Clinical characteristics and prognosis of early diagnosed Wilson's disease: A large cohort study.

BACKGROUND AND AIMS: Few studies have focused on the outcomes of Wilson's disease (WD) diagnosed before age of 5 years. This study aimed to summarize the clinical features of early diagnosed WD and analyse treatment outcomes and the risk factors associated with treatment failure. METHODS: A total of 139 children confirmed with WD before 5 years were enrolled in this study. Only patients with follow-up over 1 year were analysed with Kaplan-Meier survival analysis. The composite outcomes included death, progression to liver failure or acute hepatitis, development of renal or neurological symptoms and persistent elevation of alanine aminotransferase (ALT). The treatment failure was defined as occurrence of at least one of above outcomes. RESULTS: Among 139 WD patients at diagnosis, two (1.4%) WD patients presented with symptomatic liver disease, whereas 137 (98.6%) were phenotypically asymptomatic, including 135 with elevated ALT and 2 with normal liver function. Median serum ceruloplasmin (Cp) was 3.1 mg/dL, and urinary copper excretion was 87.4 µg/24-h. There were 71 variants identified in the the copper-transporting ATPase beta gene, and 29 were loss of function (LOF). 51 patients with LOF variant were younger at diagnosis and had lower Cp than 88 patients without LOF. Among 93 patients with over 1 year of follow-up, 19 (20.4%) received zinc monotherapy, and 74 (79.6%) received a zinc/D-penicillamine combination therapy. 14 (15.1%) patients underwent treatment failure, and its occurrence was associated with poor compliance (p < .01). CONCLUSIONS: Cp is a reliable biomarker for early diagnosis, and zinc monotherapy is an effective treatment for WD during early childhood. Good treatment compliance is critical to achieve a favourable outcome.

Assessment of the diagnostic value of serum ceruloplasmin for Wilson's disease in children.

BACKGROUND: Serum ceruloplasmin is one of the major diagnostic parameters for Wilson's disease (WD). Age and gender difference of serum ceruloplasmin remain controversy. This study aims to assess diagnostic value of serum ceruloplasmin level for WD in children up to age of 15 years. METHODS: Serum ceruloplasmin levels were measured in 317 WD patients, 21 heterozygotes, 372 healthy control children and 154 non-WD patients with other liver diseases. Receiver operating characteristic (ROC) curve was used to determine the diagnostic accuracy of serum ceruloplasmin for WD in children. RESULTS: Among healthy controls, serum ceruloplasmin level was slightly low in the infants younger than 6 months, and then maintained from 26 to 33 mg/dl after age of 6 months. A total of 8.1% of healthy children had levels of serum ceruloplasmin < 20 mg/dL. Serum ceruloplasmin level was 5.7 ± 4.7 mg/dl in WD patients, and 25.6 ± 5.9 mg/dl in heterozygous carriers. Only 1.9% of WD patients had serum ceruloplasmin levels > 20 mg/dL. Serum ceruloplasmin levels had gender difference, being higher in healthy boys than healthy girls, and higher in asymptomatic WD boys than asymptomatic WD girls (p < 0.01, p < 0.05). Serum ceruloplasmin levels also presented genotypic difference. WD patients with R778L homozygotes exhibited lower levels of serum ceruloplasmin than the patients without R778L (p < 0.05). The ROC curve revealed that serum ceruloplasmin level, at a cutoff value of 16.8 mg/dL, had the highest AUC value (0.990) with a sensitivity of 95.9% and a specificity of 93.6%. CONCLUSIONS: Serum ceruloplasmin is one of sensitive diagnostic biomarkers for WD in children. Gender and genotypic difference of serum ceruloplasmin level should be considered. The cutoff value of serum ceruloplasmin level < 16.8 mg/dL may provide the highest accuracy for diagnosis of WD in children.

Molecular diagnosis of maturity-onset diabetes of the young in a cohort of Chinese children.

