Evaluation of the clinical, biochemical, genotype and prognosis of mut-type methylmalonic acidemia in 365 Chinese cases.

BACKGROUND: Methylmalonic acidemia (MMA), which results from defects in methylmalonyl-CoA mutase (mut type) or its cofactor, is the most common inherited organic acid metabolic disease in China. This study aimed to investigate the phenotype and genotype of mut-type MMA in Chinese patients. METHODS: We recruited 365 patients with mut-type MMA; investigated their disease onset, newborn screening (NBS) status, biochemical metabolite levels, gene variations and prognosis; and explored the relationship between phenotype and genotype. RESULTS: There were 152 patients diagnosed by tandem mass spectrometry (MS/MS) expanded NBS, 209 patients diagnosed because of disease onset without NBS and 4 cases diagnosed because of sibling diagnosis. The median age of onset was 15 days old, with a variety of symptoms without specificity. Urinary levels of methylmalonic acid and methylcitric acid (MCA) decreased after treatment. Regarding the prognosis, among the 152 patients with NBS, 50.6% were healthy, 30.3% had neurocognitive impairment and/or movement disorders and 13.8% died. Among the 209 patients without NBS, 15.3% were healthy, 45.9% had neurocognitive impairment and/or movement disorders and 33.0% died. In total, 179 variants were detected in the MMUT gene, including 52 novel variations. c.729_730insTT, c.1106G>A, c.323G>A, c.914T>C and c.1663G>A were the five most frequent variations. The c.1663G>A variation led to a milder phenotype and better prognosis. CONCLUSION: There is a wide spectrum of variations in the MMUT gene with several common variations. Although the overall prognosis of mut-type MMA was poor, participation in MS/MS expanded NBS, vitamin B12 responsive and late onset are favourable factors for the prognosis.

Screening and follow-up results of fatty acid oxidative metabolism disorders in 608 818 newborns in Jining, Shandong province.

To investigate the incidence and gene mutation characteristics of fatty acid oxidative metabolism disorders in Jining area of Shandong province , and to evaluate the therapeutic effect. Blood samples of newborns were collected in Jining of Shandong province between July 14, 2014 and December 31, 2019. Tandem mass spectrometry was used to determine the levels of carnitine and acylcarnitine in the blood to screen for fatty acid oxidative metabolism disorder. For newborns with positive screening result, blood DNA was analyzed by MassARRAY and high-throughput sequencing, then verified by Sanger sequencing. The diagnosed children were given early intervention and treatment, and followed up. Forty-two children with fatty acid oxidative metabolism disorders were screened out of 608 818 newborns, with an incidence rate of 1/14 496. Primary carnitine deficiency (16 cases, 38.10%) and short-chain acyl-CoA dehydrogenase deficiency (16 cases, 38.10%) were the most common, followed by very long-chain acyl-CoA dehydrogenase deficiency (6 cases, 14.29%), medium-chain acyl-CoA dehydrogenase deficiency (4 cases, 9.53%). In children with primary carnitine deficiency, c.1400C>G (p.S467C) and c.51C>G were the most common in mutations; and c.278C>T (p.S93L), c.1049T >C (p.L350P), c.572A>G (p.K191R), c.431T>C (p.L144P) were newly discovered mutations. Ten children with carnitine replacement therapy showed normal development during the follow-up. In 6 children without carnitine replacement treatment, hypoglycemia developed during the neonatal period in 1 case, in whom the creatine kinase was increased, and the intellectual and language development delayed in the later period; the other 5 children developed normally during the follow-up period. The gene mutations c.1031A>G (p.E344G) and c.164C>T (p.P55L) were common in children with short-chain acyl-CoA dehydrogenase deficiency, and the children developed normally during the follow-up. In children with very long-chain acyl-CoA dehydrogenase deficiency, the c.1349G>A was common in gene mutations; and c.488T>A , c.1228G>T (p.D410Y), c.1276G>A (p.A426T), c.1522C>T (p.Q508*), c.1226C>T (p.T409M) were newly discovered mutations. Three children treated with milk powder rich in medium-chain fatty acids had normal development during the follow-up. The other 3 cases with combined carnitine reduction were treated with levocarnitine and milk powder enriched of medium-chain fatty acids, 1 case developed normally during the follow-up, 1 case died of acute illness at the age of and 1 case had acute illness and recovered after treatment, and developed normally during the follow-up. c.449_452del (p.T150Rfs*4) was the most common gene mutation in children with medium-chain acyl-CoA dehydrogenase deficiency, and c. 718A>G (p.M240V) was a newly discovered mutation. All children received low-fat diet, and hunger and fatigue were avoided; 1 child was supplemented with L-carnitine, and the other 3 children were not treated with drugs, and all of them developed normal during the follow-up. Primary carnitine deficiency and short-chain acyl-CoA dehydrogenase deficiency are the most common fatty acid oxidative metabolism disorders in Jining area. There are gene hotspot mutations and new discovered gene mutations in patients. Patients with early diagnosis and treatment through neonatal screening have a good prognosis.

