[Newborn screening, clinical features and genetic analysis for Citrin deficiency in Henan province].

OBJECTIVE: To explore the prevalence, clinical features, genetic characteristics and prognosis of Citrin deficiency in Henan province of China. METHODS: A total of 986 565 neonates screened by tandem mass spectrometry at the Third Affiliated Hospital of Zhengzhou University from January 2013 to December 2021 were retrospectively analyzed. Analysis of SLC25A13 gene variants and parental verification were carried out for neonates suspected for Citrin deficiency by next-generation sequencing. The clinical, biochemical and genetic characteristics of Citrin deficiency patients were integrated to guide the diet treatment and follow up the growth and development. Paired-t test was used to compare the amino acid levels in the peripheral blood samples before and after the treatment. RESULTS: Nine cases of Citrin deficiency were diagnosed among the 986 565 neonates. Specific elevation of citrulline was observed in all of the 9 cases. Six variants were detected by genetic sequencing, among which c.852_855delTATG, c.615+5G>A, c.550C>T and IVS16ins3kb were known pathogenic variants, whilst c.1111_1112delAT and c.837T>A were unreported previously. The detection rate for c. 852_855delTATG was the highest (61.6%, 11/18), followed by IVS16ins3kb (16.7%, 3/18). The clinical symptoms of all patients were relieved after the treatment, and the blood amino acid profile and biochemical parameters were significantly improved by gradually falling within the normal range. By June 2022, all patients had shown a good prognosis. CONCLUSION: The prevalence of Citrin deficiency among neonates from Henan Province by tandem mass spectrometry is 1/109 618, and the carrier rate for the pathogenic variants of the SLC25A13 gene was 1/166. The c.852_855delTATG may be a hot spot variant among the patients. Discovery of the novel variants has enriched the mutational spectrum of the SLC25A13 gene. Above results have provided a basis for the early diagnosis, treatment, prognosis and genetic counseling for the affected families.

Newborn screening and genomic analysis of duchenne muscular dystrophy in Henan, China.

BACKGROUND: Duchenne Muscular Dystrophy (DMD) is a rare disorder caused by mutations in the dystrophin gene. Recent availability in treatment for DMD raised the need of early screening in our center, but newborn screening (NBS) for DMD has not been carried out in Henan Province. OBJECTIVES: To determine an optimal cutoff value through the quantitative determination of the creatine kinase isoform MM (CK-MM) concentration dried blood spot (DBS) to identify male DMD, and to evaluate assess the detection rate and mutation spectrum of DMD in Henan, China. METHODS: The CK-MM level in DBS was measured using with a GSP® neonatal creatine kinase -MM kit from 13,110 male newborns to establish the cut-off value for CK-MM. Multiplex ligation-dependent probe amplification (MLPA) were carried out for infants with elevated CK levels to detect DMD gene deletions/ duplications, NGS and sanger sequencing were then applied to exclude MLPA-negative samples to single-nucleotide variants. Phenotype-genotype correlations were analyzed using REVEL For novel missense mutations. RESULTS: Statistical analysis of CK-MM value of the 13,110 neonates suggested that the cut-off value may be set as 472 ng/mL. 3 cases of DMD were screened among 13,110 newborns, all of whom had CK-MM levels >600 ng/mL. We detected 4 rare variants in DMD gene, including 2 exon deletions (deletion of exon 52 and deletion from exon 3 to exon 7) and 2 point variants (c.9568C>T and c.4030C>T). Two cases were all exon deletions, one case was compound heterozygous variants. CONCLUSIONS: The estimated incidence of male neonatal DMD was 1:4,370 in Henan province. NBS is of great value to the early intervention and treatment of the disease, and is fundamental to support public health decision-making. The experience from this study provided a model that will allow further expansion and facilitate establishment a universal public health screening in Henan hospital systems.

Analysis of gene mutations of medium-chain acyl-coenzyme a dehydrogenase deficiency (MCADD) by next-generation sequencing in Henan, China.

