Objective To investigate the effects of physical and chemical factors in the environment for dried blood sample (DBS) preparation of neonatal screening assay.Methods A total of 60 normal and 120 positive DBS were prepared under control and 10 different conditions.Another 30 normal and 80 positive DBS were prepared under control and 7 different concentration gradients of formaldehyde.The levels of phenylalanine (Phe),glucose-6-phosphate dehydrogenease (G6PD),thyroid stimulating hormone (TSH) and 17α-hydoxyprogesterone (17α-OHP) were tested by time-resolved fluorescence immunoassay or fluorescence assay.Statistical analysis was performed using SPSS 22.0 software.Results Compared with the control group,the results of Phe were not significantly different (P ＞ 0.05) when the samples were dried under the formaldehyde sensitive threshold (4.62 to 6.95 ppm for 18 hours).G6PD levels were significantly lowered when the samples were dried under all the conditions except for fast cold drying (2 to 8 ℃ overnight and formaldehyde condition,0.30 to 0.38 ppm for 4 hours or 0.21 to 0.24 ppm for 18 hours).TSH and 17α-OHP levels were lowered obviously when the samples were dried under the conditions of humidity,UV and formaldehyde condition (TSH:0.32 to 0.52 ppm for 4 hours,0.38 to 0.45 ppm for 18 hours,17α-OHP:4.37 to 4.62 ppm for 4 hours,0.38 to 0.45 ppm for 18 hours).The results of Phe,G6PD,TSH and 17α-OHP were not statistically different with the control group when the samples were dried under the fast cold drying and 2 to 8 ℃ overnight.Conclusion The physical and chemical factors in the environment of DBS preparation should be related to the accuracy of neonatal disease screening closely.The necessary control factors including formaldehyde,ethanol,glacial acetic acid,ultraviolet irradiation,heat,humidity and decoration pollution may exhibit significant effects on the preparation of DBS.Fast cold drying and overnight at 2 to 8 ℃ could be available for DBS preparation.

Objective: To investigate the prevalence of galactosemia(GAL), and the characteristics of genotype and phenotype of newborns who were confirmed with GAL in newborn screening in Zhejiang province. Method: The number of all live births, newborn screened infants and all clinical data of confirmed newborns with GAL from October 2013 to March 2015 were retrospectively analyzed by reviewing the data of Zhejiang Province screening center database. And the characteristics of genes and the clinical data of GAL cases who were confirmed by correlative gene test and enzyme activity measurement were analyzed. Result: The prevalence of GAL in Zhejiang province was 1/189 857. Among them, there was 1 case confirmed with GAL typeⅠ (prevalence, 1/759 428), with mutations of c. 904+ 1G>T and c. 687G>A, the enzyme activity of galactose-1-phosphate uridyltransferase (GALT) was 56.4% of controls. And there was 1 case of GAL typeⅡ(prevalence, 1/759 428), with mutations of c. 85G>T and c. 502G>A. There were 2 cases confirmed with GAL type Ⅲ(prevalence, 1/379 714), with mutations of c. 505C>T, c. 452G>A, c. 280G>A and c. 925G>A, the enzyme activity of UDP-galactose-4′-epimerase (GALE) were 42% and 38% of controls, respectively. All cases had different abnormal biochemical marks of liver function, and 1 case had combined hyperlactacidemia or hyperammonemia or increase of multiple kinds of amino acids, respectively. The newborn of GAL type Ⅱ had phacoscotasmus before treatment. All the cases were fed with lactose free milk powder, and all the abnormal parameters were improved during following up. Conclusion The disease of GAL is rare in Zhejiang province, and its genotype distribution is scattered with comparatively mind clinical manifestations, and the cases with early treatment with lactose free milk powder have good prognosis. All cases needed to be treated and followed up for a life-long time. It is recommended that the high risk cases with GAL should be screened as soon as possible.

OBJECTIVE: To explore the genetic basis for a child with mental retardation. METHODS: The child was subjected to next generation sequencing (NGS). Candidate variant was analyzed with bioinformatic software. RESULTS: NGS revealed that the child has carried a de novo heterozygous c.4035G>C (p.Gln1345His) variant of the ARID1B gene. The variant was unreported previously and may cause instability of the protein structure. CONCLUSION The de novo missense variant of ARID1B gene may underlie the mental retardation in the child. Above result has enabled genetic counseling and prenatal diagnosis for her family.

OBJECTIVETo study pantothenate kinase 2 (PANK2) gene mutations in Chinese patients with Hallervorden-Spatz syndrome (HSS).

METHODSPANK2 gene mutations were detected by PCR, DNA sequence analyses, restriction enzyme digestion and PCR-single strand conformation polymorphism in 5 patients, 3 unaffected family members and 51 unrelated healthy persons.

RESULTSNovel compound heterozygous PANK2 gene mutations, A803G and T1172A, in exons 3 and 5, respectively, were found in one patient. At the same time, 3 types of single nucleotide polymorphisms, -38 t>a in 5'-UTR, IVS1+42 c>a and G77C in exon 1, were confirmed; among them, -38 t>a, IVS1+42 c>a, were first reported.