Combining newborn metabolic and genetic screening for neonatal intrahepatic cholestasis caused by citrin deficiency.

To evaluate the feasibility of incorporating genetic screening for neonatal intrahepatic cholestasis, caused by citrin deficiency (NICCD), into the current newborn screening (NBS) program. We designed a high-throughput iPLEX genotyping assay to detect 28 SLC25A13 mutations in the Chinese population. From March 2018 to June 2018, 237 630 newborns were screened by tandem mass spectrometry at six hospitals. Newborns with citrulline levels between 1/2 cutoff and cutoff values of the upper limit were recruited for genetic screening using the newly developed assay. The sensitivity and specificity of the iPLEX genotyping assay both reached 100% in clinical practice. Overall, 29 364 (12.4%) newborns received further genetic screening. Five patients with conclusive genotypes were successfully identified. The most common SLC25A13 mutation was c.851_854del, with an allele frequency of 60%. In total, 658 individuals with one mutant allele were identified as carriers. Eighteen different mutations were observed, yielding a carrier rate of 1/45. Notably, Quanzhou in southern China had a carrier rate of up to 1/28, whereas Jining in northern China had a carrier rate higher than that of other southern and border cities. The high throughput iPLEX genotyping assay is an effective and reliable approach for NICCD genotyping. The combined genetic screening could identify an additional subgroup of patients with NICCD, undetectable by conventional NBS. Therefore, this study demonstrates the viability of incorporating genetic screening for NICCD into the current NBS program.

The intellectual disability gene PQBP1 rescues Alzheimer's disease pathology.

Early-phase pathologies of Alzheimer's disease (AD) are attracting much attention after clinical trials of drugs designed to remove beta-amyloid (Aß) aggregates failed to recover memory and cognitive function in symptomatic AD patients. Here, we show that phosphorylation of serine/arginine repetitive matrix 2 (SRRM2) at Ser1068, which is observed in the brains of early phase AD mouse models and postmortem end-stage AD patients, prevents its nuclear translocation by inhibiting interaction with T-complex protein subunit α. SRRM2 deficiency in neurons destabilized polyglutamine binding protein 1 (PQBP1), a causative gene for intellectual disability (ID), greatly affecting the splicing patterns of synapse-related genes, as demonstrated in a newly generated PQBP1-conditional knockout model. PQBP1 and SRRM2 were downregulated in cortical neurons of human AD patients and mouse AD models, and the AAV-PQBP1 vector recovered RNA splicing, the synapse phenotype, and the cognitive decline in the two mouse models. Finally, the kinases responsible for the phosphorylation of SRRM2 at Ser1068 were identified as ERK1/2 (MAPK3/1). These results collectively reveal a new aspect of AD pathology in which a phosphorylation signal affecting RNA splicing and synapse integrity precedes the formation of extracellular Aß aggregates and may progress in parallel with tau phosphorylation.