OBJECTIVE: The purpose of this study was to investigate the molecular basis of maturity-onset diabetes of the young (MODY) by whole-exome sequencing (WES) and estimate the frequency and describe the clinical characteristics of MODY in southern China. METHODS: Genetic analysis was performed in 42 patients with MODY aged 1 month to 18 years among a cohort of 759 patients with diabetes, identified with the following four clinical criteria: age of diagnosis ≤18 years; negative pancreatic autoantibodies; family history of diabetes; or persistently detectable C-peptide; or diabetes associated with extrapancreatic features. GCK gene mutations were first screened by Sanger sequencing. GCK mutation-negative patients were further analyzed by WES. RESULTS: Mutations were identified in 24 patients: 20 mutations in GCK, 1 in HNF4A, 1 in INS, 1 in ABCC8, and a 17q12 microdeletion. Four previously unpublished novel GCK mutations: c.1108G>C in exon 9, and c.1339C>T, c.1288_1290delCTG, and c.1340_1343delGGGGinsCTGGTCT in exon 10 were detected. WES identified a novel missense mutation c.311A>G in exon 3 in the INS gene, and copy number variation analysis detected a 1.4 Mb microdeletion in the long arm of the chromosome 17q12 region. Compared with mutation-negative subjects, the mutation-positive subjects had lower hemoglobin A1c and initial blood glucose levels. CONCLUSIONS: Most MODY cases in this study were due to GCK mutations, which is in contrast to previous reports in Chinese patients. Diabetes associated with extrapancreatic features should be a clinical criterion for MODY genetic analysis. Mutational analysis by WES provided a precise diagnosis of MODY subtypes. Moreover, WES can be useful for detecting large deletions in coding regions in addition to point mutations.

Clinical features and mutational analysis in 114 young children with Wilson disease from South China.

Wilson disease (WD) is a rare autosomal recessive disorder caused by mutations in the ATP7B gene. Clinical features and mutational analysis of Chinese children with WD at early age were rarely described. Herein, we retrospectively examined 114 children with WD at the mean of 5.9 years old age at diagnosis. Eight patients developed acute liver failure at mean age of 9.7 years old, 4 of whom died. Among the 114 patients, 86.0% were presymptomatic with isolated elevation of transaminases at diagnosis, 99.1% had decreased ceruloplasmin, and 68.4% had urinary copper excretion over 100 µg/24 hr. Bi-allele pathogenic ATP7B mutations were identified in all patients. Among the 60 mutations detected, 10 were novel, including 7 missense mutations (p.I566N, p.T704I, p.C980F, p.G1030 V, p.A1096Q, p.L1327P, and p.L1373F), 1 nonsense mutation (p.K866X), 1 small insertion (p.Y44LfsX2), and 1 small deletion (p.R1118PfsX10). The most frequent mutations were p.R778L, p.P992L, and p.I1148T, which affected 27.2, 25.4, and 20.2% of the 114 WD children, respectively. The patients carrying p.R778L presented a higher rate of acute liver failure than the patients without p.R778L (9.7% vs. 4.8%). These results will be helpful in establishing early diagnosis of WD at the gene level, offering beneficial information for genetic counseling and providing clues to genotype/phenotype correlation of ATP7B mutations.

Clinical and molecular characteristics of patients with Gaucher disease in Southern China.

Gaucher disease (GD) is a common lysosomal storage disorder caused by the deficiency of acid ß-glucosidase, due to mutations in the GBA gene. To explore the clinical and molecular characteristics of GD patients from Southern China, GBA gene were analyzed by nest PCR and direct Sanger-sequencing. Novel missense mutations were transiently transfected in COS-7 cells by plasmid system for functional verification. Among the 22 GD patients, 19 patients were classified as type 1 and three as type 2. Over 60% of the type 1 patient had the onset before two years of age and about 42% of them died before three years of age. Six type 1 patients with L444P homozygous genotype, presented with early onset and severe hepatosplenomegaly. Four novel mutations Y22C, F109L, L149F and c.983_990delCCCACTGG were identified. The GBA activities in vitro of novel mutants Y22C, F109L and L149F were 20.2%, 6.9% and 6.5% of the wild-type, respectively. L444P mutation accounted for 47.7% of the mutant alleles. Our results revealed that type 1 GD tends to present with a severe phenotype among southern Chinese. L444P was the most prevalent mutation and L444P homozygous genotype was associated with severe type 1 GD. Three novel missense mutations identified were pathogenic.

Clinical features, BTD gene mutations, and their functional studies of eight symptomatic patients with biotinidase deficiency from Southern China.

