[Clinical and genetic studies on 76 patients with hydrocephalus caused by methylmalonic acidemia combined with homocysteinuria].

Objective: To analyze the clinical features, genetic characteristics, treatment and follow-up results of patients with hydrocephalus caused by methylmalonic acidemia combined with homocysteinuria, and to discuss the optimal strategies for assessing and treating such patients. Methods: From January 1998 to December 2020, 76 patients with hydrocephalus due to methylmalonic acidemia combined with homocysteinuria in the Department of Pediatrics in 11 hospitals including Peking University First Hospital were diagnosed by biochemical, genetic analysis and brain imaging examination. The patients were divided into operation-group and non-operation-group according to whether they underwent ventriculoperitoneal shunt. The clinical features, laboratory examinations, genotype, and follow-up data were retrospectively analyzed. Data were compared between the two groups using rank sum test, and categorical data were compared using χ2 test. Results: Among the 76 patients (51 male, 25 female), 5 were detected by newborn screening, while 71 were diagnosed after clinical onset, 68 cases (96%) had early-onset, 3 cases (4%) had late-onset. The most common clinical manifestations of 74 cases with complete data were psychomotor retardation in 74 cases (100%), visual impairment in 74 cases (100%), epilepsy in 44 cases (59%), anemia in 31 cases (42%), hypotonia or hypertonia in 21 cases (28%), feeding difficulties in 19 cases (26%) and disturbance of consciousness in 17 cases (23%). Genetic analysis was performed in 76 cases, all of whom had MMACHC gene variations, including 30 homozygous variations of MMACHC c.609G>A. The most common variations were c.609G>A (94, 62.7%), followed by c.658_660del (18, 12.0%), c.567dupT (9, 6.0%) and c.217C>T (8, 5.3%). Therapy including cobalamin intramuscular injection, L-carnitine and betaine were initiated immediately after diagnosis. A ventriculoperitoneal shunt operation was performed in 41 cases (operation group), and 31 patients improved after metabolic intervention (non-operation group). There was no significant difference in the age of onset, the age of diagnosis, the blood total homocysteine, methionine, and urinary methylmalonic acid concentration between the two groups (all P>0.05). The symptoms of psychomotor development, epilepsy, and visual impairments improved gradually after a long-term follow-up in the operation group. Conclusions: Hydrocephalus is a severe complication of methylmalonic acidemia combined with homocysteinuria. The most common clinical manifestations are psychomotor retardation, visual impairment, and epilepsy. It usually occurs in early-onset patients. Early diagnosis and etiological treatment are very important. Hydrocephalus may improve after metabolic intervention in some patients. For patients with severe ventricular dilatation, prompt surgical intervention can improve the prognosis.

[The phenotypes and genotypes in 314 patients with isolated methylmalonic acidemia].

Objectives: To summarize the clinical and genetic characteristics of the patients with isolated methylmalonic acidemia and investigate the strategies for the diagnosis, treatment and prevention. Methods: Three hundred and fourteen patients (180 males, 134 females) with isolated methylmalonic acidemia were ascertained from 26 provinces or cities across the mainland of China during January 1998 to March 2020. Genetic analysis was performed by Sanger sequencing, gene panel sequencing, whole exome sequencing, multiplex ligation-dependent probe amplification or quantitative PCR. According to the age of onset, the patients were divided to early-onset group (≤12 months of age) and the late-onset group (>12 months of age). They were treated by cobalamin, L-carnitine and (or) special diet and symptomatic treatment. Statistical analysis was done using Chi-square test. Results: Fifty-eight of 314 (18.5%) patients were detected by Newborn screening using liquid chromatography tandem mass spectrometry. Five cases (1.6%) had a postmortem diagnosis. Two hundred and fifty-one patients (79.9%) were clinically diagnosed with an age of onset ranged from 3 hours after birth to 18 years. One hundred and fifty-nine patients (71.0%) belonged to early-onset groups, 65 patients (29.0%) belonged to the late-onset group. The most common symptoms were metabolic crises, psychomotor retardation, epilepsy, anemia and multiple organ damage. Metabolic acidosis and anemia were more common in early-onset patients than that in late-onset patients (20.8%(33/159) vs. 9.2% (6/65), 34.6% (55/159) vs. 16.9% (11/165), χ(2)=4.261, 6.930, P=0.039, 0.008). Genetic tests were performed for 236 patients (75.2%), 96.2%(227/236) had molecular confirmation. One hundred and twenty-seven variants were identified in seven genes (MMUT, MMAA, MMAB, MMADHC, SUCLG1, SUCLA2, and MCEE), of which 49 were novel. The mut type, caused by the deficiency of methylmalonyl-CoA mutase, was the most common (n=211, 93%) cause of this condition. c.729_730insTT, c.1106G>A and c.914T>C were the three most frequent mutations in MMUT gene. The frequency of c.914T>C in early-onset patients was significantly higher than that in late-onset patients (8.3% (18/216) vs. 1.6% (1/64), χ(2)=3.859, P=0.037). Metabolic crisis was more frequent in mut type than the other types (72.6% (114/157) vs. 3/13, χ(2)=13.729, P=0.001),developmental delay and hypotonia were less frequent in mut type (38.2% (60/157) vs. 9/13, 25.5% (40/157) vs. 8/13, χ(2)=4.789, 7.705, P=0.030, 0.006). Of the 58 patients identified by newborn screening, 44 patients (75.9%) who were treated from asymptomatic phase developed normally whereas 14 patients (24.1%) who received treatment after developing symptoms exhibited varying degrees of psychomotor retardation. Conclusions: The characteristics of phenotypes and genotypes among Chinese patients with isolated methylmalonic acidemia were analyzed. Expanded the mutation spectrum of the associated genes. Because of the complex clinical manifestations and severe early onset of isolated methylmalonic acidemia, Newborn screening is crucial for early diagnosis and improvement of prognosis. MMUT gene is recommended for carrier screening as an effort to move the test earlier as a part of the primary prevention of birth defects.

