Atypical phenotypes and novel OCRL variations in southern Chinese patients with Lowe syndrome.

BACKGROUND: Lowe syndrome is characterized by the presence of congenital cataracts, psychomotor retardation, and dysfunctional proximal renal tubules. This study presents a case of an atypical phenotype, investigates the genetic characteristics of eight children diagnosed with Lowe syndrome in southern China, and performs functional analysis of the novel variants. METHODS: Whole-exome sequencing was conducted on eight individuals diagnosed with Lowe syndrome from three medical institutions in southern China. Retrospective collection and analysis of clinical and genetic data were performed, and functional analysis was conducted on the five novel variants. RESULTS: In our cohort, the clinical symptoms of the eight Lowe syndrome individuals varied. One patient was diagnosed with Lowe syndrome but did not present with congenital cataracts. Common features among all patients included cognitive impairment, short stature, and low molecular weight proteinuria. Eight variations in the OCRL gene were identified, encompassing three previously reported and five novel variations. Among the novel variations, three nonsense mutations were determined to be pathogenic, and two patients harboring novel missense variations of uncertain significance exhibited severe typical phenotypes. Furthermore, all novel variants were associated with altered protein expression levels and impacted primary cilia formation. CONCLUSION: This study describes the first case of an atypical Lowe syndrome patient without congenital cataracts in China and performs a functional analysis of novel variants in the OCRL gene, thereby expanding the understanding of the clinical manifestations and genetic diversity associated with Lowe syndrome.

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.

The Role of the Gut Microbiome in the Development of Acute Pancreatitis.

With the explosion research on the gut microbiome in the recent years, much insight has been accumulated in comprehending the crosstalk between the gut microbiota community and host health. Acute pancreatitis (AP) is one of the gastrointestinal diseases associated with significant morbidity and subsequent mortality. Studies have elucidated that gut microbiota are engaged in the pathological process of AP. Herein, we summarize the major roles of the gut microbiome in the development of AP. We then portray the association between dysbiosis of the gut microbiota and the severity of AP. Finally, we illustrate the promises and challenges that arise when seeking to incorporate the microbiome in acute pancreatitis treatment.

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.

Human iPSC-derived neural stem cells with ALDH5A1 mutation as a model of succinic semialdehyde dehydrogenase deficiency.

BACKGROUND: Succinic semialdehyde dehydrogenase deficiency (SSADH-D) is an autosomal recessive gamma-aminobutyric acid (GABA) metabolism disorder that can arise due to ALDH5A1 mutations, resulting in severe, progressive, untreatable neurodegeneration. SSADH-D is primarily studied using simplified models, such as HEK293 cells overexpressing genes of interest, but such overexpression can result in protein aggregation or pathway saturation that may not be representative of actual underlying disease phenotypes. METHODS: We used a CRISPR/Cas9 approach to generate human iPSC cell lines bearing ALDH5A1 mutations. Through screening, two different mutant cell lines, NM_001080.3: c.727_735del (p.L243_S245del) and NM_001080.3: c.730_738del (p.A244_Q246del), were obtained. We induced iPSCs to neural stem cells and analyzed the characteristics of ALDH5A1 mutations in stem cells. RESULTS: The human iPSC and NSC cell lines presented typical stem cell-like morphology. We found changes in ALDH5A1 expression and GABA accumulation in the different cell lines. In addition, by analyzing the cDNA between the wild-type and the mutant cell lines, we found that the mutant cell lines had a splicing variant. CONCLUSIONS: iPSCs represent a promising in vitro model for SSADH-D that can be used to study early central nervous system developmental alterations and pathogenic mechanisms.

Two De Novo Mosaic Variants Within the Same Site of PHEX Gene in a Girl with X-Linked Hypophosphatemic Rickets.