Biotinidase (BTD) deficiency is a rare autosomal recessive metabolic disease, which develops neurological and cutaneous symptoms because of the impaired biotin recycling. Pathogenic mutations on BTD gene cause BTD deficiency. Clinical features and mutation analysis of Chinese children with BTD deficiency were rarely described. Herein, for the first time, we reported the clinical features, BTD gene mutations and their functional studies of eight symptomatic children with BTD deficiency from southern China. Fatigue, hypotonia, proximal muscular weakness, hearing deficits, rash and respiratory problems are common clinical phenotype of our patients. Seizures are observed only in patients with profound BTD deficiency. Five novel mutations were detected, among which c.637delC (H213TfsTer51) was found in 50% of our patients and might be considered as a common mutation. In vitro studies confirmed three mild mutations c.1368A>C (Q456H), c.1613G>A (R538H), and c.644T>A (L215H) which retained 10-30% of wild type enzyme activity, and six severe mutations c.235C>T (R79C), c.1271G>C (C424S), c.1412G>A (C471Y), c.637delC (H213TfsTer51), c.395T>G (M132W), c.464T>C (L155P), and c.1493dupT (L498FfsTer13) which retained <10% of wild type enzyme activity. c.1330G>C (D444H) decreased the protein expression but not activity of BTD enzyme, and H213TfsTer51 was structurally damaging while L498FfsTer13 was functionally damaging. These results will be helpful in establishing the definitive diagnosis of BTD deficiency at the gene level, offering appropriate genetic counseling, and providing clues to structure/function relationships of the enzyme.

Comparison of two different combined test strips with fluorescent microspheres or colored microspheres as tracers for rotavirus and adenovirus detection.

BACKGROUND: Rotavirus (RV) and enteric adenovirus (AdV) mainly cause infantile infectious gastroenteritis. Several separate test methods for the detection of RV or AdV are currently available, but few tests are able to simultaneously detect both RV and AdV viruses, especially in primary medical institutions. METHODS: The present study was mainly designed to compare the performance of two combined test strips for the detection of RV and AdV: a rotavirus-adenovirus strip with fluorescent microspheres for tracers (FMT); and the CerTest rotavirus-adenovirus blister strip with colored microspheres for tracers (CMT). To test the strips cultures of RV, AdV and from other enteric pathogens were used, in addition to 350 stool specimens from 45 symptomatic patients with gastrointestinal infections. RESULTS: Detection thresholds for RV and AdV cultures using serial dilutions showed that the sensitivity of FMT was significantly higher than that of CMT (both P < 0.05). Specificity evaluation demonstrated that with culture mixtures of Coxsackie (A16), ECHO (type30), and entero- (EV71) viruses there was no detection of cross reaction using the two test strips, i.e., all the results were negative. With regard to the detection of RV in 350 clinical specimens, the total coincidence rate was 92.9%, the positive coincidence rate was 98.2%, and the negative coincidence rate was 90.8%. With regard to AdV detection, the total coincidence rate was 95.4%, the positive coincidence rate was 95.2%, and the negative coincidence rate was 95.5%. CONCLUSIONS: FMT performed better than CMT with regard to the combined detection of RV and AdV.

Early transition from insulin to sulfonylureas in neonatal diabetes and follow-up: Experience from China.

BACKGROUND: Sulfonylurea therapy can improve glycemic control and ameliorate neurodevelopmental outcomes in patients suffering from neonatal diabetes mellitus (NDM) with KCNJ11 or ABCC8 mutations. As genetic testing results are often delayed, it remains controversial whether sulfonylurea treatment should be attempted immediately at diagnosis or doctors should await genetic confirmation. OBJECTIVE: This study aimed to investigate the effectiveness and safety of sulfonylurea therapy in Chinese NDM patients during infancy before genetic testing results were available. METHODS: The medical records of NDM patients with their follow-up details were reviewed and molecular genetic analysis was performed. Sulfonylurea transfer regimens were applied in patients diagnosed after May 2010, and glycemic status and side effects were evaluated in each patient. RESULTS: There were 23 NDM patients from 22 unrelated families, 10 had KCNJ11 mutations, 3 harbored ABCC8 mutations, 1 had INS mutations, 4 had chromosome 6q24 abnormalities, 1 had a deletion at chromosome 1p36.23p36.12, and 4 had no genetic abnormality identified. Sixteen NDM infants were treated with glyburide at an average age of 49 days (range 14-120 days) before genetic confirmation. A total of 11 of 16 (69%) were able to successfully switch to glyburide with a more stable glucose profile. The responsive glyburide dose was 0.51 ± 0.16 mg/kg/d (0.3-0.8 mg/kg/d), while the maintenance dose was 0.30 ± 0.07 mg/kg/d (0.2-0.4 mg/kg/d). No serious adverse events were reported. CONCLUSIONS: Molecular genetic diagnosis is recommended in all patients with NDM. However, if genetic testing results are delayed, sulfonylurea therapy should be considered before such results are received, even in infants with newly diagnosed NDM.