The role of AMPK/mTOR signal pathway in brain injury following chronic intermittent hypoxia in growing rats.

OBJECTIVE: To investigate the role of AMPK-mTOR signal pathway in brain injury induced by chronic intermittent hypoxia (CIH) in young rats. MATERIALS AND METHODS: Forty healthy male Sprague-Dawley rats (3-4 weeks old, weighing 80-100 g) were randomly assigned to four groups: 2-week-CIH group (2IH), 4-week-CIH group (4IH), 2-week-simulated air control group (2AC) and 4-week-simulated air control group (4AC). TUNEL staining was used to detect the cell apoptosis in the hippocampus and pre-frontal cortexes, respectively. The Western blot was conducted to analysis the P-AMPK (Phospho-AMP-activated protein kinase) and P-mTOR (phosphorylated mammalian target of rapamycin) protein expression. RESULTS: The neurons apoptosis in the hippocampus and pre-frontal cortex in 2IH and 4IH groups increased significantly, compared with that of in 2AC and 4AC groups, (p<0.05, respectively). Moreover, 4IH group exhibited significantly increased apoptosis rates than 2IH group (p<0.05). 2IH and 4IH groups exhibited increased protein expression levels of P-AMPK in the hippocampus and prefrontal cortexes compared with 2AC and 4AC groups (p<0.05, respectively), whereas the protein expression of P-mTOR decreased after CIH treatment (p<0.05, respectively). Higher expression levels of P-AMPK and lower levels of P-mTOR were observed in 4IH group compared to 2IH group. No difference of apoptotic cells and protein expression of P-AMPK and P-mTOR was exhibited between 2AC and 4AC groups. CONCLUSIONS: CIH induces neural apoptosis in a time-dependent manner by activating AMPK and inhibiting mTOR phosphorylation in young rats.

Identification of a novel mutation of the EDA gene in X-linked hypohidrotic ectodermal dysplasia.

This study aimed to identify the disease-causing mutation in the ectodysplasin A (EDA) gene in a Chinese family affected by X-linked hypohidrotic ectodermal dysplasia (XLHED). A family clinically diagnosed with XLHED was investigated. For mutation analysis, the coding region of EDA of 2 patients and 7 unaffected members of the family was sequenced. The detected mutation in EDA was investigated in 120 normal controls. A missense mutation (c.878T>G) in EDA was detected in 2 patients and 3 female carriers, but not in 4 unaffected members of the family. The mutation was not found in the 120 healthy controls and has not been reported previously. Our findings indicate that a novel mutation (c.878T>G) of EDA is associated with XLHED and adds to the repertoire of EDA mutations.

A novel single-base deletion mutation of the RUNX2 gene in a Chinese family with cleidocranial dysplasia.

We identified a disease-causing mutation of the RUNX2 gene in a four-generation Chinese family affected with cleidocranial dysplasia (CCD). For mutation analysis, the coding region of RUNX2 was sequenced with DNA from two patients and three unaffected family members. The RUNX2 mutation was investigated in 50 normal controls by denaturing high pressure liquid chromatography. A heterozygous single-base deletion (c.549delC) of RUNX2, which predicts a termination site at the 185th codon and leads to a stop in the runt domain of RUNX2 protein, was detected in both patients but not in the three unaffected members of the family. This mutation was also not found in 50 controls and has not been reported previously. We demonstrated that a novel mutation (c.549delC) of RUNX2 is associated with CCD in a Chinese family, adding to the repertoire of RUNX2 mutations related to CCD.

Two novel NPHS1 mutations in a Chinese family with congenital nephrotic syndrome.