BACKGROUND: Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD) is a rare inherited metabolic disorder of fatty acid ß-oxidation and one of the most common inborn errors of metabolism. The incidence of MCADD varies among regions and ethnic groups. To date, few cases of MCADD have been documented in China. OBJECTIVE: The present study aimed to find out the novel genetic pathogenic variants in the Chinese patients and evaluate the detection rate of the disease of high-frequency ACADM pathogenic variants in different regions of China. METHODS: 6 cases of MCADD were screened by tandem mass spectrometric (MS/MS) among 245 054 newborns. We performed next-generation sequencing on 6 families of infants with MCADD. We used the REVEL method to predict the protein function of the detected missense variants and used SPDBV 4.10 to predict the protein 3D structure model. We identified pathogenic variants of ACADM gene in 6 cases of MCADD, and then assessed these variants through Sanger sequencing and association analysis. RESULTS: The incidence of neonatal MCADD was 1/40,842 in Henan province. Among the 6 patients, five cases were compound heterozygous variants, one case was homozygous variants. DNA sequencing revealed 4 known (c.449_452del, c.1085G > A, c.1229 T > C, c.589A > G) and 3 novel mutations (c.849 + 5_849 + 8del, c.427A > G, c.1181C > T) in the ACADM gene. Mutation c.1085G > A (p.G362E) was most frequent among Henan people and shows obvious differences between North and South of China. CONCLUSION: MCADD is relatively rare in China, and c.1085G > A (p.G362E) is a common mutation in Henan population. Our findings, especially novel variants, will help improve the understanding of the genetic background and have facilitated clinical diagnosis and genetic counseling for the affected families.

[Analysis of GCH1 gene variant in a consanguineous Chinese pedigree affected with tetrahydrobiopterin deficiency].

OBJECTIVE: To explore the genetic basis for a child featuring tetrahydrobiopterin deficiency and global developmental delay. METHODS: Clinical and laboratory examinations were carried out for the child. Genomic DNA of the patient was subjected to high-throughput sequencing to identify genetic variants associated with hyperphenylalaninemia. Candidate variants were verified by Sanger sequencing. RESULTS: The result of blood tandem mass spectrometry showed that the Phenylalanine in the blood was 642.7 µmol/l, and the ratio of Phenylalanine/Tyrosine was 5.42. Analysis of urinary pterin: neopterin 0.09 mmol/mol Cr, biopterin 0.04 mmol/mol Cr, biopterin% 77%, which suggested tetrahydrobiopterin deficiency. The parents of the proband were first cousins. DNA sequencing revealed that the proband has harbored homozygous c.353A>T variants in exon 2 of the GCH1 gene, for which his great grandmother, grandfather, mother, uncle, father and elder brother were heterozygous carriers with normal phenotype and no clinical symptoms associated with dopa responsive dystonia. CONCLUSION: The homozygous c.353A>T variant of the GCH1 gene probably underlay the tetrahydrobiopterin deficiency in this pedigree of consanguineous marriage.

Genomic analysis of 9 infants with hypermethioninemia by whole-exome sequencing among in Henan, China.

BACKGROUND: Hypermethioninemia is an inborn error of metabolism with elevated plasma methionine (Met) caused by methionine adenosyltransferase deficiency. Methionine adenosyltransferase (MAT) I/III deficiency is the most common cause of hypermethioninemia. Except for increased blood Met, most patients have no symptoms, but a small number have nervous system complications, including cognitive impairment and mental retardation. OBJECTIVE: To investigate the gene variation of patients with hypermethioninemia in newborns in Henan province. METHODS: 9 cases of hypermethioninemia were screened for amino acids profile and acyl carnitine by tandem mass spectrometric (MS/MS) among 245 054 newborns. We performed whole-exome sequencing on 9 families of infants with hypermethioninemia. We identified mutated genes under different models of inheritance and further assessed these mutations through Sanger sequencing and association analysis. RESULTS: The incidence of neonatal hypermethioninemia was 1:27 228 in Henan province. A total of ten mutations in the MAT1A gene in the 9 patients were identified, including nine reported mutations (c.1070C > T, c.895C > T, c.100 T > A, c.315C > A, c.529C > T, c.623A > C, c.407G > T, c.1066C > T, 867G > T) and one novel mutations (c.772G > C). c.772G > C was detected in 2 families and is the most common variant. 7 infants (7/9) with hypermethioninemia were genetically autosomal dominant, and 2 infants (2/9) with hypermethioninemia were genetically autosomal recessive. CONCLUSION: Our findings expand the mutational spectrum of hypermethioninemia, with the description of one new mutation. They improve the understanding of the genetic background and clinical manifestation of MAT1A in Chinese patients.

Molecular and clinical characteristics of congenital hypothyroidism in a large cohort study based on comprehensive thyroid transcription factor mutation screening in Henan.