RpA1 ameliorates symptoms of mutant ataxin-1 knock-in mice and enhances DNA damage repair.

DNA damage and repair is a critical domain of many neurodegenerative diseases. In this study, we focused on RpA1, a candidate key molecule in polyQ disease pathologies, and tested the therapeutic effect of adeno-associated virus (AAV) vector expressing RpA1 on mutant Ataxin-1 knock-in (Atxn1-KI) mice. We found significant effects on motor functions, normalized DNA damage markers (γH2AX and 53BP1), and improved Purkinje cell morphology; effects that lasted for 50 weeks following AAV-RpA1 infection. In addition, we confirmed that AAV-RpA1 indirectly recovered multiple cellular functions such as RNA splicing, transcription and cell cycle as well as abnormal morphology of dendrite and dendritic spine of Purkinje cells in Atxn1-KI mice. All these results suggested a possibility of gene therapy with RpA1 for SCA1.

Targeting TEAD/YAP-transcription-dependent necrosis, TRIAD, ameliorates Huntington's disease pathology.

Neuronal cell death in neurodegenerative diseases is not fully understood. Here we report that mutant huntingtin (Htt), a causative gene product of Huntington's diseases (HD) selectively induces a new form of necrotic cell death, in which endoplasmic reticulum (ER) enlarges and cell body asymmetrically balloons and finally ruptures. Pharmacological and genetic analyses revealed that the necrotic cell death is distinct from the RIP1/3 pathway-dependent necroptosis, but mediated by a functional deficiency of TEAD/YAP-dependent transcription. In addition, we revealed that a cell cycle regulator, Plk1, switches the balance between TEAD/YAP-dependent necrosis and p73/YAP-dependent apoptosis by shifting the interaction partner of YAP from TEAD to p73 through YAP phosphorylation at Thr77. In vivo ER imaging with two-photon microscopy detects similar ER enlargement, and viral vector-mediated delivery of YAP as well as chemical inhibitors of the Hippo pathway such as S1P recover the ER instability and necrosis in HD model mice. Intriguingly S1P completely stops the decline of motor function of HD model mice even after the onset of symptom. Collectively, we suggest approaches targeting the signalling pathway of TEAD/YAP-transcription-dependent necrosis (TRIAD) could lead to a therapeutic development against HD.

Diagnosis and therapeutic monitoring of inborn errors of metabolism in 100,077 newborns from Jining city in China.

BACKGROUND: Mandatory newborn screening for metabolic disorders has not been implemented in most parts of China. Newborn mortality and morbidity could be markedly reduced by early diagnosis and treatment of inborn errors of metabolism (IEM). Methods of screening for IEM by tandem mass spectrometry (MS/MS) have been developed, and their advantages include rapid testing, high sensitivity, high specificity, high throughput, and low sample volume (a single dried blood spot). METHODS: Dried blood spots of 100,077 newborns obtained from Jining city in 2014-2015 were screened by MS/MS. The screening results were further confirmed by clinical symptoms and biochemical analysis in combination with the detection of neonatal deficiency in organic acid, amino acid, or fatty acid metabolism and DNA analysis. RESULTS: The percentages of males and females among the 100,077 infants were 54.1% and 45.9%, respectively. Cut-off values were established by utilizing the percentile method. The screening results showed that 98,764 newborns were healthy, and 56 out of the 1313 newborns with suspected IEM were ultimately diagnosed with IEM. Among these 56 newborns, 19 (1:5267) had amino acid metabolism disorders, 26 (1:3849) had organic acid metabolism disorders, and 11 (1:9098) had fatty acid oxidation disorders. In addition, 54 patients with IEM were found to carry mutations, and the other 2 patients had argininemia. CONCLUSIONS: Fifty-six cases of metabolic disorders in Jining were confirmed via newborn screening (NBS) by MS/MS. Early diagnosis and treatment are crucial for the survival and well-being of affected children. A nationwide NBS program using MS/MS is recommended, especially in poor areas of China.

