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.

[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.

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.

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.

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.

[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.

Plasma Amino Acid Profile in Children with Autism Spectrum Disorder in Southern China: Analysis of 110 Cases.

To retrospectively explore the characteristics of plasma amino acids (PAAs) in children with autism spectrum disorder and their clinical association via case-control study. A total of 110 autistic and 55 healthy children were recruited from 2014 to 2018. The clinical phenotypes included severity of autism, cognition, adaptability, and regression. Compared with the control group, autistic children had significantly elevated glutamate, γ-Amino-n-butyric acid, glutamine, sarcosine, δ-aminolevulinic acid, glycine and citrulline. In contrast, their plasma level of ethanolamine, phenylalanine, tryptophan, homocysteine, pyroglutamic acid, hydroxyproline, ornithine, histidine, lysine, and glutathione were significantly lower. Elevated neuroactive amino acids (glutamate) and decreased essential amino acids were mostly distinct characteristics of PAAs of autistic children. Increased level of tryptophan might be associated with severity of autism.

A sensitive liquid chromatography-tandem mass spectrometry method for determination biomarkers of monoamine neurotransmitter disorders in cerebrospinal fluid.

BACKGROUND: Cerebrospinal fluid (CSF) monoamine neurotransmitters, their precursors and metabolites are essential biomarkers in the diagnosis and follow-up of monoamine neurotransmitter disorders (MNDs). However, their extra low concentrations and potential instability challenge the detection method. Here, we present a method that enables simultaneous quantification of these biomarkers. METHOD: With propyl chloroformate /n-propanol, 16 biomarkers in 50 µL of CSF were derivatized in situ within seconds under an ambient temperature. The derivatives were extracted by ethyl acetate and separated by a reverse phase column followed by mass spectrometric detection. The method was fully validated. Optimal conditions for standard solution preparation and storage, as well as CSF sample handling, were investigated. CSF samples from 200 controls and 16 patients were analyzed. RESULTS: The derivatization reaction stabilized biomarkers and increased sensitivity. Most biomarkers were quantifiable in concentrations between 0.02 and 0.50 nmol/L that were sufficient to measure their endogenous concentrations. The intra- and inter-day imprecision were < 15% for most analytes, and accuracy ranged from 90.3% to 111.6%. The stability study showed that standard stock solutions were stable at -80 °C for six years when prepared in the protection solutions; Analytes in CSF samples were stable for 24 h on wet ice and at least two years at -80 °C; But repeated freeze-thaw should be avoided. With this method, age-dependent reference intervals for each biomarker in the pediatric population were established. Patients with MNDs were successfully identified. CONCLUSION: The developed method is valuable for MNDs diagnosis and research, benefiting from its advantages of sensitivity, comprehensiveness, and high throughput.

Diagnosis of an intermediate case of maple syrup urine disease: A case report.

BACKGROUND: Maple syrup urine disease (MSUD) is an autosomal recessive genetic disorder caused by defects in the catabolism of the branched-chain amino acids (BCAAs). However, the clinical and metabolic screening is limited in identifying all MSUD patients, especially those patients with mild phenotypes or are asymptomatic. This study aims to share the diagnostic experience of an intermediate MSUD case who was missed by metabolic profiling but identified by genetic analysis. CASE SUMMARY: This study reports the diagnostic process of a boy with intermediate MSUD. The proband presented with psychomotor retardation and cerebral lesions on magnetic resonance imaging scans at 8 mo of age. Preliminary clinical and metabolic profiling did not support a specific disease. However, whole exome sequencing and subsequent Sanger sequencing at 1 year and 7 mo of age identified bi-allelic pathogenic variants of the BCKDHB gene, confirming the proband as having MSUD with non-classic mild phenotypes. His clinical and laboratory data were retrospectively analyzed. According to his disease course, he was classified into an intermediate form of MSUD. His management was then changed to BCAAs restriction and metabolic monitoring conforming to MSUD. In addition, genetic counseling and prenatal diagnosis were provided to his parents. CONCLUSION: Our work provides diagnostic experience of an intermediate MSUD case, suggesting that a genetic analysis is important for ambiguous cases, and alerts clinicians to avoid missing patients with non-classic mild phenotypes of MSUD.

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.

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.

[Karyotypes and pelvic ultrasonography in children with Turner syndrome].

