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.

Clinical and genetic analysis of methylmalonic aciduria in 60 patients from Southern China: a single center retrospective study.

BACKGROUND: Methylmalonic aciduria (MMA) is a group of rare genetic metabolic disorders resulting from defects in methylmalonyl coenzyme A mutase (MCM) or intracellular cobalamin (cbl) metabolism. MMA patients show diverse clinical and genetic features across different subtypes and populations. METHODS: We retrospectively recruited 60 MMA patients from a single center and diagnosed them based on their clinical manifestations and biochemical assays. We then performed genetic analysis to confirm the diagnosis and identify the causal variants. RESULTS: We confirmed the common clinical manifestations of MMA reported previously. We also described four rare MMA cases with unusual symptoms or genetic variants, such as pulmonary hypertension or limb weakness in late-onset patients. We identified 15 MMACHC and 26 MMUT variants in 57 patients, including 6 novel MMUT variants. Two patients had only one MMAA variant each, and one patient had mild MMA due to mitochondrial DNA depletion syndrome caused by a SUCLA2 variant. Among 12 critically ill patients, isolated MMA was associated with higher C3, blood ammonia, and acidosis, while combined MMA was linked to hydrocephalus on skull MRI. MMACHC c.658-660delAAG and MMUT c.1280G > A variants were correlated with more severe phenotypes. CONCLUSIONS: Our study demonstrates the clinical and genotypic heterogeneity of MMA patients and indicates that metabolic screening and genetic analysis are useful tools to identify rare cases.

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.

Newborn Screening for Inborn Errors of Metabolism by Next-Generation Sequencing Combined with Tandem Mass Spectrometry.

The aim of this study was to observe the outcomes of newborn screening (NBS) in a certain population by using next-generation sequencing (NGS) as a first-tier screening test combined with tandem mass spectrometry (MS/MS). We performed a multicenter study of 29,601 newborns from eight screening centers with NBS via NGS combined with MS/MS. A custom-designed panel targeting the coding region of the 142 genes of 128 inborn errors of metabolism (IEMs) was applied as a first-tier screening test, and expanded NBS using MS/MS was executed simultaneously. In total, 52 genes associated with the 38 IEMs screened by MS/MS were analyzed. The NBS performance of these two methods was analyzed and compared respectively. A total of 23 IEMs were diagnosed via NGS combined with MS/MS. The incidence of IEMs was approximately 1 in 1287. Within separate statistical analyses, the positive predictive value (PPV) for MS/MS was 5.29%, and the sensitivity was 91.3%. However, for genetic screening alone, the PPV for NGS was 70.83%, with 73.91% sensitivity. The three most common IEMs were methylmalonic academia (MMA), primary carnitine deficiency (PCD) and phenylketonuria (PKU). The five genes with the most common carrier frequencies were PAH (1:42), PRODH (1:51), MMACHC (1:52), SLC25A13 (1:55) and SLC22A5 (1:63). Our study showed that NBS combined with NGS and MS/MS improves the performance of screening methods, optimizes the process, and provides accurate diagnoses.

Clinical manifestations and genetic mutation analysis of patients with mucopolysaccharidosis type VII in China.

OBJECTIVE: This study aimed to explore the clinical and genetic features of Chinese patients with mucopolysaccharidosis type VII (MPS VII), thereby improving early detection, disease management, and patient outcomes. METHODS: A retrospective review of medical records for five patients presenting with coarse facial features, rib protrusion, chest deformities, and scoliosis was conducted. Exome sequencing was employed to identify causative genetic mutations. RESULTS: The study comprised five patients (four males, one female) with disease onset at six months of age (range: 0-1.5 years). Common symptoms included coarse facial features, skeletal abnormalities, delayed motor and language development, and intellectual disability. Approximately 80% of the patients exhibited multiple skeletal dysplasias, enlarged adenoids or tonsils, and snoring; 60% had hernias; 40% reported hearing loss and hepatosplenomegaly. Less frequent manifestations were short stature, valvular heart disease, non-immune hydrops fetalis, and corneal opacity. All patients demonstrated elevated urine glycosaminoglycans levels and absent ß-glucuronidase activity in leukocytes. Exome sequencing identified compound heterozygous mutations in the GUSB gene in all four tested patients, uncovering seven mutations in total, three of which were novel (c.189G > A, c.869C > T, and c.1745 T > C). Furthermore, prenatal diagnosis through chorionic villus sampling in subsequent pregnancies of one patient's mother revealed both fetuses had normal ß-glucuronidase activity and no disease-causing mutations in the GUSB gene. CONCLUSION: The study's patients all presented with classic symptoms of MPS VII due to ß-glucuronidase deficiency, with three new pathogenic mutations identified in the GUSB gene. Genetic counseling and prenatal testing were highlighted as crucial for disease prevention.

