Biochemical and molecular analysis of pediatric patients with metachromatic leukodystrophy in South China: functional characterization of five novel ARSA variants.

Metachromatic leukodystrophy (MLD) is a rare hereditary neurodegenerative disease caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA). This study described the clinical and molecular characteristics of 24 Chinese children with MLD and investigated functional characterization of five novel ARSA variants. A retrospective analysis was performed in 24 patients diagnosed with MLD at Guangzhou Women and Children's Medical Center in South China. Five novel mutations were further characterized by transient expression studies. We recruited 17 late-infantile, 3 early-juvenile, 4 late-juvenile MLD patients. In late-infantile patients, motor developmental delay and gait disturbance were the most frequent symptoms at onset. In juvenile patients, cognitive regression and gait disturbance were the most frequent chief complaints. Overall, 25 different ARSA mutations were identified with 5 novel mutations.The most frequent alleles were p.W320* and p.G449Rfs. The mutation p.W320*, p.Q155=, p.P91L, p.G156D, p.H208Mfs*46 and p.G449Rfs may link to late-infantile type. The novel missense mutations were predicted damaging in silico. The bioinformatic structural analysis of the novel missense mutations showed that these amino acid replacements would cause severe impairment of protein structure and function. In vitro functional analysis of the six mutants, showing a low ARSA enzyme activity, clearly demonstrated their pathogenic nature. The mutation p.D413N linked to R alleles. In western blotting analysis of the ARSA protein, the examined mutations retained reduced amounts of ARSA protein compared to the wild type. This study expands the spectrum of genotype of MLD. It helps to the future studies of genotype-phenotype correlations to estimate prognosis and develop new therapeutic approach.

Sustained free chlorine-releasing polydimethylsiloxane/Ca(ClO)2 materials with long-lasting disinfection efficacy.

A novel sustained chlorine-releasing polydimethylsiloxane/Ca(ClO)2 (PDMS/Ca(ClO)2) material was fabricated by encapsulating Ca(ClO)2 in a PDMS matrix due to its high hydrophobicity and high chemical stability, which showed immediate-responsive and long-lasting antibacterial capabilities in aqueous conditions. Free chlorine could be released from the PDMS/Ca(ClO)2 after immersion in water for 2 min and could also be sustainedly released for 2 weeks, while the released concentration is negatively related to the duration time and positively with the initial Ca(ClO)2 contents. Additionally, Ca(ClO)2 powder as a filler significantly affects the crosslinking and pore size of PDMS. The PDMS/Ca(ClO)2 materials exhibited enduring antibacterial performance against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in both planktonic and multispecies-biofilm status. It is expected that this PDMS/Ca(ClO)2 material and its similar composite would be promising candidates for wide sustainable disinfection applications in biomedical and industrial fields.

Telomere length and micronuclei trajectories in APP/PS1 mouse model of Alzheimer's disease: Correlating with cognitive impairment and brain amyloidosis in a sexually dimorphic manner.

Although studies have demonstrated that genome instability is accumulated in patients with Alzheimer's disease (AD), the specific types of genome instability linked to AD pathogenesis remain poorly understood. Here, we report the first characterization of the age- and sex-related trajectories of telomere length (TL) and micronuclei in APP/PS1 mice model and wild-type (WT) controls (C57BL/6). TL was measured in brain (prefrontal cortex, cerebellum, pituitary gland, and hippocampus), colon and skin, and MN was measured in bone marrow in 6- to 14-month-old mice. Variation in TL was attributable to tissue type, age, genotype and, to a lesser extent, sex. Compared to WT, APP/PS1 had a significantly shorter baseline TL across all examined tissues. TL was inversely associated with age in both genotypes and TL shortening was accelerated in brain of APP/PS1. Age-related increase of micronuclei was observed in both genotypes but was accelerated in APP/PS1. We integrated TL and micronuclei data with data on cognition performance and brain amyloidosis. TL and micronuclei were linearly correlated with cognition performance or Aß40 and Aß42 levels in both genotypes but to a greater extent in APP/PS1. These associations in APP/PS1 mice were dominantly driven by females. Together, our findings provide foundational knowledge to infer the TL and micronuclei trajectories in APP/PS1 mice during disease progression, and strongly support that TL attrition and micronucleation are tightly associated with AD pathogenesis in a female-biased manner.