CONCLUSIONPANK2 gene mutations can cause HSS in Chinese patients.

Adolescent ; Adult ; Base Sequence ; Child ; China ; DNA Mutational Analysis ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Pantothenate Kinase-Associated Neurodegeneration ; genetics ; Pedigree ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Young Adult

Objective To investigate the prevalence of galactosemia ( GAL) , and the characteristics of genotype and phenotype of newborns who were confirmed with GAL in newborn screening in Zhejiang province .Method The number of all live births , newborn screened infants and all clinical data of confirmed newborns with GAL from October 2013 to March 2015 were retrospectively analyzed by reviewing the data of Zhejiang Province screening center database .And the characteristics of genes and the clinical data of GAL cases who were confirmed by correlative gene test and enzyme activity measurement were analyzed.Result The prevalence of GAL in Zhejiang province was 1/189 857.Among them, there was 1 case confirmed with GAL typeⅠ(prevalence,1/759 428), with mutations of c.904+1G>T and c.687G>A,the enzyme activity of galactose-1-phosphate uridyltransferase ( GALT) was 56.4% of controls.And there was 1 case of GAL typeⅡ( prevalence,1/759 428),with mutations of c.85G>T and c.502G>A. There were 2 cases confirmed with GAL type Ⅲ(prevalence, 1/379 714),with mutations of c.505C>T, c. 452G>A, c.280G>A and c.925G>A,the enzyme activity of UDP-galactose-4′-epimerase ( GALE) were 42% and 38% of controls, respectively .All cases had different abnormal biochemical marks of liver function, and 1 case had combined hyperlactacidemia or hyperammonemia or increase of multiple kinds of amino acids, respectively.The newborn of GAL type Ⅱhad phacoscotasmus before treatment .All the cases were fed with lactose free milk powder , and all the abnormal parameters were improved during following up . Conclusion The disease of GAL is rare in Zhejiang province , and its genotype distribution is scattered with comparatively mind clinical manifestations , and the cases with early treatment with lactose free milk powder have good prognosis .All cases needed to be treated and followed up for a life-long time.It is recommended that the high risk cases with GAL should be screened as soon as possible .

Objective:To analyze the oral phenotype and gene variation of children with hypophosphatasia (HPP), and explore the genotype-phenotype correlations.Methods:Eight children diagnosed with HPP from January 2008 to January 2023 in The Children′s Hospital, Zhejiang University School of Medicine were recruited in this study. The pathogenic genes of 5 of them were sequentially analyzed and all of their oral manifestations, laboratory tests and genetic variation types were retrospectively analyzed.Results:A total of 8 children were recruited in the study, 3 males and 5 females, aged from 20 to 104 months, whose main complaints were premature deciduous tooth loss. Among them, 3 children were diagnosed with odonto HPP, and the other 5 children were diagnosed with childhood HPP, including 2 children was odonto HPP at the first diagnosis and modified as childhood HPP at the age of 5. The age range of first deciduous tooth loss is 9 to 18 months, and the age range of diagnosis was 20 to 104 months. The patients of odonto HPP only showed premature loss of deciduous anterior tooth, while the patients with childhood HPP also showed premature loss of multiple deciduous molars. Panoramic radiographic film revealed enlarged pulp chambers and radicular canals in some primary and permanent teeth. The enamel hypoplasia, hypoplastic short roots, and alveolar resorption of deciduous molar were observed in some cases. The serum alkaline phosphatase (ALP) (30-107 U/L) levels of all the patients were lower than that in the normal children of same age and gender, and the ALP value of the 1-3 years old girls with childhood HPP (30-33 U/L) was lower than that of the three children with odonto HPP (61-107 U/L), but there was no significant difference in statistical analysis. There were 8 variation sites of ALP liver/bone/kidney (ALPL) gene detected in 5 children and their families, all of which were missense variation, including the new variants in the mutations of c.1334C>G (p.Ser445Cys) and c.1259G>T (p.Gly420Val) that were not reported in the literature. One case was autosomal dominant inheritance and other 4 cases were complex heterozygous variation with autosomal recessive inheritance.Conclusions:Pediatric stomatologists are often the first doctors to detect childhood and odonto HPP. Diagnosis of mild HPP is often delayed. The severity of HPP is related to serum ALP level and ALPL gene mutation sites.

