1/21 176. Ninety-seven out of 8924 cases of CH underwent genetic analysis. Gene mutations were detected in 9 CH related genes, the highest frequency mutations were found in DUOX2 gene (69.0%) with c.3329G>A (p.R1110Q) (18.2%) and c.1588A>T (p.K530X) (17.3%) as the hotspot mutations. There were 81 PAH gene variants detected in a total of 250 cases of cPKU, and c728G>A (p.R243Q) (24.4%), c.721C>T (p.R241C) (15.0%) were the hotspot mutations. Meanwhile 7 novel variants in PAH gene were detected: c.107C>A (p.S36*), c.137G>T (p.G46V), c.148A>G(p.K50E), c.285C>T (p.I95I), c.843-10delTTCC, exon4-7del and c.1066-2A>G. There were 12 PTS gene variants detected in 36 cases of BH4D, and c.259C>T (p.P87S) (31.9%) was the hotspot mutation. CONCLUSIONS: The incident of CH has increased from 1999 to 2022 in Zhejiang province, and it is higher than that of national and global levels; while the incidence of HPA is similar to the national average. DUOX2 gene variation is the most common in CH patients; c.728G>A (p.R243Q) is the hotspot mutation in cPKU patients, while c.259C>T (p.P87S) is the hotspot mutation in BH4D patients.

Analysis of genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine. / ä¸é °åŸºè‚‰ç¢±ä»£è°¢å¼‚å¸¸æ–°ç”Ÿå„¿çš„åŸºå› åž‹å’Œç”ŸåŒ–è¡¨åž‹åˆ†æž.

OBJECTIVES: To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4). METHODS: One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children's Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types. RESULTS: In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations. CONCLUSIONS: ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.

Long-term follow-up of children with carbamoyl phosphate synthase 1 deficiency detected in newborn screening. / æ–°ç”Ÿå„¿ç­›æŸ¥å‘çŽ°çš„æ°¨ç”²é °ç£·é ¸åˆæˆé ¶1ç¼ºä¹ç—‡æ‚£å„¿é•¿æœŸéšè®¿ç ”ç©¶.

OBJECTIVES: To investigate genotype-phenotype characteristics and long-term prognosis of neonatal carbamoyl phosphate synthetase 1 (CPS1) deficiency among children through newborn screening in Zhejiang province. METHODS: The clinical and follow-up data of children with CPS1 deficiency detected through neonatal screening and confirmed by tandem mass spectrometry and genetic testing in Zhejiang Province Newborn Disease Screening Center from September 2013 to August 2023 were retrospectively analyzed. RESULTS: A total of 4 056 755 newborns were screened and 6 cases of CPS1 deficiency were diagnosed through phenotypic and genetic testing. Ten different variations of CPS1 genewere identified in genetic testing, including 2 known pathogenic variations (c.2359C>T and c.1549+1G>T) and 8 unreported variations (c.3405-1G>T, c.2372C>T, c.1436C>T, c.2228T>C, c.2441G>A, c.3031G>A, c.3075T>C and c.390-403del). All patients had decreased citrulline levels (2.72-6.21 µmol/L), and varying degrees of elevated blood ammonia. The patients received restricted natural protein intake (special formula), arginine and supportive therapy after diagnosis, and were followed-up for a period ranging from 9 months to 10 years. Three patients experienced hyperammonemia, and one patient each had attention deficit hyperactivity disorder, transient facial twitching and increased muscle tone. One patient died, while the other five surviving patients had normal scores of the Ages & Stages Questionnaires (ASQ) and Griffiths Development Scales up to the present time; 4 cases had combined height or weight lag and one case was normal in height and weight. CONCLUSIONS: Low citrulline levels and hyperammonemia are common in CPS1 deficiency patients in Zhejiang. Most gene variants identified were specific to individual families, and no hotspot mutations were found. Early diagnosis through newborn screening and following standardized treatment can significantly improve the prognosis of the patients.