A pilot study of newborn screening for X-linked adrenoleukodystrophy based on liquid chromatography-tandem mass spectrometry method for detection of C26:0-lysophosphatidylcholine in dried blood spots: Results from 43,653 newborns in a southern Chinese population.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a rare X-linked disease caused by mutations of the ABCD1 gene. C26:0-lysophosphatidylcholine (C26:0-LPC) has been proved to be an accurate biomarker for X-ALD. This study aims to propose an effective method for screening of X-ALD and to evaluate the performance of the newborn screening (NBS) assay for X-ALD in Guangzhou. METHODS: C26:0-LPC in dried blood spots (DBS) was extracted by methanol solution containing isotope-labelled internal standard (C26:0-d4-LPC) and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The sensitivity of the method was assessed in eight male X-ALD patients, two female carriers and 583 healthy controls. The method was conducted on 43,653 newborns. Next generation sequencing was performed on screen-positive samples. Plasma analysis of very long-chain fatty acids and genetic counselling were performed by way of follow-up. RESULTS: Elevated C26:0-LPC were 100% sensitive for screening of X-ALD. Of 43,653 newborns, 32 (18 males, 14 females) screened positive. Of these, 14 (43.7%) were identified ABCD1 variants, including seven hemizygous males and seven heterozygous females, and two (6.3%) were diagnosed with other peroxisomal disorders. CONCLUSION: The LC-MS/MS method for screening of X-ALD can identify males, heterozygous females and other peroxisomal disorders. The incidence of X-ALD in Guangzhou is not low.

Genomic Sequencing as a First-Tier Screening Test and Outcomes of Newborn Screening.

Importance: Newborn screening via biochemical tests is in use worldwide. The availability of genetic sequencing has allowed rapid screening for a substantial number of monogenic disorders. However, the outcomes of this strategy have not been evaluated in a general newborn population. Objective: To evaluate the outcomes of applying gene panel sequencing as a first-tier newborn screening test. Design, Setting, and Participants: This cohort study included newborns who were prospectively recruited from 8 screening centers in China between February 21 and December 31, 2021. Neonates with positive results were followed up before July 5, 2022. Exposures: All participants were concurrently screened using dried blood spots. The screen consisted of biochemical screening tests and a targeted gene panel sequencing test for 128 conditions. The biochemical and genomic tests could both detect 43 of the conditions, whereas the other 85 conditions were screened solely by the gene panel. Main Outcomes and Measures: The primary outcomes were the number of patients detected by gene panel sequencing but undetected by the biochemical test. Results: This study prospectively recruited 29 601 newborns (15 357 [51.2%] male). The mean (SD) gestational age was 39.0 (1.5) weeks, and the mean (SD) birth weight was 3273 (457) g. The gene panel sequencing screened 813 infants (2.7%; 95% CI, 2.6%-2.9%) as positive. By the date of follow-up, 402 infants (1.4%; 95% CI, 1.2%-1.5%) had been diagnosed, indicating the positive predictive value was 50.4% (95% CI, 50.0%-53.9%). The gene panel sequencing identified 59 patients undetected by biochemical tests, including 20 patients affected by biochemically and genetically screened disorders and 39 patients affected by solely genetically screened disorders, which translates into 1 out of every 500 newborns (95% CI, 1/385-1/625) benefiting from the implementation of gene panels as a first-tier screening test. Conclusions and Relevance: In this cohort study, the use of gene panel sequencing in a general newborn population as a first-tier screening test improved the detection capability of traditional screening, providing an evidence-based suggestion that it could be considered as a crucial method for first-tier screening.