Early prenatal diagnosis of lysosomal storage disorders by enzymatic and molecular analysis.

OBJECTIVE: To report the 4-year experience of early prenatal diagnosis of lysosomal storage disorders (LSDs) at a center in mainland China. METHOD: Forty-seven pregnancies affected with LSDs were assed using enzymes and/or molecular studies. Prenatal studies were performed on 43 uncultured chorionic villi (CV) samples, two amniotic fluid samples, and two umbilical cord blood samples. RESULTS: Of the 47 fetuses, 23 (48.9%) were determined to normal, 13 (27.7%) to be carriers, and 11 (23.4%) diagnosed as affected. In this cohort, mucopolysaccharidoses (MPS) type II was the most common LSD, followed by Pompe disease and then metachromatic leucodystrophy. In the 17 MPS II cases, the four affected fetuses showed MPS II enzyme activity expression levels of 1.4% to 6.7%, while the enzyme activity levels of the 13 normal fetuses ranged from 72% to 240.4%. In the seven Pompe cases, three fetuses were normal with Pompe enzyme activity expression levels of 20%, 38.8%, and 77.3%, while four carrier pregnancies showed enzyme activity levels of 17.5%, 17.5%, 33.4%, and 13.8%, respectively. CONCLUSION: Based on different enzyme properties in uncultured CV, different prenatal diagnostic strategies should be adopted for MPS II and Pompe disease. Combining enzyme assay and molecular studies in uncultured CV improves the reliability of prenatal diagnosis of LSDs.

Clinical and molecular characteristics of 11 Chinese probands with GM1 gangliosidosis.

GM1 gangliosidosis is an autosomal recessive lysosomal storage disease caused by the deficiency of ß-galactosidase activity, precisely due to mutations in the GLB1 gene. To explore the clinical and molecular characteristics of GM1 gangliosidosis patients from China, GLB1 gene were analyzed in 11 probands with GM1 gangliosidosis by exploiting direct Sanger-sequencing. Among them, five patients were classified as the infantile type and the remaining six as the late-infantile or juvenile type. In these probands, eight novel mutations p.Y50N, p.Y237C, p.S267F, p.G453R, p.K578 N, c.618delC, c.475_478delGACA and c.1979_1980insG have been identified. Among them, three novel missense mutations p.Y50N, p.S267F and p.G453R were transiently transfected in COS-7 cells by plasmid system for functional verification. In vitro GLB1 activities carrying the aforesaid missense mutants p.Y50N, p.S267F and p.G453R were 0.11%, 0 and 0.55% of wild-type, respectively. Mutation c.495_497delTCT and p.S149F accounted for 22.7 and 13.6% of the mutant alleles, respectively. Our results expand the spectrum of GLB1 gene, provide new insights into the clinical and molecular characteristics of GM1 gangliosidosis in China.

Genetic and clinical characteristics of Chinese children with Glucokinase-maturity-onset diabetes of the young (GCK-MODY).

BACKGROUND: There is scarcity of information on the clinical features and genetics of glucokinase-maturity-onset diabetes of the young (GCK-MODY) in China. The aim of the study was to investigate the clinical and molecular characteristics of Chinese children with GCK-MODY. METHODS: Eleven children with asymptomatic hyperglycemia and clinically suspected GCK-MODY were identified from the database of children with diabetes in the biggest children's hospital in South China. Clinical data were obtained from medical records. Blood was collected from the patients and their parents for glucokinase (GCK) gene analysis. Parents without diabetes were tested for fasting glucose and HbA1c. Clinical information and blood for GCK gene analysis were obtained from grandparents with diabetes. GCK gene mutational analysis was performed by polymerase chain reaction and direct sequencing. Patients without a GCK gene mutation were screened by targeted next-generation sequencing (NGS) technology for other MODY genes. RESULTS: Nine children tested positive for GCK gene mutations while two were negative. The nine GCK-MODY patients were from unrelated families, aged 1 month to 9 years and 1 month at first detection of hyperglycaemia. Fasting glucose was elevated (6.1-8.5 mmol/L), HbA1c 5.2-6.7% (33.3-49.7 mmol/mol), both remained stable on follow-up over 9 months to 5 years. Five detected mutations had been previously reported: p.Val182Met, c.679 + 1G > A, p.Gly295Ser, p.Arg191Gln and p.Met41Thr. Four mutations were novel: c.483 + 2 T > A, p.Ser151del, p.Met57GlyfsX29 and p.Val374_Ala377del. No mutations were identified in the other two patients, who were also tested by NGS. CONCLUSIONS: GCK gene mutations are detected in Chinese children and their family members with typical clinical features of GCK-MODY. Four novel mutations are detected.