Congenital nephrotic syndrome of the Finnish type (CNF) is a lethal, autosomal recessive disorder mainly caused by mutations in the NPHS1 gene; it is found at a relatively high frequency in Finns. We investigated the disease-causing mutations in a Chinese family with CNF and developed a prenatal genetic diagnosis for their latest pregnancy. Mutation analysis was made of all exons and exon/intron boundaries of NPHS1 in the fetus, parents and 50 unrelated controls using PCR and direct sequencing. A heterozygous nonsense mutation within exon 20 (c.2783C>A) and a missense mutation within exon 17 (c.2225T>C) in NPHS1 were detected in the proband's father and mother, respectively, but were not found in the fetus or in 50 unrelated controls. Two novel mutations of c.2783C>A and c.2225T>C in NPHS1 were found to be causative in this Chinese CNF family with no known Finnish ancestry. The most recent sibling did not inherit these two mutations and hence was unaffected with CNF. Determining the cumulative number and ethnic distribution of known mutations can help expedite further study of the pathogenesis of CNF.

Mutation L437P in the 2B domain of keratin 1 causes diffuse palmoplantar keratoderma in a Chinese pedigree.

Diffuse palmoplantar keratoderma (DPPK) is an autosomal dominant genodermatosis characterized by uniform hyperkeratosis of the palm and sole epidermis. This disorder can be caused by mutations in the genes keratin 1, keratin 9, keratin 16, desmoglein 1 and plakoglobin. Here we present a DPPK Chinese pedigree and identify the aetiology as a novel missense mutation, L437P, located in a highly conserved helix motif in domain 2B of KRT1. Functional analysis shows that overexpression of the L437P mutant in cultured cells leads to abnormal intermediate filament networks and filament aggregation. This gain-of-function mutation highlights the role of domain 2B in mediating filament assembly.

An improved mechanical technique for assisted hatching.

BACKGROUND: Varied clinical outcomes of assisted hatching (AH) have been reported. We attempt to investigate whether the size of the zona opening created by AH is adequate for blastocyst hatching, and, if not, set up a new method to improve it. METHODS: A new AH technique, long zona dissection (LZD), was established, and experiments were performed to compare the effects of different sizes of zona opening on complete hatching of blastocysts in mouse and human embryos in vitro. RESULTS: The LZD technique can create a long zona slit on early embryos, even blastocysts, with the slit size beyond two-thirds of zona diameter. Compared with three-dimensional partial zona dissection, LZD can significantly enhance the hatching speed and the rate of complete hatching of mouse blastocysts (93.9%). All (100%) human blastocysts completely hatched following LZD; however, when the slit size after AH was about two-fifths of zona diameter, more of the larger inner cell masses (ICM) became trapped by the zona opening during hatching than the smaller ICM (53.3 versus 12.5%, P = 0.01). CONCLUSIONS: Zona opening of moderate size following AH is inadequate for the completion of blastocyst hatching in vitro; in some cases, however, it can be significantly improved by LZD.

[Effect of Ganoderma polysaccharides on cAMP in murine peritoneal macrophages].

OBJECTIVE: Investigating the effect of GLB7 on cAMP in murine peritoneal macrophages to provide a scientific evidence for the immunomodulatory mechanism. METHOD: Cell culture and radio-immunological assay of cAMP were used. RESULT: GLB7 increased the production of cAMP in a concentration and time dependent manner in murine peritoneal macrophages. CONCLUSION: The immunopotentiating effect of GLB7 may be due to the activation of macrophages that leads to the increase of cAMP.

Determination of berberine in Rhizoma coptidis and its preparations by non-aqueous capillary electrophoresis.

A non-aqueous capillary electrophoretic method was established for the determination of berberine in Rhizoma coptidis and its preparations. The effects of organic solvent and the concentrations of sodium acetate were studied, which showed that berberine in extracts of traditional Chinese medicine can be separated successfully in a buffer solution of 75 mmol/L of sodium acetate in methanol containing 1 mol/L of acetic acid. The simple and rapid method was linear in the range 25-200 microgram/mL of berberine and had a good reproducibility, with the relative standard deviation below 2%. Non-aqueous capillary electrophoresis is a satisfactory system for the analysis of alkaloids in traditional Chinese medicine.

Determination of tetrahydropalmatine in Chinese traditional medicine by nonaqueous capillary electrophoresis.

Tetrahydropalmatine in Rhizoma corydalis and its preparations were separated and determined with no pretreatment in the buffer solution of 50 mmol/L of sodium acetate in methanol containing 2 mol/L acetic acid.

Determination of ferulic acid in Angelica sinensis and Chuanxiong by capillary zone electrophoresis.