Objective To investigate the feasibility of Region 4 Stork(R4S)project used for newborn screening by tandem mass spectrometry in China.Methods This retrospective study was performed among 362 822 neonates screened by tandem mass spectrometry from May 2015 to April 2016 in Zhejiang newborn screening center.Infants were grouped by screening result category: 83 true positive cases,360 554 true negative cases and 2 185 false positive cases.Raw data was uploaded into R4S website to perform postanalytical interpretive tools, then results were analyzed with interpretation rules.The comparisons of normal population percentiles were done at five selected percentiles between Zhejiang newborn screening center and R4S project with min-max normalization.Results Compared with cutoff system by using R4S project with interpretation rules,the positive predictive value increased from 3.7%to 8.3%,the specificity increased from 99.40%to 99.75%, and the false positive rate declined from 0.6% to 0.2%. The two cases of true positive hyperprolinemia were reported negative, and one case of β-ketothiolase deficiency was misdiagnosis.Totally 311 638 cases in true negative group were resolved by postanalytical interpretive tools,and the remaining 48 916 cases were excluded with interpretation rules.False positive cases were reduced to 897 cases.Results of percentiles comparison showed that levels of some markers were significantly different between zhejiang newborn screening center and R 4S project.Conclusions R4S project effectively improved the newborn screening performance, whereas leaded to a small number of misdiagnosis and missed diagnosis.Besides,many true negative cases should be excluded with interpretation rules.Optimization should be achieved based on local normal population.(Chin J Lab Med,2018,41:300-304)

Objective:To investigate the application feasibility of Region 4 Stork (R4S) system, an international collaborative newborn screening data platform, combined with cut-off value analysis in the neonatal screening for very long chain acyl-CoA dehydrogenase deficiency (VLCADD) by tandem mass spectrometry (MS/MS).Methods:The retrospective study was performed in 2, 040 072 neonates screened by MS/MS in Neonatal Screening Center of Zhejiang Province, China from October 2013 to July 2018. Nine hundred and ten cases were determined and identified as suspected positive VLCADD neonates by traditional cut-off method of tandem mass spectrometry. The original data of these 910 screened neonates were further analyzed by R4S system. Based on clinical diagnosis and ACADL gene test results, the screening efficiency between two methods was statistically compared.Results:The data of 910 suspected VLCADD-positive cases interpreted by cut-off method were further analyzed by R4S system, and the positive interpretation was reduced to 238 cases (including 9 confirmed positive cases). A total of 16 different mutations were found in ACADL gene sequencing among the confirmed children. The screening false positive rate (FPR) declined from 0.44‰ (901/2 040 072) to 0.11‰ (229/2 040 072), the rate of positive predictive value (PPV) increased from 0.99% (9/910) to 3.78% (9/238), and the specificity increased from 99.96% (2 039 162/2 040 063) to 99.99% (2 039 834/2 040 063). There was a statistically significant difference between cut-off method alone and cut-off method combined R4S system analysis (χ2=393.5, P<0.05). Conclusions:The R4S system combined with cut-off method applied in VLCADD neonatal screening by MS/MS can effectively improve screening performance, reduce false positive rate, and has certain value in clinical application.

Hypophosphatemia rickets is a rare disease that is divided into two categories,namely,hereditary and ac-quirability.Its clinical manifestations include growth disorders,limb deformities and dysfunction,poor mineralization of the teeth,and growth retardation in children as well as hyperparathyroidism,osteoarthritis,osteomalacia,and pseudofrac-ture in adults.Oral manifestations include non-carious teeth with recurrent apical periodontitis,periapical abscess and even cellulitis,periodontitis,and early tooth loss.X-linked hypophosphatemia rickets(XLHR)accounts for approximate-ly 80％of all hypophosphatemia rickets.We report a 3-year-old child with XLHR whose first diagnosis was apical peri-odontitis of multiple non-carious and non-traumatic teeth.Through medical history,clinical examination,laboratory ex-amination,radiographic findings,genotype testing,and literature analysis,we analyze the pathogenesis,clinical manifes-tations,radiographic features,diagnosis and differential diagnosis,treatment,and follow-up.This work provides refer-ence for clinical diagnosis and treatment and reduces missed diagnosis and misdiagnosis by dentists.

Objective:To investigate the efficiency of biochemical screening and hotspot gene screening in the detection of neonatal inherited metabolic diseases.Methods:This was a prospective multi-center study.The study was carried out on 21 442 neonatal samples collected from 12 hospitals in 10 provinces from November 2020 to November 2021.The results of biochemical screening and hotspot gene screening were analyzed jointly.Biochemical screening methods included glucose-6-phosphate dehydrogenase deficiency enzyme activity assay and neonatal tandem mass spectrometry.Genetic screening analysis involved 135 genes associated with 75 neonatal diseases.Results:Of all the 21 442 neonates enrolled in the study, 21 205 were subject to biochemical screening.A total of 813 cases were positive in the initial screening, and 0.45% of them (95 cases) were diagnosed after recall.All the 21 442 neonates underwent gene screening.About 168 positive cases were detected in the initial screening, and 0.73% (156 cases) of them were confirmed finally.Biochemical and genetic screening improved the detection sensitivity of such diseases as primary carnitine deficiency, neonatal intrahepatic cholestasis caused by citrin deficiency, and 2-methylbutyrylglycinemia.Moreover, biochemical and genetic screening enabled the detection of more diseases, including the common single-gene genetic diseases such as thalassemia and Wilson disease.Conclusions:In neonatal screening, the combination of biochemical screening and gene screening expands the number of diseases detected and improve screening efficiency.