Epidermal growth factor and tumor necrosis factor α cooperatively promote the motility of hepatocellular carcinoma cell lines via synergistic induction of fibronectin by NF-κB/p65.

BACKGROUND: The interaction between hepatocellular carcinoma (HCC) cells and their microenvironment plays a fundamental role in tumor metastasis. The HCC microenvironment is rich in epidermal growth factor (EGF) and tumor necrosis factor α (TNFα), which may cooperatively, rather than individually, interact with tumor cells to influence their biological behavior. METHODS: Immunohistochemistry was performed to study the expression of EGF and TNFα in HCCs. Western blotting, immunofluorescence, qRT-PCR, wound healing scratch and invasion assay, and chromatin immunoprecipitation assays were used to study the combined roles of EGF and TNFα in the motility of HCC cells in vitro. RESULTS: We demonstrated that both EGF and TNFα were highly expressed in HCCs, and HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. In vitro, EGF and TNFα cooperatively promoted the motility of HCC cells mainly via synergistic induction of an extracellular matrix glycoprotein fibronectin (FN). Mechanistically, EGF and TNFα jointly increased the nuclear translocation and PKC mediated phosphorylation of NF-κB/p65 which could bind to the -356bp to -259bp fragment of the FN promoter, leading to a markedly increased activity of the FN promoter in HCC cells. CONCLUSIONS: HCCs with higher expression of both EGF and TNFα were more frequently rated as high-grade tumors. EGF and TNFα cooperatively promoted the motility of HCC cells mainly through NF-κB/p65 mediated synergistic induction of FN in vitro. GENERAL SIGNIFICANCE: These findings highlight the crosstalk between EGF and TNFα in promoting HCC, and provide potential targets for HCC prevention and treatment.

Two homozygous mutations in the exon 5 of BCKDHB gene that may cause the classic form of maple syrup urine disease.

Maple syrup urine disease (MSUD) is a rare autosomal recessive genetic disorder caused by defects in the catabolism of the branched-chain amino acids (BCAAs). Classic form of MSUD (CMSUD) is caused by mutations in BCKDHA, BCKDHB, DBT genes mostly. In this study, we analyzed the clinical and genetic characteristics of two patients with CMSUD. Two homozygous mutations, c.517G > T (p.Asp173Tyr) and c.503G > A (p.Arg168His), both in the exon 5 of BCKDHB were detected respectively. The novel mutation p.Asp173Tyr of patient A, inherited from his parents, is predicted to affect conformation of protein by computer analysis. The reported mutation p.Arg168His observed in patient B seemed to occur in a maternal uniparental disomy inheritance manner. Review of related literature revealed that most missense mutations in exon 5 of BCKDHB in homozygous genotype often result in CMSUD because of its incorrect conformation, and exon 5 of BCKDHB might be a susceptible region. Thus the novel homozygous mutation p.Asp173Tyr and the founder homozygous mutation p.Arg168His may be responsible for the clinical presentation of the two CMSUD patients, facilitating the future genetic counselling and prenatal diagnosis.

Clinical and molecular analysis of 6 Chinese patients with isoleucine metabolism defects: identification of 3 novel mutations in the HSD17B10 and ACAT1 gene.