Association Analysis of Gene Sequencing by NeoSeq Combined with Tandem Mass Spectrum and Four Neonatal Diseases.

BACKGROUND: NeoSeq is a new method of gene sequencing for newborn screening. The goal is to explore the relationship between gene sequencing by NeoSeq combined with tandem mass spectrum (TMS) and four neonatal diseases. METHODS: A total of 1,989 newborns from August 2010 to December 2021 were enrolled. The case number of congenital hypothyroidism, phenylketonuria, adrenocortical hyperplasia, and glucose-6-phosphate dehydrogenase deficiency was counted, and the results of gene sequencing by NeoSeq and TMS were analyzed. RESULTS: The proportion of male newborns was higher than that of female newborns (51.68% vs. 48.32%). The detection rate of glucose-6-phosphate dehydrogenase deficiency was higher than that of the other three diseases (0.60% vs. 0.05%, 0.05%, 0.15%). A total of 121 newborns were recalled from 1989 newborns by traditional screening technique, and TMS detected phenylketonuria, citrullinemia, glutaric acidemia type I, and 3-methylcro-tonyl-CoA carboxylase deficiency in 1 newborn each. Gene sequencing by NeoSeq of newborns with positive TMS results confirmed the presence of susceptibility genes, and 17 of 1,868 newborns with normal biochemical tests had pathogenic genes. CONCLUSIONS: The incidence of glucose-6-phosphate dehydrogenase deficiency is relatively higher in four neonatal diseases, and the detection rate of gene sequencing by NeoSeq combined with TMS is high.

Clinical, biochemical characteristics and genotype-phenotype analysis of congenital hypothyroidism diagnosed by newborn screening in China.

BACKGROUND: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder worldwide. However, the underlying etiology remains unclear in most patients. METHODS: The newborn screening was performed for TSH in dried blood spots. Serum TSH, T3, T4, free T3(FT3) and free T4 (FT4) were detected for the recalled children. High-throughput sequencing were applied to detect 29 known CH genes. The statistical analyses were performed to analyze the differences between biochemical data, thyroid volume, clinical prognosis and genetic results for 97 patients who had one or more variants in CH related genes. RESULTS: DUOX2 gene had the highest variant rate, followed by TG, TPO and TSHR gene. The "DUOX2 biallelic variants" group was associated with "Goiter", while "DUOX2 monoallelic variants" group was associated with "Agenesis". In addition, the TSH levels and initial L-T4 dose were significantly higher in "TPO biallelic variants" group than those in "DUOX2 and TSHR biallelic variants" groups. CONCLUSIONS: Our study showed dyshormonogenesis (DH) might be the leading pathophysiology of CH in Chinese populations. DUOX2 gene mostly caused goiter, but also could be associated with hypoplasia. TPO might play a more irreplaceable role than DUOX2. The digenic variants combination indicated the complexity of genetic etiology in CH.

[A prospective study of genetic screening of 2 060 neonates by high-throughput sequencing].

OBJECTIVE: To assess the value of genetic screening by high-throughput sequencing (HTS) for the early diagnosis of neonatal diseases. METHODS: A total of 2 060 neonates born at Ningbo Women and Children's Hospital from March to September 2021 were selected as the study subjects. All neonates had undergone conventional tandem mass spectrometry metabolite analysis and fluorescent immunoassay analysis. HTS was carried out to detect the definite pathogenic variant sites with high-frequency of 135 disease-related genes. Candidate variants were verified by Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA). RESULTS: Among the 2 060 newborns, 31 were diagnosed with genetic diseases, 557 were found to be carriers, and 1 472 were negative. Among the 31 neonates, 5 had G6PD, 19 had hereditary non-syndromic deafness due to variants of GJB2, GJB3 and MT-RNR1 genes, 2 had PAH gene variants, 1 had GAA gene variants, 1 had SMN1 gene variants, 2 had MTTL1 gene variants, and 1 had GH1 gene variants. Clinically, 1 child had Spinal muscular atrophy (SMA), 1 had Glycogen storage disease II, 2 had congenital deafness, and 5 had G6PD deficiency. One mother was diagnosed with SMA. No patient was detected by conventional tandem mass spectrometry. Conventional fluorescence immunoassay had revealed 5 cases of G6PD deficiency (all positive by genetic screening) and 2 cases of hypothyroidism (identified as carriers). The most common variants identified in this region have involved DUOX2 (3.93%), ATP7B (2.48%), SLC26A4 (2.38%), GJB2 (2.33%), PAH (2.09%) and SLC22A5 genes (2.09%). CONCLUSION: Neonatal genetic screening has a wide range of detection and high detection rate, which can significantly improve the efficacy of newborn screening when combined with conventional screening and facilitate secondary prevention for the affected children, diagnosis of family members and genetic counseling for the carriers.