Features of liver injury in 138 Chinese patients with NICCD.

OBJECTIVES: To find biochemical and molecular markers can assist in identifying serious liver damage of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) patients. METHODS: 138 patients under 13 days to 1.1 year old diagnosed of NICCD in our center from 2004 to 2020. Base on the abnormal liver laboratory tests, we divided 138 patients into three groups: acute liver failure (ALF), liver dysfunction, and non-liver dysfunction groups, then compared their clinical, biochemical and, molecular data. RESULTS: 96 % of 138 patients had high levels of citrulline and high ratio of threonine to serine, which is the distinctive feature of plasma amino acid profile for NICCD. A total of 18.1 % of 138 patients had evidence of ALF who presented the most severity hepatic damage, 51.5 % had liver dysfunction, and the remaining 30.4 % presented mild clinical symptoms (non-liver dysfunction). In ALF group, the levels of citrulline, tyrosine, TBIL, ALP, and γ-GT was significantly elevated, and the level of ALB and Fisher ratio was pronounced low. Homozygous mutations of 1,638_1660dup, IVS6+5G.A, or IVS16ins3kb in SLC25A13 gene were only found in ALF and liver dysfunction groups. Supportive treatment including medium-chain triglyceride supplemented diet and fresh frozen plasma could be life-saving and might reverse ALF. CONCLUSIONS: High level of citrulline, tyrosine, TBIL, ALP, γ-GT, and ammonia, low level of albumin, and low Fisher ratio were predictors to suggest severe liver damage in NICCD patients who may go on to develop fatal metabolic disorder. Early identification and proper therapy is particularly important for these patients.

Presenilin-1, mutated in familial Alzheimer's disease, maintains genome stability via a γ-secretase dependent way.

Mutations in Presenilin-1 (PS1) account for over 80 % mutations linked to familial Alzheimer's disease (AD). However, the mechanisms of action of PS1 mutations in causing familial AD are not fully understood, limiting opportunities to develop targeted disease-modifying therapies for individuals carrying PS1 mutation. To gain more comprehensive insights into the impact of PS1 mutations on genome stability, we knocked down PS1 in SH-SY5Y, HMC3 and A549 cells. This revealed that PS1 knockdown (KD) dramatically induces genome instability (GIN) in all cell types, as indicated by the increased incidence of micronuclei, nucleoplasmic bridges and/or nuclear buds. Although amyloid ß (Aß) was able to induce GIN, PS1-KD was associated with decreased expression of Aß in SH-SY5Y cells, suggesting Aß is not the primary cause of GIN in PS1-KD cells. In contrast, inhibiting the PS1 γ-secretase activity by DAPT recapitulated GIN phenotype as seen in PS1-KD cells, indicating that the induction of GIN following PS1 KD can be attributed to the loss of γ-secretase activity. PS1 KD or γ-secretase inhibition markedly sensitizes SH-SY5Y to the genotoxicity of mitomycin C. Interestingly, overexpression of the wildtype PS1 dramatically increased GIN in SH-SY5Y. Collectively, our study demonstrates the potential of PS1 and its γ-secretase activity in maintaining genome stability, highlighting a novel potential link between PS1 loss-of-function or gain-of-function mutations and familial AD through GIN. Several mechanisms by which GIN induced by PS1 dys-expression may contribute to AD are discussed.

Modified nusinersen intrathecal injection method: inclusion of a septal needle-free closed infusion connector.