Hydroxysteroid (17ß) dehydrogenase 10 (HSD10) and mitochondrial acetoacetyl-CoA thiolase (ß-KT) are two adjacent enzymes for the degradation of isoleucine, thus HSD10 and ß-KT deficiencies are confusing at an early stage because of nearly the same elevation of typical metabolites in urine, such as 2-methyl-3-hydroxybutyric acid (2M3HBA) and tiglylglycine (TG). In order to better understand the differences between these two disorders, we described the clinical and molecular characteristics of two HSD10 deficiency patients and four ß-KT deficiency patients. ß-KT deficiency patients had a much more favorable outcome than that of HSD10 deficiency patients, indicating that the multifunction of HSD10, especially neurosteroid metabolic activity, other than only enzymatic degradation of isoleucine, is involved in the pathogenesis of HSD10 deficiency. Two different mutations, a novel mutation p.Ile175Met and a reported mutation p.Arg226Gln, were detected in the HSD17B10 gene of HSD10 deficiency patients. Six different mutations, including four known mutations: p.Ala333Pro, p.Thr297Lys, c.83_84delAT, c.1006-1G > C, and two novel mutations: p.Thr277Pro and c.121-3C > G were identified in the ACAT1 gene of ß-KT deficiency patients. In general, DNA diagnosis played an important role in distinguishing between these two disorders.

Clinical,biochemical and molecular analysis of five Chinese patients with Sandhoff disease.

Sandhoff disease (SD) is a rare autosomal recessive lysosomal storage disorder of sphingolipid metabolism resulting from the deficiency of ß-hexosaminidase (HEX). Mutations of the HEXB gene cause Sandhoff disease. In order to improve the diagnosis and expand the knowledge of the disease, we collected and analyzed relevant data of clinical diagnosis, biochemical investigation, and molecular mutational analysis in five Chinese patients with SD. The patients presented with heterogenous symptoms of neurologic deterioration. HEX activity in leukocytes was severely deficient. We identified seven different mutations, including three known mutations: IVS12-26G > A, p.T209I, p.I207V, and four novel mutations: p.P468PfsX62, p.L223P, p.Y463X, p.G549R. We also detected two different heterozygous mutations c.-122delC and c.-126C > T in the promoter which were suspected to be deleterious mutations. We attempted to correlate these mutations with the clinical presentation of the patients. Our study indicates that the mutation p.T209I and p.P468PfsX62 may link to the infantile form of SD. Our study expands the spectrum of genotype of SD in China, provides new insights into the molecular mechanism of SD and helps to the diagnosis and treatment of this disease.

Phenotype and molecular characteristics in 45 Chinese children with 5α-reductase type 2 deficiency from South China.

CONTEXT: Affected by steroid 5α-reductase type 2 deficiency (5α-RD2), 46, XY individuals present divergent phenotypes characterized by undervirilization of male external genitalia. To identify the disorder, mutational analysis of 5α-reductase type 2 gene (SRD5A2) is a reliable approach. The genotype-phenotype relationship has not been elucidated. OBJECTIVE: To improve the diagnosis and expand the knowledge of the disease, we collected and analysed relevant data of clinical diagnosis, biological investigation and molecular determination in 45 children with the SRD5A2 gene mutations from South China in our centre. SUBJECTS AND METHODS: We studied a cohort of 45 Chinese children with SRD5A2 gene mutations. RESULTS: Isolated microphallus (35·6%) and microphallus associated with various degrees of hypospadias (55·6%) were frequent phenotype. Female external genitalia with clitoromegaly (8·9%) were rare. 16 of 18 (88·9%) cases had hCG-stimulated T/DHT ratio above 10. In 45 patients, we identified 15 different mutations. Five have never been described: p.His90ThrfsX31, p.Gly21Arg, p.Gly149Asp, p.Arg145Leu and p.Gly66Arg. The p.Arg227Gln mutation was detected in 41 (91·1%) patients. The p.Leu89Val polymorphism was found in 38 (84·4%) patients. Homozygous mutations were presented in 16 (35·6%) patients, compound heterozygous mutations in 20 (44·4%) patients, compound heterozygous mutations alone with the p.Leu89Val polymorphism in nine (20·0%) patients. Exons 1 and 4 were most affected, and the number of mutant alleles per exon was 78·1% and 12·2%, respectively. CONCLUSIONS: The study demonstrated a wide spectrum of phenotypes, biological profiles and genotypes in the children with 5α-RD2 from South China. The heterozygous mutation p.Arg227Gln is presumably a hot spot mutation and suggests a founder effect in the population of South China that may explain a moderate phenotype among our patients. Our report provides new insights into the molecular mechanism of 5α-RD2 and help to the diagnosis and treatment of this disease.

Clinical, biochemical, neuroimaging and molecular findings of X-linked Adrenoleukodystrophy patients in South China.