Clinical features and outcomes of 31 children with congenital hypothyroidism missed by neonatal screening.

OBJECTIVE: To investigate the clinical features and outcomes of children with congenital hypothyroidism (CH) missed by neonatal screening. METHODS: The clinical and laboratory date of 31 children with CH missed by neonatal screening from February 2015 to February 2022 in Guangzhou Women and Children's Medical Center were retrospectively analyzed. Whole-exome high-throughput sequencing analysis was performed in 17 patients. RESULTS: Among the 31 patients, 19 cases (61.3%) were preterm, 12 cases (38.7%) were term neonates. The median value of gestation age was 36 (26-40) weeks, birth weight was 2.35 (0.75-3.70) kg, diagnosed age was 20 d (7 d-4 years), dry blood spot thyrotropin was 4.18 (0.34-8.97) mU/L. Nine cases (29.0%) were same-sex twins and 4 cases (12.9%) had a family history of hypothyroidism. The initial clinical symptoms were growth retardation in 11 cases (35.5%), prolonged jaundice in 7 cases (22.6%), short stature, abdominal distension, fetal edema and goiter in 1 case (3.2%), respectively. Genetic analysis of the 17 children showed that DUOX2 gene mutations were detected in 10 cases (6 cases with biallelic mutations and 4 cases with monoallelic mutations), of whom 3 had a family history of hypothyroidism. A total of 22 patients were reevaluated at the age of 2-3 years, of whom 17 cases (77.3%) were transient CH and 5 cases (22.7%) were permanent CH. Among the 10 cases with DUOX2 gene mutations, 6 cases were transient CH, 1 case was permanent CH, and 3 cases (< 3 years old) were still under treatment with L-thyroxine. CONCLUSIONS: False negative results on neonatal screening for CH often occurs in preterm birth, low birth weight, same-sex twins, family history of hypothyroidism, and DUOX2 defects are the common molecular pathogenesis, most of whom are transient CH. Thyroid function should be evaluated in time for children with unexplained slow growth and delayed jaundice regression.

A pilot study of newborn screening for Duchenne muscular dystrophy in Guangzhou.

Background: To estimate the overall situation of Duchenne muscular dystrophy (DMD) screening in newborns in Guangzhou, China. Method: A total of 62553 newborns including 44268 males and 18285 females were screened for DMD by measuring muscle specific creatine kinase isoform (CK-MM) concentrations using the GSP® Neonatal CK-MM kit based on time-resolved immunofluorescence. We recalled positive cases and recollected dried blood spots (DBS) for retest of CK-MM. The newborns with retest positive result were recalled again for serum creatine kinase (CK) and multiplex ligation-dependent probe amplification (MLPA) test. Whole exon sequencing was performed when MLPA test was negative. Results: Four males were diagnosed with DMD. The incidence of males was 1/11067. No DMD patient was found in female. There were significant differences of CK-MM concentration between male and female newborns. Among gestational age (GA), birth weight (BW) and age at sampling, linear regression analysis showed that CK-MM concentration was much more closely correlated with GA and age at sampling. Conclusions: CK-MM concentration is affected by gender, GA, BW and age at sampling. The efficiency of DMD screening might be improved by adjusting a multitier cut-off value according to GA and age at sampling. DMD newborn screening should be male priority.

Incidence tendency, etiological classification and outcome of congenital hypothyroidism in Guangzhou, China: an 11-year retrospective population-based study.