X-linked hypophosphatemic rickets (XLH) is the most common form of hypophosphatemic rickets, which is caused by the deficiencies of PHEX gene with an X-linked dominant inheritance pattern. As at least several thousands of XLH patients have been diagnosed, only several males and fewer females with mosaicism of PHEX gene were found. Here we describe an XLH girl with two de novo mosaic variants within the same site of PHEX gene. To rapidly screen all of the causative genes of hypophosphatemic rickets and rule out other diseases, DNA samples were initially analyzed using whole exome sequencing (WES). Interestingly, two different pathogenic mosaic variants, a known c.1809G > A(p.W603*) variant and a novel c.1809G > T(p.W603C) variant within the same site of PHEX gene, were identified in the proband by WES. Subsequent Sanger sequencing confirmed the presence and de novo pattern of these two mosaic variants in the proband, which were absent in her healthy parents. This is the first case to report two different mosaic variants of PHEX gene in an XLH individual. This XLH girl has a de novo mosaic genotype of c.1809 = /G > T/G > A in PHEX gene. Our report adds an unusual mocaicism case for XLH and expands the mutational event and spectrum of PHEX gene. Our report also alerts clinicians and geneticists to be cautious about mocaicism and detection methods.

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.

Features of chinese patients with sitosterolemia.

BACKGROUND: Sitosterolemia is a lipid disorder characterized by the accumulation of phytosterols in plasma and organs, caused by mutations in the ABCG5 and/or ABCG8 genes. The disease is frequently misdiagnosed and mistreated as familial hypercholesterolemia (FH). To gain a better understanding of the disease, the current status of diagnosis and treatment of Chinese patients with sitosterolemia was reviewed and summarized. METHOD: Literature search was performed. The clinical features and molecular characteristics of Chinese patients with sitosterolemia were analysed. Four children with sitosterolemia and the treatment experience were described. RESULTS: Fifty-five patients with sitosterolemia have been reported in China. These patients were aged from 3 months to 67 years at diagnosis, and the median was 8 years of age. Several complications, such as xanthomas in 47 patients (85%), thrombocytopenia in 17 patients (31%), anemia in 14 patients (25%), and cardiovascular damage in 12 patients (22%), were observed. Thirty-nine patients (71%) exhibited mutations in the ABCG5 gene, 15 patients (27%) showed mutations in ABCG8, and variations in both genes occurred in one patient (2%). A patient with two clinically rare diseases, namely, sitosterolemia and glycogen storage disease type VI (GSD VI)), is reported here for the first time. The four reported patients were treated with low cholesterol and phytosterol-limited diet alone or combined with cholestyramine. Even though decreases were observed for total plasma cholesterol (TC) and low-density-lipoprotein cholesterol (LDL-C), and these levels were as low as normal in some patients, the levels of plant sterols remained above the normal range. However, TC, LDL-C and plant sterol levels remained at high levels in patients treated with a control diet control only. CONCLUSIONS: The analysis reveals that different from Caucasians carrying mainly variations in ABCG8, most Chinese patients have mutations in the ABCG5 gene, and Arg446Ter, Gln251Ter, anArg389His might be hot-spot mutations in Chinese patients. The current survey provides clinical data to enable the development of a standardized protocol for the diagnosis and treatment of sitosterolemia in China.

Thyroid function in children with Prader-Willi syndrome in Southern China: a single-center retrospective case series.

BACKGROUND: To investigate hypothalamic-pituitary-thyroid function in children of different ages, nutritional phases, and genotypes that were diagnosed with Prader-Willi syndrome (PWS), as well as the effects of recombinant human growth hormone (rhGH) treatment on thyroid hormones in PWS patients. METHODS: One hundred and thirty PWS patients (87 boys and 43 girls) aged from newborn to 15 years (y) (median 1.25 y, mean, SD: 2.95 ± 3.45 y), were surveyed in this study. Serum thyroid hormone levels were examined at least once per3-6 months during the 2 years follow-up study. Central hypothyroidism (C-HT) was identified as low/normal thyroid-stimulating hormone (TSH) and low free thyroxine 4 (FT4). RESULTS: All study participants had normal neonatal TSH screening test results. The prevalence of C-HT is 36.2% (47/130). No C-HT cases were diagnosed in PWS either below 1 month (m) or above 12 y. The prevalence of C-TH would be increased with age before 3 y until reaching the peak, followed by a gradual decline over the years. The prevalence of C-HT varies significantly at different ages (Pearson's χ2 = 19.915; p < 0.01). However, there is no correlation between the C-HT prevalence and nutritional phases (Pearson's χ2 = 4.992; p = 0.288), genotypes (Pearson's χ2 = 0.292; p = 0.864), or rhGH therapy (Pearson's χ2 = 1.799; p = 0.180). CONCLUSIONS: This study suggests the prevalence of C-TH was increased with the age before 3 y, and reached the peak in the 1 to 3 y group, then gradually declined over the years. There is no correlation between C-HT prevalence and nutritional phases, genotypes, or rhGH treatment.