A multicenter prospective study of next-generation sequencing-based newborn screening for monogenic genetic diseases in China.

BACKGROUND: Newborn screening (NBS) is an important and successful public health program that helps improve the long-term clinical outcomes of newborns by providing early diagnosis and treatment of certain inborn diseases. The development of next-generation sequencing (NGS) technology provides new opportunities to expand current newborn screening methodologies. METHODS: We designed a a newborn genetic screening (NBGS) panel targeting 135 genes associated with 75 inborn disorders by multiplex PCR combined with NGS. With this panel, a large-scale, multicenter, prospective multidisease analysis was conducted on dried blood spot (DBS) profiles from 21,442 neonates nationwide. RESULTS: We presented the positive detection rate and carrier frequency of diseases and related variants in different regions; and 168 (0.78%) positive cases were detected. Glucose-6-Phosphate Dehydrogenase deficiency (G6PDD) and phenylketonuria (PKU) had higher prevalence rates, which were significantly different in different regions. The positive detection of G6PD variants was quite common in south China, whereas PAH variants were most commonly identified in north China. In addition, NBGS identified 3 cases with DUOX2 variants and one with SLC25A13 variants, which were normal in conventional NBS, but were confirmed later as abnormal in repeated biochemical testing after recall. Eighty percent of high-frequency gene carriers and 60% of high-frequency variant carriers had obvious regional differences. On the premise that there was no significant difference in birth weight and gestational age, the biochemical indicators of SLC22A5 c.1400C > G and ACADSB c.1165A > G carriers were significantly different from those of non-carriers. CONCLUSIONS: We demonstrated that NBGS is an effective strategy to identify neonates affected with treatable diseases as a supplement to current NBS methods. Our data also showed that the prevalence of diseases has significant regional characteristics, which provides a theoretical basis for screening diseases in different regions.

[Analysis of clinical features, biochemical indices and genetic variants among children with Short/branched-chain acyl-CoA dehydrogenase deficiency detected by neonatal screening].