Objective: Nusinersen, an extremely expensive biologic drug (around 100,000 US$ per dose) that needs to be administered intrathecally, is approved for the treatment of 5q-spinal muscular atrophy (SMA). Because of the low muscle tone of the back muscles of pediatric SMA patients, especially type 1 SMA patients, the safe, effective, and fast execution of sheath injection is needed. Therefore, a modified intrathecal injection method was developed accordingly. This paper aims to describe the applicability and safety of this modified method. Methods: The modified intrathecal injection method (MIIM) mainly includes a septal needle-free closed infusion connector between the lumbar puncture needle and the syringe, besides the procedures of routine lumbar puncture. Its applicability and safety were evaluated through clinical observation. Results: A total of 92 children with SMA have successfully received nusinersen treatment at our hospital using the modified method since 2019 without obvious adverse events related to the modified injection method. Based on the clinical feedback of operators, the advantages of the modified method include successfully injecting the total dose of nusinersen with constant injection rate and a more stable fixation of the puncture needle, as well as making the operator more relaxed. However, compared with the routine method, the procedure of the modified method has additional steps. Conclusion: The modified intrathecal injection method is an effective and safe method to inject nusinersen when weighing the pros and cons, and it may also be used for administering intrathecal injections of other expensive medicines or for patients with other strict requirements for intrathecal injection.

Mitochondrial FAD shortage in SLC25A32 deficiency affects folate-mediated one-carbon metabolism.

The SLC25A32 dysfunction is associated with neural tube defects (NTDs) and exercise intolerance, but very little is known about disease-specific mechanisms due to a paucity of animal models. Here, we generated homozygous (Slc25a32Y174C/Y174C and Slc25a32K235R/K235R) and compound heterozygous (Slc25a32Y174C/K235R) knock-in mice by mimicking the missense mutations identified from our patient. A homozygous knock-out (Slc25a32-/-) mouse was also generated. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice presented with mild motor impairment and recapitulated the biochemical disturbances of the patient. While Slc25a32-/- mice die in utero with NTDs. None of the Slc25a32 mutations hindered the mitochondrial uptake of folate. Instead, the mitochondrial uptake of flavin adenine dinucleotide (FAD) was specifically blocked by Slc25a32Y174C/K235R, Slc25a32K235R/K235R, and Slc25a32-/- mutations. A positive correlation between SLC25A32 dysfunction and flavoenzyme deficiency was observed. Besides the flavoenzymes involved in fatty acid ß-oxidation and amino acid metabolism being impaired, Slc25a32-/- embryos also had a subunit of glycine cleavage system-dihydrolipoamide dehydrogenase damaged, resulting in glycine accumulation and glycine derived-formate reduction, which further disturbed folate-mediated one-carbon metabolism, leading to 5-methyltetrahydrofolate shortage and other folate intermediates accumulation. Maternal formate supplementation increased the 5-methyltetrahydrofolate levels and ameliorated the NTDs in Slc25a32-/- embryos. The Slc25a32K235R/K235R and Slc25a32Y174C/K235R mice had no glycine accumulation, but had another formate donor-dimethylglycine accumulated and formate deficiency. Meanwhile, they suffered from the absence of all folate intermediates in mitochondria. Formate supplementation increased the folate amounts, but this effect was not restricted to the Slc25a32 mutant mice only. In summary, we established novel animal models, which enabled us to understand the function of SLC25A32 better and to elucidate the role of SLC25A32 dysfunction in human disease development and progression.

A very rare case report of glycogen storage disease type IXc with novel PHKG2 variants.

BACKGROUND: Pathogenic mutations in the PHKG2 are associated with a very rare disease-glycogen storage disease IXc (GSD-IXc)-and are characterized by severe liver disease. CASE PRESENTATION: Here, we report a patient with jaundice, hypoglycaemia, growth retardation, progressive increase in liver transaminase and prominent hepatomegaly from the neonatal period. Genetic testing revealed two novel, previously unreported PHKG2 mutations (F233S and R320DfsX5). Functional experiments indicated that both F223S and R320DfsX5 lead to a decrease in key phosphorylase b kinase enzyme activity. With raw cornstarch therapy, hypoglycaemia and lactic acidosis were ameliorated and serum aminotransferases decreased. CONCLUSION: These findings expand the gene spectrum and contribute to the interpretation of clinical presentations of these two novel PHKG2 mutations.

High-dose metformin induces a low-glucose dependent genotoxic stress.