[Dual mediating effects of positive and negative emotions on fertility stress and fertility quality of life among infertile men in China].

Objective: To explore the effects of fertility stress on the quality of life (QOL) of infertile men and the dual mediating roles of positive and negative emotions in fertility stress and fertility QOL. METHODS: Using the Fertility Problem Inventory, Fertility Quality of Life Questionnaire and Positive and Negative Affect Scale, we conducted a cross-sectional survey among 304 infertile men. We established a structural equation model for analysis of the relationship between the four variables of fertility stress, fertility QOL, positive emotion and negative emotion. RESULTS: The scores of the patients in fertility stress, fertility QOL, positive emotion and negative emotion were (158.42 ± 21.725), (60.72 ± 10.926), (32.15 ± 6.294) and (19.48 ± 6.378), respectively. The root mean square error approximation (RMSEA) of the direct effect model, positive emotion separate mediation model and negative emotion separate mediation model was >0.08, and the dual mediation model showed optimum fit indexes, with χ2 / df = 1.959, goodness of fit index (GFI) = 0.950, adjusted GFI (AGFI) = 0.919, normed fit index (NFI) = 0.899, incremental fit index (IFI) = 0.948, Tucker-Lewis index (TLI) = 0.926, comparative fit index (CFI) = 0.947, and RMSEA = 0.056. The results of bootstrap test indicated that the positive and negative emotions had significant mediating effects, both incomplete, on fertility stress and fertility QOL. Moreover, the separate mediation of positive emotion exhibited no statistically significant difference from that of negative emotion (95% CI: －0.063 to 0.028). CONCLUSIONS: Positive emotion and negative emotions are part of the intermediary in fertility stress and fertility QOL. Fertility stress can affect fertility QOL through the dual mediating effect of positive emotion and negative emotions in infertile men.

[The level of the stress biomarker salivary alpha amylase is correlated with semen quality in infertile young men].

Objective: To investigate the relationship between the level of the stress biomarker salivary alpha amylase (SAA) and semen quality in infertile young men. METHODS: Totally, 313 infertile and 96 normal healthy men, aged 20－40 years old, were enrolled in this study. The SAA levels and semen parameters of the subjects were measured and compared between the two groups. RESULTS: Compared with the normal healthy controls, the young infertility patients showed a significantly higher SAA level (ï¼»141.04 ± 44.13ï¼½ vs ï¼»151.48 ± 38.42ï¼½ µmol/L, P < 0.05) and percentage of immotile sperm (IMS) (ï¼»39.98 ± 14.53ï¼½% vs ï¼»64.48 ± 26.32ï¼½%, P < 0.05), but lower sperm concentration (ï¼»44.23 ± 21.63ï¼½ vs ï¼»32.42 ± 23.07ï¼½ ×106/ml, P < 0.05) and percentage of progressively motile sperm (PMS) (ï¼»52.13 ± 15.42ï¼½% vs ï¼»27.91 ± 21.22ï¼½%, P < 0.05). Sperm concentration (ï¼»26.33 ± 31.83ï¼½ vs ï¼»35.28 ± 27.70ï¼½ ×106/ml, P < 0.05) and the percentage of PMS were remarkably lower in the infertile men with a high than in those with a low SAA level (ï¼»19.85 ± 21.55ï¼½% vs ï¼»31.70 ± 20.02ï¼½%, P < 0.05), while the percentage of IMS was higher in the former than in the latter group (ï¼»74.19 ± 26.84ï¼½% vs ï¼»59.92 ± 24.85ï¼½%, P < 0.05). The SAA level in the young infertility patients was correlated positively with the percentage of IMS (r = 0.170, P < 0.01), but negatively with sperm concentration (r = －0.227, P < 0.01) and the percentage of PMS (r = －0.468, P < 0.01). CONCLUSIONS: The stress biomarker salivary alpha amylase level in infertile young men is negatively correlated with semen quality, and therefore semen parameters can be improved by reducing the stress level.