OBJECTIVE: To investigate the clinical manifestations, biochemical abnormalities and pathogenic variants among children with Short/branched-chain acyl-CoA dehydrogenase (SBCAD) deficiency detected by neonatal screening. METHODS: A total of 2 730 852 newborns were screened from January 2016 to December 2021 with liquid chromatography tandem mass spectrometry. Suspected SBCAD deficiency patients were diagnosed by urine organic acid analysis and high-throughput gene sequencing analysis. The clinical, biochemical and genetic changes of the confirmed cases were analyzed, in addition with guidance for diet and life management, L-carnitine supplement, and survey of growth and intellectual development. RESULTS: Twelve cases of SBCAD deficiency were diagnosed, which yielded a prevalence of 1/227 571. The lsovaleryl carnitine (C5) of primary screening blood samples was between 0.6 and 2.1 µmol/L, all exceeded the normal range. C5/acety1 carnitine (C2) was between 0.02 and 0.12, with 6 cases exceeding the normal range. C5/propionyl carnitine (C3) was between 0.1 and 1.16, with 5 cases exceeding the normal range. Free carnitine (C0) was between 18.89 and 58.12 µmol, with 1 case exceeding the normal range. Three neonates with abnormal screening results were recommended to have appropriate restriction for protein intake and two were given L-carnitine. During follow-up, their C5 has ranged from 0.22 to 2.32 µmol/L, C5/C2 has ranged from 0.01 to 0.31, C5/C3 has ranged from 0.14 to 1.7. C5 or C5/C2 and C5/C3 were transiently normal in all patients except for case 8 during the neonatal screening and follow-up. C0 was 17.42 ∼ 76.83 µmol/L Urine organic acid analysis was carried out in 9 of the 12 cases, and 2-methylbutyroglycine was elevated in 8 cases. Urine organic acid analysis was carried out in 9 cases, and 2-methylbutyrylglycine was increased in 8 cases. Genetic analysis was carried out for 11 children, and in total 6 ACADSB gene variants were identified, which included 4 missense variants (c.655G>A, c.923G>A, c.461G>A, c.1165A>G), 1 frameshift variant (c.746del) and 1 nonsense variant (c.275C>G). Among these, the C.461G>A variant was unreported previously. The most common variants were c.1165A>G (40.9%) and C.275C>G (22.7%). The patients were followed up for 18 days to 55 months. Only one patient had mental retardation, with the remainders having normal physical and mental development. CONCLUSION: SBCAD deficiency is a rare disease. The detection rate of newborn screening in this study was 1/227 571. Early intervention can be attained in most asymptomatic patients through neonatal screening. In this study, the common gene variants are c.1165A>G and c.275C>G.

Dynamic changes of metabolic characteristics in neonatal intrahepatic cholestasis caused by citrin deficiency.

Introduction: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a pan-ethnic complicated inborn error of metabolism but the specific mechanism is not fully understood. Methods: A total of 169 patients with NICCD who have biallelic pathogenic SLC25A13 variants detected by targeted next-generation sequencing were collected. They were divided into the "Newborn-screen Group" and "Clinical diagnosed Group" depending on the newborn screening results. Amino acid and acylcarnitine profiles were measured by MS/MS. The total bile acids, blood amino acids and acylcarnitines, general biochemistry, blood count, and coagulation parameters were monitored every 2-3 months. We compared the differences in metabolic indices and their dynamic changes between these two groups. The Mann-Whitney test and orthogonal partial least squares discrimination analysis (OPLS-DA) were used for statistical analysis. Results: At the onset of NICCD, we found that the "Clinical diagnosed Group" had higher levels of intermediate products of the urea cycle, free carnitine, and short-chain and long-chain acylcarnitines than those in the "Newborn-screen Group," but the levels of ketogenic/glucogenic amino acids and several medium-chain acylcarnitines were lower. Furthermore, concentrations of direct bilirubin, total bile acid, lactate, prothrombin time, and several liver enzymes were significantly higher while total protein, amylase, and hemoglobin were lower in the "Clinical diagnosed Group" than in the "Newborn-screen Group." Dynamic change analysis showed that direct bilirubin, albumin, arginine, and citrulline were the earliest metabolic derangements to reach peak levels in NICCD groups, followed by acylcarnitine profiles, and finally with the elevation of liver enzymes. All abnormal characteristic metabolic indicators in the "Newborn-screen Group" came back to normal levels at earlier ages than the "Clinical diagnosed Group." c.852_855del (41.2%), IVS16ins3kb (17.6%), c.615 + 5G>A (9.6%), 1638_1660dup (4.4%), and c.1177 + 1G>A (3.7%) accounted for 76.5% of all the mutated SLC25A13 alleles in our population. Conclusion: Argininosuccinate synthesis, gluconeogenesis, ketogenesis, fatty acid oxidation, liver function, and cholestasis were more severely affected in the "Clinical diagnosed Group." The "Newborn-screen Group" had a better prognosis which highlighted the importance of newborn screening of NICCD.

Application of a next-generation sequencing (NGS) panel in newborn screening efficiently identifies inborn disorders of neonates.