Comprehensive phosphoproteome analysis unravels the core signaling network that initiates the earliest synapse pathology in preclinical Alzheimer's disease brain.

Using a high-end mass spectrometry, we screened phosphoproteins and phosphopeptides in four types of Alzheimer's disease (AD) mouse models and human AD postmortem brains. We identified commonly changed phosphoproteins in multiple models and also determined phosphoproteins related to initiation of amyloid beta (Aß) deposition in the mouse brain. After confirming these proteins were also changed in and human AD brains, we put the proteins on experimentally verified protein-protein interaction databases. Surprisingly, most of the core phosphoproteins were directly connected, and they formed a functional network linked to synaptic spine formation. The change of the core network started at a preclinical stage even before histological Aß deposition. Systems biology analyses suggested that phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by overactivated kinases including protein kinases C and calmodulin-dependent kinases initiates synapse pathology. Two-photon microscopic observation revealed recovery of abnormal spine formation in the AD model mice by targeting a core protein MARCKS or by inhibiting candidate kinases, supporting our hypothesis formulated based on phosphoproteome analysis.

Interleukin-1 receptor accessory protein organizes neuronal synaptogenesis as a cell adhesion molecule.

Interleukin-1 receptor accessory protein (IL-1RAcP) is the essential component of receptor complexes mediating immune responses to interleukin-1 family cytokines. IL-1RAcP in the brain exists in two isoforms, IL-1RAcP and IL-1RAcPb, differing only in the C-terminal region. Here, we found robust synaptogenic activities of IL-1RAcP in cultured cortical neurons. Knockdown of IL-1RAcP isoforms in cultured cortical neurons suppressed synapse formation as indicated by decreases of active zone protein Bassoon puncta and dendritic protrusions. IL-1RAcP recovered the accumulation of presynaptic Bassoon puncta, while IL-1RAcPb rescued both Bassoon puncta and dendritic protrusions. Consistently, the expression of IL-1RAcP in cortical neurons enhances the accumulation of Bassoon puncta and that of IL-1RAcPb stimulated both Bassoon puncta accumulation and spinogenesis. IL-1RAcP interacted with protein tyrosine phosphatase (PTP) δ through the extracellular domain. Mini-exon peptides in the Ig-like domains of PTPδ splice variants were critical for their efficient binding to IL-1RAcP. The synaptogenic activities of IL-1RAcP isoforms were diminished in cortical neurons from PTPδ knock-out mice. Correspondingly, PTPδ required IL-1RAcPb to induce postsynaptic differentiation. Thus, IL-1RAcPb bidirectionally regulated synapse formation of cortical neurons. Furthermore, the spine densities of cortical and hippocampal pyramidal neurons were reduced in IL-1RAcP knock-out mice lacking both isoforms. These results suggest that IL-1RAcP isoforms function as trans-synaptic cell adhesion molecules in the brain and organize synapse formation. Thus, IL-1RAcP represents an interesting molecular link between immune systems and synapse formation in the brain.

Variable phenotypes and outcomes associated with the MMACHC c.482G > A mutation: follow-up in a large CblC disease cohort.