BACKGROUND: Congenital hypothyroidism (CH), the most common neonatal endocrine disorder worldwide, can be caused by variants in thyroid transcription factor (TTF) genes including NKX2-1, FOXE1, PAX8, NKX2-5 and HHEX. This study aims to perform targeted next-generation sequencing (NGS) panel for comprehensive mutation screening on these genes in a cohort of 606 CH patients with various types from Henan Province, China, to investigate the mutation rate of TTF genes, and to analyze the clinical, biochemical and molecular characteristics of our CH cohort. METHODS: High-throughput sequencing combined with statistical calculation were applied for mutation screening and analyses of the clinical data. RESULTS: Twenty-two likely disease-causing monoallelic mutations in the TTF genes were identified in our cohort (3.63%, 22/606). Mutated PAX8 was the most predominant genetic alteration among these TTF mutations. Interestingly, PAX8 defects were only found in TD cases and variants in the five TTF genes were detected in gland in situ (GIS) patients. CH patients with the same genotype may have significant phenotypic variability and permanent CH (PCH) patients in the GIS group were significantly fewer than those in the TD group. CONCLUSIONS: Our study showed the estimated TTF mutation rate among CH cases was 3.63% in Henan Province and genetic alternations in TTF genes played a role not only in TD but also in GIS, especially in goiter. Although we speculated that the five TTF genes may be involved in certain steps of thyroid hormone biosynthesis, more researches are needed to verify the conclusions of the present study.

[Clinical and genetic characteristics of primary carnitine deficiency identified by neonatal screening].

OBJECTIVE: To study the prevalence, clinical and genetic characteristics of primary carnitine deficiency (PCD). METHODS: From January 2013 to December 2017, 720 667 newborns and their mothers were tested for PCD by tandem mass spectrometry. Potential mutations of carnitine transporter gene SLC22A5 among suspected PCD patients were analyzed. Dietary guidance and L-carnitine supplementation were provided to the parents. Growth and intelligence development were surveyed during follow-up. RESULTS: In total 21 neonates and 6 mothers were diagnosed with PCD, which yielded an incidence of 1 in 34 317. Eighteen SLC22A5 mutations were detected, which included 4 novel mutations, namely c.1484T>C, c.394-1G>T, c.431T>C and c.265-266insGGCTCGCCACC. Eighteen patients were found to carry compound heterozygous mutations and 3 have carried homozygous SLC22A5 mutations. Three mothers carried compound heterozygous mutations and 2 carried homozygous mutations. Common mutations included c.1400C>G (42.3%), c.760C>T (11.5%) and c.51C>G (7.7%). During the 8-42 month follow-up, neonates with PCD showed no clinical symptoms but normal growth. Blood level of free carnitine was raised in all mothers after the treatment. CONCLUSION: The incidence of neonatal PCD in Henan is 1 in 34 317, with the most common mutation being c.1400C>G. Above finding has enriched the spectrum of SLC22A5 gene mutations.

[Genotype and phenotype correlation of phenylalanine hydroxylase deficiency among patients from Henan].

OBJECTIVE: To delineate the mutation spectrum of phenylalanine hydroxylase (PAH) gene among patients affected with phenylalanine hydroxylase deficiency (PAHD) in Henan Province of China, and to explore the correlation between the genotype and the phenotype. METHODS: A total of 155 affected children were recruited. Potential mutation of the PAH gene were analyzed by direct sequencing. The genotype-phenotype correlation was analyzed by matching the expected and observed phenotypes. RESULTS: Over 72 mutations and 108 genotypes have been identified. There were 7 homozygous mutations, including 1 case with EX6-96A>G/EX6-96A>G, 1 with R241C/R241C, 1 with R413P/R413P, and 4 with R243Q/R243Q. Among these, 6 patients have presented classic PKU phenotypes, except for a R241C/R241C genotype which has led to mild PKU. In 104 patients carrying compound PAH mutations, 52 were classic, 34 were mild and 39 had mild HPA. Patients who were heterozygous for EX6-96A>G/R241C, R243Q/A434D, EX6-96A>G/R413P and EX6-96A>G/ R241C were found with both the classic PKU and mild PKU phenotypes. Common mutations associated with mild HPA have included R53H, R243Q, V399V and H107R. The common mutations associated with mild PKU included R243Q, R241C, EX6-96A>G, and IVS4-1G>A. The prevalent mutations in classic PKU were R243Q, EX6-96A>G and V399V. The consistency between prediction of the biochemical genotype and observed phenotype was 77.78%, especially in classic PKU, the consistency was up to 82.14%. Significant correlations were disclosed between pretreatment levels of phenylalanine and AV sum (r=-0.6729, P < 0.01). CONCLUSION: The mutation spectrum of PAH gene in Henan seems to differ from that of other regions. Independent assortment of mutant alleles may result in a complex genotype-phenotype correlation, but the genotypes of PAHD patients have correlated with the phenotype.