X-linked adrenoleukodystrophy is a common X-linked recessive peroxisomal disorder caused by the mutations in the ABCD1 gene. In this study, we analyzed 19 male patients and 9 female carriers with X-linked adrenoleukodystrophy in South China. By sequencing the ABCD1 gene, 13 different mutations were identified, including 7 novel mutations, and 6 known mutations, and 1 reported polymorphism. Mutation c.1180delG was demonstrated to be de novo mutation. 26.3 % (5/19) patients carried the deletion c.1415_16delAG, which may be the mutational hot spot in South China population. In addition, 73.7 % (14/19) patients were type of childhood cerebral adrenoleukodystrophy, 26.3 %(5/19) were in Addison only. Half of the childhood cerebral adrenoleukodystrophy patients had the adrenocortical insufficiency preceded the onset of neurological symptoms. Furthermore, 5 of 19 cases underwent hematopoietic stem cell transplantation. Our data showed that hematopoietic stem cell transplantation performed at an advanced stage of the cerebral X- linked adrenoleukodystrophy would accelerate the progression of the disease. Good clinical outcome achieved when hematopoietic stem cell transplantation performed at the very early stage of the disease.

[Kniest dysplasia due to mutation of COL2A1 gene].

OBJECTIVE: To detect potential mutation of COL2A1 gene in two children suspected for Kniest dysplasia. METHODS: The 54 exons and splicing regions of the COL2A1 gene were amplified with PCR and the product was subjected to direct sequencing. RESULTS: A missense mutation (c.905C>T, p.Ala302Val) was found in the coding region of the COL2A1 gene, which has been previously reported in abroad. The patients appeared to have short trunk dwarfism, enlarged joints and midface hypoplasia. CONCLUSION: The probands are the first cases of Kniest dysplasia described in China, and so was the p.Ala302Val mutation.

[Identification of a novel pathogenic mutation in PDHA1 gene for pyruvate dehydrogenase complex deficiency].

OBJECTIVE: To study the molecular genetic mechanism and genetic diagnosis of pyruvate dehydrogenase complex deficiency (PHD), and to provide a basis for genetic counseling and prenatal genetic diagnosis of PHD. METHODS: Polymerase chain reaction (PCR) was performed to amplify the 11 exons and exon junction of the PDHA1 gene from a child who was diagnosed with PHD based on clinical characteristics and laboratory examination results. The PCR products were sequenced to determine the mutation. An analysis of amino acid conservation and prediction of protein secondary and tertiary structure were performed using bioinformatic approaches to identify the pathogenicity of the novel mutation. RESULTS: One novel duplication mutation, c.1111_1158dup48bp, was found in the exon 11 of the PDHA1 gene of the patient. No c.1111_1158dup48bp mutation was detected in the sequencing results from 50 normal controls. The results of protein secondary and tertiary structure prediction showed that the novel mutation c.1111 _1158dup48bp led to the duplication of 16 amino acids residues, serine371 to phenylalanine386, which induced a substantial change in protein secondary and tertiary structure. The conformational change was not detected in the normal controls. CONCLUSIONS: The novel duplication mutation c.1111_1158dup48bp in the PDHA1 gene is not due to gene polymorphisms but a possible novel pathogenic mutation for PHD.

Mechanism of DNA trapping in nanoporous structures during asymmetric pulsed-field electrophoresis.

We investigate the trapping mechanism of individual DNA molecules in ordered nanoporous structures generated by crystalline particle arrays. Two requisites for trapping are revealed by the dynamics of single trapped DNA, fully-stretched U/J shapes and hernia formation. The experimental results show there is a stronger possibility for hernias to lead the reorientation upon switching directions of the voltage at high field strengths, where trapping occurs. Fully stretched DNA has longer unhooking times than expected by a classic rope-on-pulley model. We propose a dielectrophoretic (DEP) force reduces the mobility of segments at the apex of the U or J, where field gradients are highest, based on simulations and observations of the trapping force dependence on field strength. A modified model for unhooking time is obtained after the DEP force is introduced. The new model explains the unhooking time data by predicting an infinite trapping time when the ratio of arm length differences (of the U or J) to molecule length Δx/L < ß, where ß is a DEP parameter that is found to strongly increase with electric field. The DNA polarizability calculated with the DEP model and experimental value of ß is of the same magnitude of reported value. The results indicate the tension at the apex of U/J shape DNA is the primary reason for DNA trapping during pulsed field separation, instead of hernias.