BACKGROUND: The aim of this study was to characterize the variable phenotypes and outcomes associated with the methylmalonic aciduria and homocystinuria type C protein gene (MMACHC) c.482G > A mutation in 195 Chinese cases with CblC disease. METHODS: We carried out a national, retrospective multicenter study of 195 Chinese patients with CblC disease attributable to the MMACHC c.482G > A variant either in a homozygous or compound heterozygous state. The control group consisted of 200 patients diagnosed with CblC disease who did not possess the c.482G > A mutation. Clinical features, including disease onset, symptoms, biochemical metabolites, gene mutation, and follow-up outcomes were reviewed and analyzed in detail. The median follow-up period spanned 3 years and 8 months, with a range of 1 year and 2 months to 12 years and 10 months. RESULTS: Among 195 patients carrying the c.482G > A variant, 125 (64.1%) cases were diagnosed by newborn screening (NBS), 60 (30.8%) cases were detected due to disease onset, and 10 (5.1%) cases were identified from sibling diagnoses. One hundred and seventeen (93.6%) individuals who were diagnosed by NBS, and nine patients who came from sibling diagnoses remained asymptomatic in this study. From 69 symptomatic patients of the c.482G > A group, more patients presented with later onset, and the top six common clinical symptoms at disease onset were developmental delay (59.4%), lower limb weakness and poor exercise tolerance (50.7%), cognitive decline (37.7%), gait instability and abnormal posture (36.2%), seizures (26.1%), and psychiatric and behavioral disturbances (24.6%). In the 159 symptomatic patients lacking c.482G > A variants, the most frequently observed clinical manifestations at disease onset included developmental delay (81.8%), lethargy and feeding difficulty (62.9%), lower limb weakness and poor exercise tolerance (54.7%), prolonged neonatal jaundice (51.6%), vomiting (47.2%), and seizures (32.7%). Before treatment, the levels of blood propionylcarnitine, propionylcarnitine/acetylcarnitine ratio, and homocysteine in the c.482G > A group were significantly lower (P < 0.05) than those in the non-c.482G > A group, while the concentration of urinary methylmalonic acid was slightly lower (P > 0.05). The degree of decline in the above metabolites after treatment in different groups significantly differed in both plasma total homocysteine values and urinary methylmalonic acid levels (P < 0.05). In patients carrying the c.482G > A variant compared with the non-c.428G > A group, there were markedly lower rates of mortality (0.5% vs. 2.0%) and developmental delay (20.5% vs. 65.5%). When compared with individuals diagnosed due to disease onset, those identified through NBS in either group exhibited a reduced proportion of disease onset (6.7% vs. 100% in the c.482G > A group, 54.4% vs. 100% in the non-c.482G > A group), lower mortality (0.0% vs. 1.7% in the c.482G > A group, 0.0% vs. 3.6% in the non-c.482G > A group), and had a higher percentage of patients exhibiting normal psychomotor and language development (99.3% vs. 33.3% in the c.482G > A group, 58.9% vs. 10.9% in the non-c.482G > A group). CONCLUSIONS: The c.482G > A variant in MMACHC is associated with late-onset and milder phenotypes of CblC disease. Patients with this mutation tend to have a relatively better response to hydroxocobalamin, better metabolic control, and more favorable neurological outcomes. NBS and other appropriate pre-symptomatic treatments seem to be helpful in early diagnosis, resulting in favorable clinical outcomes. Video Abstract (MP4 136794 kb).

Protein tyrosine phosphatase σ regulates the synapse number of zebrafish olfactory sensory neurons.

The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) σ, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTPσ enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTPσ, indicating that presynaptic PTPσ is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTPσ in OSNs enhanced the accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPσ. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTPσ controls the number of OSN-mitral cell synapses by suppressing their excessive increase.

Reference Standards for Newborn Screening of Metabolic Disorders by Tandem Mass Spectrometry: A Nationwide Study on Millions of Chinese Neonatal Populations.

Introduction: The major clinical problem presently confronting the Chinese newborn screening (NBS) programs by tandem mass spectrometry (MS/MS) is the lack of comprehensive reference intervals (RIs) for disease biomarkers. To close this gap, the Chinese National Center for Clinical Laboratories (NCCL) launched a nationwide study to investigate the dynamic pattern of 35 MS/MS NBS biomarkers and establish accurate and robust RIs. Methods: Blood spot samples from 4,714,089 Chinese neonates were tested in participating centers/laboratories and used for study analysis. MS/MS NBS biomarker trends were visually assessed by their concentrations over age. Specific partitions were determined arbitrarily by each day and sex or by the statistical method of Harris and Boyd. RIs, corresponding to the 2.5th and 97.5th percentiles, as well as the 1th, 25th, 75th and 99th percentiles were calculated for each reference partition using a non-parametric rank approach. Results: Most MS/MS NBS biomarkers fluctuated during the first week of life, followed by a relatively stable concentration. Age and sex-specific RIs were established and presented an improved specificity over the RIs used in participating centers/laboratories. Females demonstrated higher 2.5th and 97.5th percentiles in all amino acids except arginine and ornithine than males, whereas males showed higher 2.5th and 97.5th percentiles in most acylcarnitines. Conclusion: The present study determined the dynamic trends of 35 MS/MS biomarkers and established age and sex-specific RIs, valuably contributing to the current literature and timely evaluation of neonatal health and disease.