Epidemiological studies have demonstrated that metformin (a cornerstone of diabetes treatment) has anticancer activity, but the underlying mechanism remains elusive. We aimed to investigate whether metformin elicits anticancer activity via increasing genotoxic stress, a state of increased genome damage that becomes tumor-suppressing if it goes beyond an intolerable threshold. We found that metformin (1-16 mM) suppressed proliferation and colony formation in a panel of cancer cell lines (HeLa, A375, A549 and QGY). Metformin induced a dose-dependent increase of genotoxic stress (including micronucleus, nucleoplasmic bridge and nuclear bud) and the increase of genotoxic stress correlated well with metformin's anticancer potential. Metformin deregulated the expression of BUBR1 and MAD2, two core genes of spindle assembly checkpoint (SAC) that surveillances chromosome segregation. Metformin had weakened antiproliferative effect and a corresponding attenuated genotoxic effect in HeLa cells cultured in high glucose (16 mg/ml). Meanwhile, metformin significantly increased genotoxicity in non-cancer cells (NCM460 and HUVECs). Metformin became non-genotoxic to HUVECs in high-glucose (8 and 16 mg/ml) conditions and reduced the genotoxicity of high glucose. Overall, these results infer a new mechanism of high-dose metformin, whereby low-glucose dependent genotoxic stress derived from SAC dysfunction might mediate some of the anticancer effect of this drug.

Glucose-6-Phosphate dehydrogenase deficiency associated hemolysis in a cohort of new onset type 1 diabetes children in Guangdong province, China.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human genetic abnormalities, with a high prevalence in Guangdong, China. The purpose of this study was to explore the characteristics of newly diagnosed type 1 diabetes (T1D) patients with G6PD deficiency in a cohort of Chinese children and to investigate the relationship between the diabetic ketoacidosis (DKA) and hemolysis due to G6PD deficiency in these patients. METHODS: A total of 503 newly diagnosed T1D children aged 6 months-18 years were collected and their G6PD enzyme activity were measured. Fasting plasma glucose (FPG), hemoglobin A1c (HbA1c), and G6PD gene were analysed. The pH, HCO3, and plasma osmotic pressure between DKA patients with and without hemolysis at the presentation were compared. RESULTS: In the present study, G6PD deficiency accounted for 5.3% of newly diagnosed T1D children. There were no significant differences in FPG/HbA1c and HbA1c levels between T1D children alone and T1D children with G6PD deficiency. Hemolysis appeared in five of the twenty-two DKA patients with G6PD deficiency. Two patients had fever at onset and were given ibuprofen and cefazolin. The other three patients did not have infection or ingestion of hemolytic drugs. There were no significant difference in pH, HCO3, and osmotic pressure between the children with DKA with and without hemolysis at the presentation. The hemolysis occurred between 2 and 7 days after admission and the hyperglycaemia had been corrected by the time hemolysis occurs. Four G6PD gene mutations were found in the diabetes with G6PD deficiency patients: c.1376G > T, c.1388G > A, c.95A > G, and c.871G > A, all of which were genes with high frequency of G6PD deficiency in Guangdong Province. No correlation between genotype and hemolysis was found. CONCLUSION: In the present study, we found the frequency of G6PD deficiency among newly diagnosed T1D children was similar to that of the general population. However, DKA children with G6PD deficiency are prone to occur hemolytic anemia, and these hemolysis usually occurs when DKA is corrected and blood glucose is in homeostatic state, which is easy to be ignored. To reduce the risk of this complication, especially in areas with high incidence of G6PD deficiency, screening for G6PD activity in people with newly diagnosed diabetes should be considered.

A ruptured giant mediastinal mature teratoma mimicking an encapsulated empyema.

A female patient with a right-sided encapsulated pleural effusion was misdiagnosed preoperatively as having an encapsulated empyema. However, a giant mass in the anterior mediastinum was found via thoracoscopy, and a mature teratoma was detected based on the pathological result. Herein we report this case and provide lessons for cardiothoracic surgeons.

Clinical and biochemical characteristics of patients with ornithine transcarbamylase deficiency.

BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is pleomorphic congenital hyperammonemia, in which the prognosis of the patient is determined both by genotype and environmental factors. This study investigated the clinical and biochemical characteristics of OTCD patients with different prognosis. METHOD: Of 35 OTCD patients, six males deceased at the first disease-onset, 17 males survived and had controllable ammonia levels after treatment, and 12 females survived through the first disease-onset but had intractable hyperammonemia and high mortality. Fasting blood samples from patients collected at three disease stages were used for the analysis of amino acid (AA) profile, acylcarnitine profile, and micronutrients. Differences in profiles between patients and healthy controls and within patient groups were studied. RESULTS: All OTCD patients had accumulation of glutamine, homocitrulline, lysine, glutamate, cystathionine, and pipecolic acid, as well as deficiency of citrulline, tryptophan, threonine, and carnitine. For male non-survivors, most other AAs and long-chain acylcarnitines were elevated at disease onset, of which the levels of creatine, N-acetylaspartic acid, and homoarginine were remarkably high. Male survivors and female patients had most other AAs at low to normal levels. Compared with male survivors, female patients had much lower protein-intolerance, as indicated by significantly lower levels of protein consumption indicators, including essential AAs, 1-methylhistidine, acylcarnitines et al., but high levels of ammonia. Female patients still had significantly higher levels of citrulline, homocitrulline, and citrulline/arginine compared to male survivors. CONCLUSION: Unique profiles were observed in each group of OTCD patients, indicating specific physiological changes that happened to them.

Clinical Characteristics of Elderly Patients with COVID-19 in Hunan Province, China: A Multicenter, Retrospective Study.

BACKGROUND: The aim of this study was to analyze and summarize the clinical characteristics of elderly patients with coronavirus disease 2019 (COVID-19) and compare the differences of young-old patients (60-74 years old) and old-old patients (≥75 years old). METHODS: In thisretrospective, multicenter study, the medical records of elderly patients who were diagnosed with COVID-19 in Hunan province, China, from January 21 to February 19, 2020 were reviewed. The characteristics of young-old patients and old-old patients were compared. RESULTS: Of the 105 elderly patientsconfirmed withCOVID-19, 81.0% were young-old patients, and 19.0% were old-old patients; 54.3% of elderly patients were females. Overall, 69.5% of elderly patients had underlying diseases, and the most common comorbidities included hypertension (43.8%), diabetes (25.7%), and cardiac disease (16.2%). Of the elderly patients, 22.9% were severe and 10.5% were critical severe cases. On admission, the most frequent symptoms in elderly patients included fever (66.7%), cough (64.8%), and fatigue (33.3%). Lymphopenia (31.4%), increased D-dimer (38.1%), depressed albumin (36.2%), elevated lactate dehydrogenase (41.0%), and a high level of C-reactive protein (79.0%) were common among elderly patients with COVID-19. The median prothrombin time (PT) and the activated partial thromboplastin time (APTT) were longer in old-old patients than young-old patients (PT median 12.3 vs. 13.1 s, p = 0.007; APTT median 39.0 vs. 33.5 s, p = 0.045). Young-old patients showed fewer complications (14.1%) than old-old patients (40.0%; p = 0.0014) and fewer received invasive ventilator support (3.5 vs. 25.0%, p = 0.006). As of March 11, 2020, 85.7% of elderly patients had been discharged, 3 deaths had occurred, and 11.4% were still hospitalized. CONCLUSIONS: Elderly patients usually have chronic medical illness and are likely to have a severe or critically severe condition. They could show atypical symptoms without fever or cough and multiple organ dysfunction. Old-old patients tend to have more complications than young-old patients during hospitalization. Careful nursing, observation, and systemic treatment are very important in elderly patients.

Novel mutations in a Chinese family with two patients with succinic semialdehyde dehydrogenase deficiency.

Background: A considerable proportion of pediatric disease burden is mainly caused by inborn errors of metabolism. Succinic semi-aldehyde dehydrogenase (SSADH) deficiency is an unusual disorder of the gamma-aminobutyric acid metabolism. Till date, very few cases have been reported in China.Case presentation: Trio-WES was used to characterize the ALDH5A1 gene in two children of a Chinese family, who presented with seizures, psychomotor delay, development regression, borderline cognition, hypotonia, and harbored the compound heterozygotes NM_001080.3: c.1321G > A (p. Gly441Arg) and c.727_735del (p. Leu243_Ser245del). The former has been reported earlier (rs1041467895), whereas the latter is novel. Amino acid coding at highly conserved amino acid residues was observed to be altered by both mutations. This structural impairment influenced the enzyme structure as indicated by the in silico protein modeling. Cerebral magnetic resonance imaging of the proband and her brother showed excessive gap in the cerebrum and abnormal signals in the bilateral frontal lobe, bilateral basal ganglia, and cerebral foot. Elevated levels of Gamma-hydroxybutyric aciduria were found in their patients on urine organic acid analysis.Conclusion: Our findings contribute to the current knowledge of missense and deletion mutations associated with SSADH deficiency.