OBJECTIVES: An increased incidence of congenital hypothyroidism (CH) has been described worldwide over the years. In this study, we aimed to investigate the epidemiologic characteristics of CH, the iodine status in Guangzhou, China and to investigate which factors might influence the CH incidence during the period 2010-2020. METHODS: We retrospectively reviewed all cases of CH detected by newborn screening during the period 2010-2020. CH was classified as either suspected thyroid dyshormonogenesis (SDH) or thyroid dysgenesis (TD) based on thyroid ultrasound at first diagnosis. Patients were re-evaluated after 4 weeks of L-thyroxine withdrawal at age of 2-3 years to confirm the diagnosis of permanent CH (PCH) or transient CH (TCH). RESULTS: From 2010 to 2020, 1,655 patients with CH were confirmed from 2,400,383 newborns (1:1,450). The CH incidence increased from 1:2,584 in period [2010-2014] to 1:1,086 in period [2015-2020]. Among the 1,337 patients with thyroid ultrasound, 84.29% were SDH whereas 15.71% had TD. Further analysis revealed that more SDH (78.32%) were TCH whereas more TD (87.12%) turned to be PCH. The proportion of blood spot thyrotropin values >5 mIU/L ranged from 8.03 to 20.46%, indicating iodine deficiency. The prevalence of preterm infants increased from 5.50% in period [2010-2014] to 7.06% in period [2015-2020] (p<0.001). CONCLUSIONS: In the past decade, the CH incidence has increased progressively. SDH was the majority of CH, most of which were TCH, while most patients with TD were PCH. The increased incidence might be mainly due to iodine deficiency and increased rates of preterm infants in our study.

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

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

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.

AAV9-coGLB1 Improves Lysosomal Storage and Rescues Central Nervous System Inflammation in a Mutant Mouse Model of GM1 Gangliosidosis.

BACKGROUND: GM1 gangliosidosis (GM1) is an autosomal recessive disorder characterized by the deficiency of beta-galactosidase (ß-gal), a ubiquitous lysosomal enzyme that catalyzes the hydrolysis of GM1 ganglioside. OBJECTIVE: The study aims to explore the application of the AAV9-coGLB1 for effective treatment in a GM1 gangliosidosis mutant mouse model. METHODS: We designed a novel adeno-associated virus 9 (AAV9) vector expressing ß-gal (AAV9- coGLB1) to treat GM1 gangliosidosis. The vector, injected via the caudal vein at 4 weeks of age, drove the widespread and sustained expression of ß-gal for up to 32 weeks in the Glb1G455R/G455R mutant mice (GM1 mice). RESULTS: The increased levels of ß-gal reduced the pathological damage occurring in GM1 mice. Histological analyses showed that myelin deficits and neuron-specific pathology were reduced in the cerebral cortex region of AAV9-coGLB1-treated mice. Immunohistochemical staining showed that the accumulation of GM1 ganglioside was also reduced after gene therapy. The reduction of the storage in these regions was accompanied by a decrease in activated microglia. In addition, AAV9 treatment reversed the blockade of autophagic flux in GM1 mice. CONCLUSION: These results show that AAV9-coGLB1 reduces the pathological signs of GM1 gangliosidosis in a mouse model.

Screening for neonatal inherited metabolic disorders by tandem mass spectrometry in Guangzhou.

To analyze the screening results for inherited metabolic disorders (IMD) in newborns by tandem mass spectrometry (MS/MS) in Guangzhou.A total of 272 117 newborns in Guangzhou from Jan 2015 to Dec 2020 were screened for IMD by MS/MS in Guangzhou Newborn Screening Center. When the primary screening was positive, the newborns and their mothers were recalled. For those with positive in re-examination, the biochemical and related genetic analysis were required for confirmation. The screening results, clinical characteristics and outcomes of the confirmed cases were retrospectively analyzed and the performance was optimized. Among 272 117 neonates, 1808 (0.66%) cases were positive in primary screening, and 1738 cases (96.13%) were recalled for review. The median clinical diagnosis time was 15 d after birth. A total of 79 cases of IMD were diagnosed, including 23 with aminoacidopathy, 17 with disorder of organic acid metabolism and 39 with fatty acid oxidation disorders, involving 21 diseases. The incidence rate was 1/3444 in newborns, and the positive predictive value of 4.5%. Four false negative cases were found, all of them were citrin deficiency. The common diseases were primary carnitine deficiency (26.6%), methylmalonic aciduria (12.7%) and phenylalanine hydroxylase deficiency (11.4%). The mothers of 32 cases were confirmed, including 30 cases of primary carnitine deficiency, 1 case of isobutyl-coenzyme A dehydrogenase deficiency and 1 case of 3-methylcromaryl coenzyme A carboxylase deficiency. The detection rate was 1/2451 in total population. During the follow-up, most patients remain asymptomatic, except for 5 severe cases who died early (1 case of maple syrup urine disease, 2 cases of isolated methylmalonic acidurmia, and 2 cases of carnitine-acylcarnitine translocase deficiency); and 10 cases of organic acid metabolism disorders developed mild psychomotor developmental retardation. After optimizing the screening indicators, the number of initial screening positives dropped to 903, and the positive predictive value increased to 9.1%, and no confirmed cases were missed. The incidence rate of fatty acid oxidation disorders is high in Guangzhou. A variety of IMD can be effectively screened out by MS/MS, and the screening performance can be improved by optimizing screening indicators.