A rare mutation c.1663G > A (p.A555T) in the MMUT gene associated with mild clinical and biochemical phenotypes of methylmalonic acidemia in 30 Chinese patients.

BACKGROUND: Methylmalonic acidemia is an inherited organic acid metabolic disease. It involves multiple physiological systems and has variable manifestations. The primary causative gene MMUT carries wide range of mutations, and one of them, c.1663G > A (p.A555T), is considered to be a rare type, which is seen more frequently in Asian than other populations. So far, little is known about the clinical features of patients carrying this mutation. In the present study, we aimed to define the clinical and biochemical features of the patients with this genotype. METHODS: Among 328 mut type methylmalonic acidemia patients from multiple hospitals in China, we collected 30 compound heterozygous patients sharing the mutation c.1663G > A (p.A555T) in the MMUT gene. Their clinical characteristics and biochemical index were described in detail and compared with methylmalonic acidemia patients without this variant. RESULTS: Most of these patients were diagnosed via newborn screening (26/30), treated in a timely manner, and kept healthy (24/30). Disease onset occurred in 7 patients. Developmental delay or intellectual impairment occurred in 4 patients. 100% of these patients (29/29) were responsive to Vitamin B12 administration. The blood propionylcarnitine, blood propionylcarnitine/acetylcarnitine ratio, urinary methylmalonic acid, urinary methylcitric acid before and after treatment in c.1663G > A (p.A555T) carrying patients were much lower than those in non-c.1663G > A (p.A555T) carrying patients. CONCLUSION: Compared to patients with other mutations in the MMUT gene, patients with the c.1663G > A (p.A555T) mutation showed later onset, milder clinical phenotype, lighter biochemical abnormalities, better vitamin B12 responsiveness, lower morbidity, easier metabolic control, and thereby better prognosis. Newborn screening project plays an important role in early diagnosis, treatment, and prognosis of these patients.

Different mutations in the MMUT gene are associated with the effect of vitamin B12 in a cohort of 266 Chinese patients with mut-type methylmalonic acidemia: A retrospective study.

BACKGROUND: To summarize the relationship between different MMUT gene mutations and the response to vitamin B12 in MMA. METHODS: This was a retrospective study of patients diagnosed with mut-type MMA. All patients with mut-type MMA were tested for responsiveness to vitamin B12. RESULTS: There were 81, 27, and 158 patients in the completely responsive, partially responsive, and nonresponsive groups, respectively, and the proportions of symptom occurrence were 30/81 (37.0%), 21/27 (77.8%), and 131/158 (82.9%), respectively (p < .001). The median levels of posttreatment propionyl carnitine (C3), C3/acetyl carnitine (C2) ratio in the blood, and methylmalonic acid in the urine were all lower than pretreatment, and the median level of C3/C2 ratio in the completely responsive group was within the normal range. In 266 patients, 144 different mutations in the MMUT gene were identified. Patients with the mutations of c.1663G>A, c.2080C>T, c.1880A>G, c.1208G>A, etc. were completely responsive and with the mutations of c.1741C>T, c.1630_1631GG>TA, c.599T>C, etc. were partially responsive. The proportions of healthy/developmental delay outcomes in the three groups were 63.0%/23.5%, 33.3%/40.7%, and 13.3%/60.1%, respectively (p < .001). CONCLUSION: Different mutations in the MMUT gene are associated with the effect of vitamin B12 treatment.

Effect of PBNA on the NO content and NOS activity in ischemia/reperfusion injury in the rat retina.