Ferulic acid in extracts of raw herbs was separated by capillary zone electrophoresis in the buffer solution of 10 mmol/L Na(2)B(4)O(7). The simple and rapid method was linear, ranging from 5 to 100 microg/mL, and had a good reproducibility with the RSD below 2%. The contents of ferulic acid in Angelica sinensis and Chuanxiong could be easily determined within 15 min with no pretreatment and no interference.

Separation and determination of anthraquinone derivatives in rhubarb and its preparations by micellar electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatographic method was set up for the quality control of rhubarb and its preparations. Anthraquinone derivatives were separated successfully within 10 min in the buffer solution of 50 mmol/L H3BO3-NaOH (pH 11) containing 25 mmol/L sodium deoxycholate. The established method, with a recovery of extraction of over 90%, has good linear relationship and reproducibility. The contents of anthraquinone derivatives in rhubarb and a tablet of Niu-huang-jie-du differed significantly, showing that the quality control of rhubarb and its preparations is necessary.

The separation of anthraquinone derivatives of rhubarb by miceller electrokinetic capillary chromatography.

A micellar electrokinetic capillary chromatographic method was set up for the separation of five anthraquinone derivatives in rhubarb. Optimization of pH and sodium deoxycholate(SDC) concentrations was studied, which showed that 50 mM H3BO3-NaOH(pH 11) containing 25 mM SDC could separate the five ingredients completely within 10 min with good reproducibility of elution time.

[Stroke and thyroid hormones].

The levels of thyroid hormones were determined by RIA in 65 cases in with stroke, in which 42 cases showed low T3 levels (64.6%). The rates of abnormal T3 and rT3 were much higher in hemorrhage group (72.5%) than those in infarction group (52%). T3 and rT3 abnormalities were apparent in the acute stage compared to those in the chronic stage (P less than 0.01), and closely correlated with size of lesions, that is, size of lesions had negative correlation with T3 and T3/rT3 but positive correlation with rT3 when the volume of hemorrhage was more than 30 ml or the area of infarction more than 20 cm2. Determination of T3 and rT3 levels in patients with stroke may be helpful in assessing the severity and prognosing.

[Induction of retarded exoerythrocytic schizonts by chloroguanide resulting in delayed parasitaemia of Plasmodium inui in Macaca mulatta].

Three Rhesus monkeys inoculated with a large number of sporozoites of Plasmodium inui dissected from the salivary glands of infected mosquitoes, Anopheles dirus. Two of the monkeys (inoculated with 8.06 x 10(5) and 1.3 x 10(7) sporozoites, respectively) were treated with chloroguanide base 6 mg/(kg.d) x 4 d starting from 24 h after the inoculations and the other one (inoculated with 5.93 x 10(6) sporozoites) was not treated with chloroguanide as control. The primary parasitaemia attacks occurred in the former 2 monkeys were 31 and 25 d respectively after inoculation, while in the control was 7.5 d. Liver biopsies were done in all of the 3 monkeys, normal schizonts were seen in the control monkey on d 8 after inoculation, while none were detected in the 2 monkeys treated with chloroguanide. However, the retarded exoerythrocytic schizonts were found by indirect fluorescent antibody test (IFAT) in liver sections of the monkey inoculated with 1.3 x 10(7) sporozoites. Therefore, it is evident that moderate doses of chloroguanide retarded the formation of schizonts and thus delayed the primary parasitaemia.

Observations on early and late post-sporozoite tissue stages in primate malaria. V. The effect of pyrimethamine and proguanil upon tissue hypnozoites and schizonts of Plasmodium cynomolgi bastianellii.

Two rhesus monkeys were each infected with 2.1 x 10(6) sporozoites of Plasmodium cynomolgi bastianellii; one was treated with 1.0 mg of pyrimethamine base per kg body weight for 5 d after sporozoite inoculation. A further 2 monkeys were each infected with 9.75 x 10(6) sporozoites of the same parasite; one was treated with 10 mg of proguanil per kg body weight for 4 out of 5 d after inoculation. The treated monkeys showed a delayed primary parasitaemia and relapses. In sections of liver biopsies taken 7.5 d after sporozoite inoculation, all monkeys showed numerous hypnozoites. However, there were no full grown schizonts and only rare retarded schizonts in the treated monkeys, in contrast to the untreated monkeys which had many mature or nearly mature schizonts. Later biopsies confirmed the continued presence of hypnozoites in all monkeys.

Qinghaosu-induced changes in the morphology of Plasmodium inui.

The ultrastructural changes induced by the administration of the antimalarial drug, qinghaosu, were studied in monkeys (Macaca assamensis) infected with Plasmodium inui. Significant changes, notably mitochondrial swelling within the parasites but not within host cells, were first observed 2.5 hr after exposure to qinghaosu. This suggests that the target of qinghaosu may be the parasite's mitochondria, as occurs with primaquine. This is in contrast to the most widely used antimalarial drug, chloroquine.