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.

Stress increases the risk of pregnancy failure in couples undergoing IVF.

Stress plays an important role in reproductive health and likely is one of the psychological factors affecting ART success. This study was designed to examine the relationship between the stress level as inferred from the amount of the enzyme alpha-amylase secreted in saliva (SAA) and pregnancy outcome in infertile couples undergoing in vitro fertilization and embryo transplantation (IVF-ET). A prospective cohort study was conducted in the Reproductive Medicine Centre of Zhengzhou University Hospital in Henan, China. Four hundred fifty-seven infertile couples undergoing in vitro fertilization and embryo transplantation (IVF-ET) for the first time participated in the study. Couples collected saliva samples the morning before the start of their first treatment cycle for the measurement of SAA. We found that the level of SAA (and hence, the amount of stress) in female partners, male partners, and couples analyzed together significantly affected IVF-ET outcome. Cutoff levels of SAA that predicted pregnancy failure were 136 µmol/L, 149 µmol/L, and 288 µmol/L in female partners, male partners, and couples, respectively. Female partners, male partners, and couples with high SAA levels had increased risk of pregnancy failure compared to those with low SAA levels. The SAA level directly correlated with the follicle-stimulating hormone level and was inversely proportional to the anti-Müllerian hormone level and endometrial thickness. Some semen parameters of male partners, such as density, survival rate, sperm rapid progressive motility (A%), and progressive motility [(A + B)%], were significantly lower in the high-SAA than in the low-SAA group. Furthermore, couples in the high SAA group had fewer transferable and high-quality embryos. We concluded that a high SAA level, known to be an objective indicator of high stress, increases the risk of pregnancy failure in infertile couples undergoing IVF-ET. Lay summary To explore the relationship between stress, as measured by the levels of the stress biomarker salivary alpha-amylase (SAA), and pregnancy outcome in infertile couples undergoing in vitro fertilization, a prospective cohort study was conducted in the Reproductive Medicine Centre of Zhengzhou University Hospital in Henan, China. Four hundred fifty-seven infertile couples undergoing IVF-ET collected saliva samples the morning before the start of their first treatment cycle for the measurement of SAA. Results of this study demonstrated that a high SAA level, known to be an objective indicator of high stress, increases the risk of pregnancy failure in infertile couples undergoing IVF-ET.

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.

The effect of fertility stress on endometrial and subendometrial blood flow among infertile women.

BACKGROUND: To investigate the effect of fertility stress on endometrial and subendometrial blood flow among infertile women. METHODS: This case-control study was conducted in The First Affiliated Hospital of Zhengzhou University. The fertility problem inventory (FPI) was adopted to evaluate fertility stress. Three-dimensional power Doppler ultrasonography (3D PD-US) was performed during the proliferative phase of the menstrual cycle (days 5-11) to measure endometrial thickness, pattern, endometrial and subendometrial volume (V), the vascularization index (VI), the flow index (FI) and the vascularization-FI (VFI) index. Then, 300 infertile women were separated into two groups (high-score group and low-score group) based on total FPI scores and 80 healthy women were selected as controls. RESULTS: No differences were found among all three groups with regard to general characteristics, endometrial thickness, pattern, endometrial and subendometrial V, VI and VFI. The endometrial and subendometrial FIs associated with different stress levels significantly differed among the three groups (F = 33.95, P < 0.001; F = 44.79, P < 0.001, respectively). The endometrial and subendometrial FIs in the control group were significantly higher than those in the high-score group and low-score groups. The endometrial and subendometrial FIs in the low-score group were significantly higher than those in the high-score group. The total FPI score was closely related to the endometrial and subendometrial FIs (r = -0.304, P < 0.001; r = -0.407, P < 0.001, respectively). CONCLUSION: Fertility stress was associated with endometrial and subendometrial flow index. Whether fertility stress might affect pregnancy outcome by reducing endometrial and subendometrial blood flow requires further research.

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, 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.