OBJECTIVE: To investigated the effect of Polygonum Bistorta L. n-butyl Alcohol (PBNA) extract on the NO content and NOS activity in ischemia/reperfusion (I/R) injury in the rat retina. METHODS: The model of retinal I/R injury in SD rats was made by reperfusion for 1 h after occlusion of common carotid artery (CCA) for 1 h. The rats were randomly divided into four groups: control group, retinal I/R injury group, low-dosage PBNA treated group and high-dosage treated PBNA group. The control group was injected with 1 ml/kg NS through sublingual vein after CCA was dissociated. Other groups were treated with normal saline or PBNA before occlusion of CCA. After occlusion of CCA for 1 h following reperfusion for 1 h, blood was collected and serum was separated to determine the contents of NO, the activity of T-NOS, iNOS and eNOS. RESULT: (1) The contents of NO in I/R group showed lower values than in control group (P<0.001) and low-dosage PBNA treated group (P<0.05). (2) The activities of T-NOS in both low-dosage PBNA group and high-dosage group increased, compared with I/R group (P<0.01). (3) The activity of serum iNOS in I/R group increased compared with control group (P<0.05) and low-dosage PBNA treated group evidently (P<0.05). (4) The activity of serum eNOS in I/R group decreased compared with control group (P<0.05), both low-dosage (P<0.05) and high-dosage PBNA (P<0.01) treated group markedly. CONCLUSION: The date suggest that PBNA have a therapeutic effect on retinal ischemia/reperfusion injury by increasing the activities of T-NOS and eNOS, decreasing the activity of iNOS, elevating the content of NO, enhancing the Anti-oxidation and expanding the blood vessel.

Newborn screening and diagnosis of inborn errors of metabolism: A 5-year study in an eastern Chinese population.

Inborn errors of metabolism (IEMs) can cause intellectual disability or even death in children. To evaluate the disease spectrum and genetic characteristics of IEMs in Jining City of Shandong Province in East China, we used tandem mass spectrometry (MS/MS) technology for IEMs screening combined with genetic analysis. Newborns were screened from July 14, 2014, to December 31, 2018. Amino acid and carnitine contents were detected by MS/MS. According to the results for normal newborns, the reference range of our laboratory was established with the percentile method. The suspected positive newborns were further diagnosed using next-generation sequencing. A total of 514,234 newborns were screened, and 265 were diagnosed with IEMs, with a detection rate of 1:1941. Of the 265 patients, 130 (49.06%) had organic acid disorders, 83 (31.32%) had amino acid disorders, 34 (12.83%) had fatty acid oxidation disorders, and 18 (6.79%) had urea circulatory disorders. PAHD and MMA were the two most common disorders. IEMs-associated genes were identified in 233 patients. Our data indicated that IEMs are never uncommon in Jining, and the disease spectrum and genetic background were clearly elucidated, contributing to the treatment and prenatal genetic counseling of these disorders in the region.

Methods to Image Macroautophagy in the Brain In Vivo.

Macroautophagy is the process to remove intracellular organelles or proteins by using autophagosome that is composed of autophagy proteins such as atg3, atg7, and atg8/LC3 (Mizushima, et al. Annu Rev Cell Dev Biol. 27:107-132, 2011). Here, we develop a useful method for in vivo imaging of autophagosome under the two-photon microscopy. Time-lapse imaging of LC3-ECFP enables us to quantify the dynamics of number, size, and signal intensity of autophagosomes in neurons or in other types of cells in the brain.

Newborn Screening for Spinal Muscular Atrophy in China Using DNA Mass Spectrometry.