Possible Vicarious Traumatization Among Psychiatric Inpatients During the Remission Phase of the COVID-19: A Single-Center Cross-Sectional Study.

Background: Far from being a clinical disease, the COVID-19 pandemic has become a threatening social event worldwide exerting long-term impacts on human beings. Objective: This study was designed to determine if and to what extent psychiatric inpatients during the remission phase of the pandemic suffered from vicarious traumatization. Method: Totally 266 eligible participants from psychiatric and psychological wards in a hospital were recruited during October 26th, 2020 to February 4th, 2021 to finish a self-made online questionnaire consisting of Impact of Event Scale-Revised (IES-R), Self-Rating Depression Scale (SDS), Self-Rating Anxiety Scale (SAS), Obsessive-Compulsive Inventory-Revised (OCI-R), Pittsburgh Sleep Quality Index-Revised (PSQI-R), Social Support Rating Scale (SSRS), Beck Suicide Ideation Scale (SSI), 12-Item Short-Form Health Survey (SF-12). Meanwhile, some socio-demographics and information related to the pandemic were also recorded. Results: The detection rate of vicarious traumatic symptoms (VTS) was 80.83%, including 40.98% for mild ones, 25.56% for moderate ones, and 14.29% for severe ones, among whom 98.14% possessed all three phenotypes. 27.07% of the sample were considered possible vicarious traumatization (pVT). Having acquaintances infected with or died from COVID-19, worries on re-outbreak of COVID-19, a higher score of OCI-R or lower score of SF-12, and long latency of VTS were independent risk factors of pVT. Conclusion: Our study showed that COVID-19 could have profound mental influences on psychiatric inpatients. It is high time we did some screening in the wards to seek for patients at risk.

A GM1 gangliosidosis mutant mouse model exhibits activated microglia and disturbed autophagy.

GM1 gangliosidosis is a rare lysosomal storage disease caused by a deficiency of ß-galactosidase due to mutations in the GLB1 gene. We established a C57BL/6 mouse model with Glb1G455R mutation using CRISPR/Cas9 genome editing. The ß-galactosidase enzyme activity of Glb1G455R mice measured by fluorometric assay was negligible throughout the whole body. Mutant mice displayed no marked phenotype at eight weeks. After 16 weeks, GM1 ganglioside accumulation in the brain of mutant mice was observed by immunohistochemical staining. Meanwhile, a declining performance in behavioral tests was observed among mutant mice from 16 to 32 weeks. As the disease progressed, the neurological symptoms of mutant mice worsened, and they then succumbed to the disease by 47 weeks of age. We also observed microglia activation and proliferation in the cerebral cortex of mutant mice at 16 and 32 weeks. In these activated microglia, the level of autophagy regulator LC3 was up-regulated but the mRNA level of LC3 was normal. In conclusion, we developed a novel murine model that mimicked the chronic phenotype of human GM1. This Glb1G455R murine model is a practical in vivo model for studying the pathogenesis of GM1 gangliosidosis and exploring potential therapies.

Detection of glucosylsphingosine in dried blood spots for diagnosis of Gaucher disease by LC-MS/MS.