Simultaneous quantification of 48 plasma amino acids by liquid chromatography-tandem mass spectrometry to investigate urea cycle disorders.

Urea cycle disorders (UCD) are inborn errors of ammonia detoxification in which early diagnosis and treatment are critical to prevent metabolic emergencies. Unfortunately, the diagnosis was often and pronounced delayed. To improve diagnosis, we developed herein a liquid chromatography-tandem mass spectrometry method to investigate the disturbance of amino acid profile caused by UCD. The method enabled absolute quantification of 48 amino acids (AAs) within 20 min. Only 2.5 µL plasma was required for the analysis. The lower limits of quantification for most AAs were 0.01 µmol/L. Method accuracies ranged from 89.9% to 113.4%. The within- and between-run coefficients of variation were 0.8-7.7% and 2.6-14.5%, respectively. With this method, age-specific reference values were established for 42 AAs by analyzing 150 samples from normal controls, and patients with different subtypes of UCD were successfully distinguished. The data of patients revealed that UCD not only disturbed the metabolism of urea cycle AAs and induced accumulation of ammonia detoxification AAs, but also interfered the metabolism of some nervous system related AAs, such as pipecolic acid and N-acetylaspartic acid. This data may provide new insight into pathogenesis for UCD.

Clinical features, molecular characteristics, and treatments of a Chinese girl with sitosterolemia: A case report and literature review.

Sitosterolemia is a rare autosomal recessive disease characterized by a significant increase in blood plant sterol levels. Clinical manifestations usually include xanthomas, hypercholesterolemia,premature atherosclerosis and hematological abnormalities. We report here a sitosterolemia patient who presented with multiple xanthomas and profound hypercholesterolemia since 3 years old. The girl was mistreated as familial hypercholesterolemia for 6 years until correct diagnosis was made by detecting serum plant cholesterol levels. Sequence analysis revealed compound heterozygous mutations in ABCG5 gene, including the previously reported mutation c.904+1G＞A and a novel missense mutation c.1528C＞A. Although cholestyramine therapy reduced cholesterol level in association with marked regress of the xanthomas, serum plant sterol levels still remain high. Our study suggests that patients develop severe hypercholesterolemia and xanthomas at early age should be suspected of sitosterolemia. In addition, we also describe a novel missense mutation in exon 11 of the ABCG5 gene, which enriches the genetic mutation spectrum of sitosterolemia.

Genetic analysis of 63 Chinese patients with mucopolysaccharidosis type II: Functional characterization of seven novel IDS variants.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disorder resulting from the deficiency of the enzyme iduronate-2-sulfatase (IDS).This study described the molecular characteristics of 63 Chinese children with MPS II and investigated functional characterization of seven novel IDS variants. We analyzed mutations in the IDS gene of 63 children with MPS II. Seven novel mutations were further characterized by transient expression studies. 49 different mutations were identified in the IDS gene including 33 previously reported and 16 novel mutations. The mutation p.R443X and c.1122C > T(p.G374G) may be link to attenuated type. The novel missense mutations were predicted damaging in silico. The bioinformatic structural analysis of the novel missense mutations showed that these amino acid replacements would cause a severe impairment of protein structure and function. In vitro functional analysis of the seven novel mutants, showing a very low IDS activity, clearly demonstrated their pathogenic nature. In western blotting analysis of the IDS protein, the examined mutations showed a similar or slightly lower molecular mass of precursor without mature forms being detected. Our study expands the spectrum of genotype of MPS II, provides new insights into the molecular mechanism of MPS II and helps to the future studies of genotype-phenotype correlations to estimate prognosis and develop new therapeutic approach.

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.

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.