OBJECTIVE: To study the relationship of karyotypes and gonadal development in children with Turner syndrome. METHODS: Fifty-one children with Turner syndrome were studied. Pelvic ultrasonography was performed on the children. Twenty healthy age-matched girls were used as control group. RESULTS: Eleven kinds of karyotypes were detected in 51 children with Turner syndrome. Children were classified into two groups based on karyotypes: Group 1 (45,XO; n= 24) and Group 2 (karyotypes other than 45, XO; n=27). 45,XO karyotype was the most common (47.1%), followed by 46,X,i (Xq)/45, XO (21.6%), 46,XX/45, XO (7.8%) and 47,XXX/45, XO (5.9%). Pelvic ultrasonography showed a primordial uterus in 41 cases, infantile uterus in 7 cases, congenital absence of uterus and ovary in 3 cases, simple anovarism in 42 cases and ovarian dysgenesis in 6 cases. Uterine size in both Groups 1 and 2 were significantly smaller than those of the control group (P<0.05). Group 1 showed smaller size of uterus than Group 2 (P<0.05). Ovaries were not detected in all Group 1 patients. CONCLUSIONS: The dysplasia and atrophy of ovaries and uterus exist in children with Turner syndrome. The patients with 45,XO karyotype had poorer gonadal development than those with other karyotypes.

A novel mutation of estrogen receptor gene detected in girls with precocious puberty.

Female precocious puberty is caused by premature activation of the hypothalamic-pituitary-gonadal axis, exposure to exogenous sex steroid hormones, and the presence of endogenous sex steroids caused by various factors. Estrogen is the final key factor to start onset of puberty. However,in some cases of precocious puberty in girls estrogen elevation could not be detected. The raised sensitivity of estrogen receptor, which may caused by ESR1 mutation or polymorphism, has been frequently mentioned for interpreting the etiology of sporadic low estrogen type cases. But no case evidence has been found in clinical practice. For the purpose of screening possible mutations in estrogen receptor gene, leukocyte genomic DNA were collected from 16 girls with precocious puberty of sporadic low estrogen,and exons of ESR1 were amplified and analysized using PCR-SSCP/silver staining method. A single strand conformation change in exon 8 was found in one of the patients (No. 14). The suspected fragment were cloned to a T vector and sequenced for analysis. Sequencing of these clones revealed that this conformation change is caused by a C to T transition. This mutation results in the replacement of arginine by cystine at position 548 of ESR1 protein. The mutation created an extra Btsl digest site and made it can be readily identified by PCR-PFLP method. Further detection using this method, and sequencing of cloned exon8 colonies from patients proved that the patient No. 14 is Arg548/Cys548 heterozagous in genotype. This mutation increased hydrophobility of the area dramatically. The position and the conservative of this residue in vertebrates suggested Arg548 may play an important role in ESR1 function. For study the role of this mutation in the onset of precocious puberty, a firefly luciferase reporter plasmid pGL3-promoter-ERE was constructed,and a pCR3. 1-hermut pisimid expressing Cys548 ER was constructed based on wild type pCR3. 1her. Co-transfection of reporter and pCR3. 1 -hermut in CMF-7 cell strain proved that Cys548 mutant can significantly increase the transcription activity over the Arg548 wild type.

Combination of a Haploidentical Stem Cell Transplant With Umbilical Cord Blood for Cerebral X-Linked Adrenoleukodystrophy.

BACKGROUND: Childhood cerebral X-linked adrenoleukodystrophy is a rapidly progressive neurodegenerative disorder that affects central nervous system myelin and the adrenal cortex. Hematopoietic stem cell transplantation is the best available curative therapy if performed during the early stages of disease. Only 30% of patients who might benefit from a hematopoietic stem cell transplant will have a full human leukocyte antigen-matched donor, which is considered to be the best choice. PATIENT DESCRIPTION: We present a 5-year-old boy with cerebral X-linked adrenoleukodystrophy whose brain magnetic resonance imaging severity score was 7 and who needed an immediate transplantation without an available full human leukocyte antigen-matched donor. We combined haploidentical and umbilical cord blood sources for transplantation and saw encouraging results. After transplantation, the patient showed neurological stability for 6 months and the level of very long chain fatty acids had decreased. By 1 year, the patient appeared to gradually develop cognition, motor, and visual disturbances resulting from possible mix chimerism. CONCLUSION: Transplantation of haploidentical stem cells combined with the infusion of umbilical cord blood is a novel approach for treating cerebral X-linked adrenoleukodystrophy. It is critical to monitor posttransplant chimerism and carry out antirejection therapy timely for a beneficial clinical outcome.

A novel ABCD1 gene mutation in a Chinese patient with X-linked adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) (OMIM: 300100) is a recessive neurodegenerative disorder caused by defects in the ABCD1 gene on chromosome Xq28. Childhood cerebral ALD (CCALD) is the most frequent phenotype. OBJECTIVE: We describe an affected boy who developed normally until he was 8 years old then suffered progressive neurological deficits that ultimately led to death. METHODS: Diagnosis was based on clinical symptoms, an abnormal very long chain fatty acid profile in plasma, typical CCALD MRI pattern, and molecular analysis. RESULTS: Direct sequencing of the ABCD1 gene in this patient identified a novel splicing mutation (IVS1+1G>A) in intron 1, which is considered to be the pathogenic mutation. CONCLUSION: We have identified a novel ABCD1 mutation as the likely cause of CCALD in a Chinese patient.