INTRODUCTION: Gaucher disease (GD) is caused by a deficiency of ß-glucosidase (GCase), leading to accumulation of glucosylceramide (GlcC) and glucosylsphingosine (Lyso-Gb1). Lyso-Gb1 is a reliable biomarker for GD. OBJECTIVES: This study aims to develop a simple, effective and accurate method for the screening and diagnosis of GD using dried blood spot (DBS) samples. METHODS: Lyso-Gb1 in DBS was extracted by 50% acetonitrile aqueous solution containing isotope-labeled internal standard and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A reference interval was established by analyzing samples from 277 healthy controls. Lyso-Gb1 was detected in the residual DBS samples from 142 high-risk patients with splenomegaly and/or thrombocytopenia. Based on GCase activity in DBS, samples were classified into four groups: confirmed GD patients (n = 52), GD carriers (n = 5), false positive (n = 36) and negative (n = 49). RESULTS: The optimized Lyso-Gb1 assay showed intra- and inter-assay variations ranged between 2.0%-8.2% and 3.8%-10.2%, respectively. Accuracies ranged from 93.5% to 112.6%. The lowest limit of quantification was 1 ng/mL. The normal reference interval of Lyso-Gb1 in DBS ranged from 2.1 to 9.9 ng/mL. Among the 142 subjects, except for one GD patient (Lyso-Gb1 > 2500 ng/mL), the Lyso-Gb1 concentrations in 51 GD patients ranged from 190.5 to 2380.6 ng/mL (the median 614.8 ng/mL). Also, one negative patient was found to have an elevated Lyso-Gb1 level (684.5 ng/mL), while the other patients were normal. The negative case was then confirmed to be an atypical GD patient with a c.1091A > G (p.Y364C) homozygous variant in PSAP gene by next generation sequencing. CONCLUSIONS: The optimized method to determine Lyso-Gb1 in DBS was demonstrated as a useful tool for the screening and diagnosis of GD.

Clinical and GAA gene mutation analysis in 21 Chinese patients with classic infantile pompe disease.

Pompe disease is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Early and precise diagnosis can be highly important for the treatment, genetic counselling and prenatal diagnosis of this disease in potential candidates. Considering that Pompe disease studies have not been frequently conduced in China, to better understand the clinical course and molecular defects among this group, our study examined 21 Chinese patients with classic infantile Pompe disease. The median age of symptom onset in the patients was 2.5 months (0-7 months), and the median age of confirmed diagnosis was 5.6 months (2-12 months). GAA gene mutation analysis revealed 17 different mutations, two of which were novel (c.538C>A and c.2096T>C). The most frequent mutation in these patients was c.1935C>A, accounting for 40.5% (17/42 alleles) of the mutations. These results confirm the high prevalence of the c.1935C>A mutation in Chinese patients with classic infantile Pompe disease. Furthermore, identification of the novel alterations in the GAA gene will help to broaden the spectrum of the GAA mutations causing Pompe disease and to better understand the potential pathogenic role of each change.

High risk screening for Gaucher disease in patients with splenomegaly and/or thrombocytopenia in China: 55 cases identified.

Gaucher disease (GD) is a common lysosomal storage disorder caused by deficiency of glucocerebrosidase (GCase) due to the pathogenic variants in the GBA gene. The aim of this study was to evaluate the performance of high risk screening program for GD by measuring the enzyme activities of GCase and chitotriosidease in dried blood spots of patients with splenomegaly and/or thrombocytopenia. A total of 787 subjects (364 females and 423 males) with unexplained splenomegaly and/or thrombocytopenia were enrolled in this study from May 2016 to Aug 2019. The cutoff value of GCase activity was set as less than 3.0 pmol/punch/h for screening positive. The diagnosis of GD was confirmed by Sanger sequencing of the GBA gene. Among 131 screening positive cases, 49 patients were confirmed GD. The positive predictive value was 37.4%.Three patients with boundary values (GCase 3-4 pmol/punch/h) and other three splenectomic patients with normal GCase activity were confirmed GD by GBA genetic analysis because of increased chitotriosidase or Gaucher cells in bone marrow. A total of 55 GD cases were identified. The sensitivity and specificity of the high risk screening were 98.2% and 89.5%, respectively. These 55 GD patients presented splenomegaly (100%), hepatomegaly (70.9%), thrombocytopenia (83.6%). The level of GCase in GD patients was (1.7 ± 1.6) pmol/punch/h. The increased chitotriosidase (383.8 ± 130.2 pmol/punch/h) was found in 42 (76.4%) patients with GD. Molecular genetic analysis identified 44 variants in the GBA gene, including 11 novel variants. The results showed the high risk screening for GD is accurate, rapid and cost-effective.