BACKGROUND: Newborn screening (NBS) has been implemented for neonatal inborn disorders using various technology platforms, but false-positive and false-negative results are still common. In addition, target diseases of NBS are limited by suitable biomarkers. Here we sought to assess the feasibility of further improving the screening using next-generation sequencing technology. METHODS: We designed a newborn genetic sequencing (NBGS) panel based on multiplex PCR and next generation sequencing to analyze 134 genes of 74 inborn disorders, that were validated in 287 samples with previously known mutations. A retrospective cohort of 4986 newborns was analyzed and compared with the biochemical results to evaluate the performance of this panel. RESULTS: The accuracy of the panel was 99.65% with all samples, and 154 mutations from 287 samples were 100% detected. In 4986 newborns, a total of 113 newborns were detected with biallelic or hemizygous mutations, of which 36 newborns were positive for the same disorder by both NBGS and conventional NBS (C-NBS) and 77 individuals were NBGS positive/C-NBS negative. Importantly, 4 of the 77 newborns were diagnosed currently including 1 newborn with methylmalonic acidemia, 1 newborn with primary systemic carnitine deficiency and 2 newborns with Wilson's disease. A total of 1326 newborns were found to be carriers with an overall carrier rate of 26.6%. CONCLUSION: Analysis based on next generation sequencing could effectively identify neonates affected with more congenital disorders. Combined with C-NBS, this approach may improve the early and accurate identification of neonates with inborn disorders. Our study lays the foundation for prospective studies and for implementing NGS-based analysis in NBS.

Long-term prognosis of 35 patients with methionine adenosyltransferase deficiency based on newborn screening in China.

Methionine adenosyltransferase deficiency (MATD) is a rare metabolic disorder caused by mono- or biallelic MAT1A mutations that are not yet well understood. Of the 4,065,644 neonates screened between November 2010 and December 2021, 35 individuals have been diagnosed with an estimated incidence of 1: 116,161 by a cutoff value of methionine 82.7 µmol/L and follow-up over 11 years. MATD patients with autosomal recessive (AR) type had higher clinical and genetic heterogeneity than those with autosomal dominant (AD) type. Fifteen unrelated AD patients harbored one well-known dominant variant, c.791 G>A or c.776 C>T, and were clinically unaffected with a mean plasma methionine (Met) value <300 µmol/L. Twenty AR cases have unique genotypes and presented a wide range of clinical abnormalities from asymptomatic to white matter lesions. Of them, 10 AR patients displayed severe manifestations, such as verbal difficulty, motor delay, development delay, and white matter lesions, with mean Met >500 µmol/L and thereby were treated with a methionine-restricted diet alone or in combination with betaine, folate, or vitamin B6, and were healthy finally. Neurological abnormalities were evidenced in two patients (P16 and P27) with Met values >800 µmol/L by MRI scan. Neurological abnormalities were reversed here by liver transplantation or by the determination of S-adenosylmethionine supplementation. Additionally, 38 variants of MAT1A were distributed within patients and carriers, of which 24 were novel and mostly predicted to be damaged. Our findings with an extensive clinical and genetic dataset provided new insights into its diagnosis and treatment and will be helpful for its optimal management in the future.

FMR1 allele frequencies in 51,000 newborns: a large-scale population study in China.

BACKGROUND: Fragile X syndrome (FXS), caused by CGG-repeat expansion in FMR1 promoter, is one of the most common causes of mental retardation. Individuals with full mutation and premutation alleles have a high risk of psychophysiological disorder and of having affected offspring. Frequencies of FMR1 alleles in general newborns have been reported in Caucasians but have not been investigated in the large-scale population in  the mainland of China. METHODS: The sizes of FMR1 CGG-repeats were analyzed in 51,661 newborns (28,114 males and 23,547 females) and also in a cohort of 33 children diagnosed with developmental delay using GC-rich polymerase chain reaction (PCR) and triple repeat primed PCR. RESULTS: The frequency of CGG repeats > 100 was 1/9371 in males and 1/5887 in females, and the frequency of CGG repeats > 54 was 1/1561 in males and 1/1624 in females. FMR1 full mutation and premutation were identified in 27.27% of children who had Ages and Stages Questionnaire scores less than two standard deviations from the cutoff value. CONCLUSIONS: Our study revealed the prevalence of FXS in China and improved the sample databases of FXS, suggesting that the prevalence of FXS in Chinese is higher than estimated previously and that FXS screening can be advised to high-risk families.