Background: Spinal muscular atrophy (SMA) is the most common neurodegenerative disorder and the leading genetic cause of infant mortality. Early detection of SMA through newborn screening (NBS) is essential to selecting pre-symptomatic treatment and ensuring optimal outcome, as well as, prompting the urgent need for effective screening methods. This study aimed to determine the feasibility of applying an Agena iPLEX SMA assay in NBS for SMA in China. Methods: We developed an Agena iPLEX SMA assay based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and evaluated the performance of this assay through assessment of 167 previously-genotyped samples. Then we conducted a pilot study to apply this assay for SMA NBS. The SMN1 and SMN2 copy number of screen-positive patients were determined by multiplex ligation-dependent probe amplification analysis. Results: The sensitivity and specificity of the Agena iPLEX SMA assay were both 100%. Three patients with homozygous SMN1 deletion were successfully identified and conformed by multiplex ligation-dependent probe amplification analysis. Two patients had two SMN2 copies, which was correlated with severe SMA type I phenotype; both of them exhibited neurogenic lesion and with decreased muscle power. Another patient with four SMN2 copies, whose genotype correlated with milder SMA type III or IV phenotype, had normal growth and development without clinical symptoms. Conclusions: The Agena iPLEX SMA assay is an effective and reliable approach for population-based SMA NBS. The first large-scale pilot study using this assay in the Mainland of China showed that large-scale implementation of population-based NBS for SMA is feasible.

Chronic cerebral hypoperfusion shifts the equilibrium of amyloid ß oligomers to aggregation-prone species with higher molecular weight.

Epidemiological studies have shown that atherosclerotic risk factors accelerate the pathological process underlying Alzheimer's disease (AD) via chronic cerebral hypoperfusion. In this study, we aimed to clarify the mechanisms by which cerebral hypoperfusion may exacerbate AD pathology. We applied bilateral common carotid artery stenosis (BCAS) to a mice model of AD and evaluated how the equilibrium of amyloid ß oligomers respond to hypoperfusion. BCAS accelerated amyloid ß (Aß) convergence to the aggregation seed, facilitating the growth of Aß plaques, but without changing the total Aß amount in the brain. Furthermore, Aß oligomers with high molecular weight increased in the brain of BCAS-operated mice. Considering Aß is in an equilibrium among monomeric, oligomeric, and aggregation forms, our data suggest that cerebral hypoperfusion after BCAS shifted this equilibrium to a state where a greater number of Aß molecules participate in Aß assemblies to form aggregation-prone Aß oligomers with high molecular weight. The reduced blood flow in the cerebral arteries due to BCAS attenuated the dynamics of the interstitial fluid leading to congestion, which may have facilitated Aß aggregation. We suggest that cerebral hypoperfusion may accelerate AD by enhancing the tendency of Aß to become aggregation-prone.

Expanded Newborn Screening for Inborn Errors of Metabolism and Genetic Characteristics in a Chinese Population.

The incidence of inborn errors of metabolisms (IEMs) varies dramatically in different countries and regions. Expanded newborn screening for IEMs by tandem mass spectrometry (MS/MS) is an efficient approach for early diagnosis and presymptomatic treatment to prevent severe permanent sequelae and death. To determine the characteristics of IEMs and IEMs-associated mutations in newborns in Jining area, China, 48,297 healthy neonates were recruited for expanded newborn screening by MS/MS. The incidence of IEMs was 1/1178 in Jining, while methylmalonic acidemia, phenylketonuria, and primary carnitine deficiency ranked the top 3 of all detected IEMs. Thirty mutations in nine IEMs-associated genes were identified in 28 confirmed cases. As 19 cases with the mutations in phenylalanine hydroxylase (PAH), solute carrier family 22 member 5 (SLC22A5), and methylmalonic aciduria (cobalamin deficiency) cblC type with homocystinuria (MMACHC) genes, respectively, it suggested that mutations in the PAH, SLC22A5, and MMACHC genes are the predominant causes of IEMs, leading to the high incidence of phenylketonuria, primary carnitine deficiency, and methylmalonic acidemia, respectively. Our work indicated that the overall incidence of IEMs is high and the mutations in PAH, SLC22A5, and MMACHC genes are the leading causes of IEMs in Jining area. Therefore, it is critical to increase the coverage of expanded newborn screening by MS/MS and prenatal genetic consulting in Jining area.