Application of genetic screening processor in screening neonatal glucose-6-phosphate dehydrogenase deficiency.

To evaluate the performance of genetic screening processor (GSP analyzer) in neonatal screening for glucose-6-phosphate dehydrogenase (G6PD)deficiency. The accuracy and precision of GSP analyzer was evaluated with the control materials from National Center for Clinical Laboratories and the low and high quality G6PD control kit (fluorescence analysis). GSP analyzer and semi-automatic fluorescence immunoanalyzer (1420 analyzer) were simultaneously used to detect 2622 neonatal screening samples and 41 confirmed samples to analyze the correlation and consistency of the test results; 78 floating samples and 78 non-floating samples were detected to compare the result. A total of 1 100 384 neonatal screening samples from January 2017 to December 2018 and 855 856 neonatal screening samples from January 2019 to December 2020 were detected with 1420 analyzer and GSP analyzer, respectively. Referring to the percentile method and the expert consensus, the new cut-off value of GSP analyzer for G6PD deficiency in screening was established. The relative bias of GSP analyzer in detecting G6PD was 0.71%-4.23%; the intra assay precision was 4.34%-4.91%, the inter assay precision was 0.85%-2.12%, and the total coefficient of variation was 5.44%-5.72%. There was a significant positive correlation between G6PD activity detected by GSP analyzer and 1420 analyzer (=0.740, <0.01). Forty-one clinical confirmed patients were identified by both 1420 analyzer and GSP analyzer (=0.945). The G6PD activity in floating dry blood spots detected by 1420 analyzer was significantly lower than that in non-floating dry blood spots (<0.05), but there was no significant difference in G6PD activity between floating and non-floating dry blood spots detected by GSP analyzer (>0.05). The sensitivities of GSP analyzer and 1420 analyzer in screening G6PD deficiency were both 100.00%, and the specificities were both more than 99.80%. Compared with 1420 analyzer, the positive predictive value, positive rate and prevalence of G6PD deficiency detected by GSP analyzer were increased, and the false positive rate was decreased (all <0.01). The new cut-off value was 26.1 U/dL for male and 29.1 U/dL for female according to the 99.1% percentile of the population. GSP analyzer has better detection performance with high automation, efficiency and throughput, which can be used in large-scale screening for neonatal G6PD deficiency.

Screening of multiple acyl-CoA dehydrogenase deficiency in newborns and follow-up of patients.

To investigate the incidence rate, clinical and gene mutation characteristics of multiple acyl-CoA dehydrogenase deficiency (MADD) in newborns in Zhejiang province. A total of 3 896 789 newborns were screened for MADD using tandem mass spectrometry in Zhejiang Neonatal Screening Center during January 2009 and December 2020. Patients of MADD were confirmed by urine organic acid and electron transferring flavoprotein (or electron transferring flavoprotein dehydrogenase () gene detection. MADD patients were given diet and life management, supplemented with L-carnitine, riboflavin and coenzyme Q 10 treatment, and their growth and intellectual development were evaluated during the followed up.Thirteen patients with MADD were diagnosed, with an incidence of 1/299 753. One patient was type Ⅱ, and the rest were type Ⅲ. Patients were followed up for 1 case died, 4 cases had acute metabolic disorders with hypoglycemia as the main manifestation due to infection, 1 case had hypotonia, and the rest 7 cases developed well. Patients had raised levels of C4-C18:1 acylcarnitines in the initial screening. Thirteen children were genetically tested, 1 case with compound heterozygous mutation in the gene, 1 case with homozygous mutation in the gene, 1 case with compound heterozygous mutation in the gene, 8 cases with compound heterozygous mutation and 1 case with homozygous mutation in the gene, 1 case that only 1 locus of gene was detected. The c.250G>A was the hotspot mutation in this study.The clinical manifestations of MADD are highly heterogeneous. The neonatal-onset form is serious, and late onset form usually has no obvious clinical symptoms. C4-C18:1 acylcarnitines usually increased in the initial screening, and the hotspot gene mutation is c.250G>A.

Case Report: Expanding the Digenic Variants Involved in Thyroid Hormone Synthesis-10 New Cases of Congenital Hypothyroidism and a Literature Review.

Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder. Although it has been understood to be a monogenic disease, some CH patients are reported to carry two or more variants at different genes. Here, ten permanent congenital hypothyroidism (PCH) patients were retrospectively reviewed, with elevated levels of serum thyroid-stimulating hormone and levothyroxine dependence during follow-up between 2015 and 2019. Each affected individual carried digenic variants, which were heterozygous at two of pathogenic genes. In total, five pathogenic genes, TSHR, TG, TPO, DUOX2 and DUOXA2, were simultaneously identified in subjects that were involved in the same metabolic pathway: thyroid hormone biosynthesis. There were digenic variants at TSHR and DUOX2 combined in three patients, DUOX2 and TG combined in two patients, DUOX2 and DUOXA2 combined in two patients, TG and DUOXA2 combined in two patients, and TG and TPO combined in one patient. Additionally, seven novel variants, TSHR c.679G>A, DUOX2 c.127A>T, c.608-619del, c.959T>C, TG c.2307G>A, and c.6759_6765del, and DUOXA2 c.93T>G, were identified in these PCH patients. Along with a literature review on digenic variants in patients with CH, our findings illustrated the complexity of genetic etiology in CH.

[Expert consensus for the diagnosis and treatment of glutaricacidemia type 1].

Glutaricacidemia type 1(GA1) is an autosomal recessive disease caused by reduced or missing glutaryl-CoA dehydrogenase activity which hamps metabolism of lysine, hydroxylysine and tryptophan. The catabolic products of glutarylcarnitine and glutaric acid are abnormally accumulated in the body, resulting in metabolic disorders which primarily lead to damage to the nervous system. Clinical manifestations of patients include macrocephaly, dystonia, dyskinesia, and developmental retardation. Acute encephalopathy may be induced in infants and young children due to infection, vaccination and surgery. For GA1 is a rare disease and its clinical manifestations are similar to other neurological diseases, it may be easily missed or misdiagnosed. To facilitate early diagnosis and treatment and improve the prognosis, this consensus was formulated by pediatric experts from the fields of endocrinology and genetic metabolism through full discussion and reference to the latest literature and guidelines home and abroad.

Genetic and epigenetic mechanisms in the development of congenital heart diseases.

Congenital heart disease (CHD) is the most common of congenital cardiovascular malformations associated with birth defects, and it results in significant morbidity and mortality worldwide. The classification of CHD is still elusive owing to the complex pathogenesis of CHD. Advances in molecular medicine have revealed the genetic basis of some heart anomalies. Genes associated with CHD might be modulated by various epigenetic factors. Thus, the genetic and epigenetic factors are gradually accepted as important triggers in the pathogenesis of CHD. However, few literatures have comprehensively elaborated the genetic and epigenetic mechanisms of CHD. This review focuses on the etiology of CHD from genetics and epigenetics to discuss the role of these factors in the development of CHD. The interactions between genetic and epigenetic in the pathogenesis of CHD are also elaborated. Chromosome abnormalities and gene mutations in genetics, and DNA methylations, histone modifications and on-coding RNAs in epigenetics are summarized in detail. We hope the summative knowledge of these etiologies may be useful for improved diagnosis and further elucidation of CHD so that morbidity and mortality of children with CHD can be reduced in the near future.