GM2 gangliosidosis is a group of rare lysosomal storage disorders (LSDs) including Tay-Sachs disease (TSD) and Sandhoff disease (SD), caused by deficiency in activity of either β-hexosaminidase A (HexA) or both β-hexosaminidase A and β-hexosaminidase B (HexB). Methods for screening and diagnosis of TSD and SD include measurement and comparison of the activity of these two enzymes. Here we report a novel method for duplex screening of dried blood spots (DBS) for TSD and SD by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method requires incubation of a single 3 mm DBS punch with the assay cocktail followed by the injection into the LC-MS/MS. The performance of the method was evaluated by comparing the confirmed TSD and SD patient DBS to random healthy newborn DBS which showed easy discrimination between the three cohorts. The method is multiplexable with other LSD MS/MS enzyme assays which is critical to the continued expansion of the NBS panels.

The pathological accumulation of GM2 ganglioside associated with Tay-Sachs disease (TSD) and Sandhoff disease (SD) occurs in individuals who possess mutant forms of the heterodimer β-hexosaminidase A (Hex A) because of mutation of the HEXA and HEXB genes, respectively. With a lack of approved therapies, patients experience rapid neurological decline resulting in early death. A novel bicistronic vector carrying both HEXA and HEXB previously demonstrated promising results in mouse models of SD following neonatal intravenous administration, including significant reduction in GM2 accumulation, increased levels of Hex A, and a 2-fold extension of survival. The aim of the present study was to identify an optimal dose of the bicistronic vector in 6-week-old SD mice by an intrathecal route of administration along with transient immunosuppression, to inform possible clinical translation. Three doses of the bicistronic vector were tested: 2.5e11, 1.25e11, and 0.625e11 vector genomes per mouse. The highest dose provided the greatest increase in biochemical and behavioral parameters, such that treated mice lived to a median age of 56 weeks (>3 times the lifespan of the SD controls). These results have direct implications in deciding a human equivalent dose for TSD/SD and have informed the approval of a clinical trial application (NCT04798235).

Amyloid β (Aβ) oligomers are the most neurotoxic forms of Aβ, and Aβ(1-42) is the prevalent Aβ peptide found in the amyloid plaques of Alzheimer\'s disease patients. Aβ(25-35) is the shortest peptide that retains the toxicity of Aβ(1-42). Aβ oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aβ(1-42) concentration range. Aβ and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aβ(1-42). The formation of His6/Aβ(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aβ(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aβ(1-42) and Aβ(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aβ peptides toward selected cellular targets.

Diabetes has been listed as one of the three major diseases that endanger human health. Accurately injecting insulin (Ins) depending on the level of blood glucose (LBG) is the standard treatment, especially controlling LBG in the long-term by a single injection. Herein, the pH-responsive hexa-histidine metal assembly (HmA) encapsulated with enzymes (GOx and CAT) and Ins (HmA@GCI) is engineered as the vehicle for glucose-mediated insulin delivery. HmA not only shows high proteins loading efficiency, but also well retained proteins activity and protect proteins from protease damage. Within HmA, the biocatalytic activities of enzymes and the efficiency of the cascade reaction between GOx and CAT are enhanced, leading to a super response to the change of LBG with insulin release and efficient clearance of harmful byproducts of GOx (H2 O2 ). In the treatment of diabetic mice, HmA@GCI reduces LBG to normal in half an hour and maintains for more than 5 days by a single subcutaneous injection, and nearly 24 days with four consecutive injections. During the test period, no symptoms of hypoglycemia and toxicity to tissues and organs are observed. These results indicate that HmA@GCI is a safe and long-acting hypoglycemic agent with prospective clinical application.

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Tay-Sachs disease is a lethal lysosomal storage disorder caused by mutations in the HexA gene encoding the α subunit of the lysosomal β-hexosaminidase enzyme (HEXA). Abnormal GM2 ganglioside accumulation causes progressive deterioration in the central nervous system in Tay-Sachs patients. Hexa-/- mouse model failed to display abnormal phenotype. Recently, our group generated Hexa-/-Neu3-/- mouse showed severe neuropathological indications similar to Tay-Sachs patients. Despite excessive GM2 ganglioside accumulation in the brain and visceral organs, the regulation of autophagy has not been clarified yet in the Tay-Sachs disease mouse model. Therefore, we investigated distinct steps of autophagic flux using markers including LC3 and p62 in four different brain regions from the Hexa-/-Neu3-/- mice model of Tay-Sachs disease. Our data revealed accumulated autophagosomes and autophagolysosomes indicating impairment in autophagic flux in the brain. We suggest that autophagy might be a new therapeutic target for the treatment of devastating Tay-Sachs disease.

Tay-Sachs disease (TSD), an autosomal recessively inherited neurodegenerative lysosomal storage disease, reported worldwide with a high incidence among population of Eastern European and Ashkenazi Jewish descent. Mutations in the alpha subunit of HEXA that encodes for the β-hexosaminidase-A lead to deficient enzyme activity and TSD phenotype. This study is the first to highlight the HEXA sequence variations spectrum in a cohort of Egyptian patients with infantile TSD.
This study involved 13 Egyptian infant/children patients presented with the infantile form of TSD, ten of the 13 patients were born to consanguineous marriages. β-hexosaminidase-A enzyme activity was markedly reduced in the 13 patients with a mean activity of 3 µmol/L/h ± 1.56. Sanger sequencing of the HEXA\' coding regions and splicing junctions enabled a detection rate of ~ 62% (8/13) in our patients revealing the molecular defects in eight patients; six homozygous-mutant children (five of them were the product of consanguineous marriages) and two patients showed their mutant alleles in heterozygous genotypes, while no disease-causing mutation was identified in the remaining patients. Regulatory intragenic mutations or del/dup may underlie the molecular defect in those patients showing no relevant pathogenic sequencing variants or in the two patients with a heterozygous genotype of the mutant allele. This research identified three novel, likely pathogenic variants in association with the TSD phenotype; two missense, c.920A > C (E307A) and c.952C > G (H318D) in exon 8, and a single base deletion c.484delG causing a frameshift E162Rfs*37 (p.Glu162ArgfsTer37) in exon 5. Three recurrent disease-causing missense mutations; c.1495C > T (R499C), c.1511G > A(R504H), and c.1510C > T(R504C) in exon 13 were identified in five of the eight patients. None of the variants was detected in 50 healthy Egyptians\' DNA. Five variants, likely benign or of uncertain significance, S3T, I436V, E506E, and T2T, in exons 1, 11,13, & 1 were detected in our study.
For the proper diagnostics, genetic counseling, and primary prevention, our study stresses the important role of Next Generation Sequencing approaches in delineating the molecular defect in TSD-candidate patients that showed negative Sanger sequencing or a heterozygous mutant allele in their genetic testing results. Interestingly, the three recurrent TSD associated mutations were clustered on chromosome 13 and accounted for 38% of the HEXA mutations detected in this study. This suggested exon 13 as the first candidate for sequencing screening in Egyptian patients with infantile TSD. Larger studies involving our regional population are recommended, hence unique disease associated pathogenic variations could be identified.

GM2-Gangliosidosis are a group of inherited lysosomal storage pathologies characterized by a large accumulation of GM2 ganglioside in the lysosome. They are caused by mutation in HEXA or HEXB causing reduced or absent activity of a lysosomal β-hexosaminidase A, or mutation in GM2A causing defect in GM2 activator protein (GM2AP), an essential protein for the activity of the enzyme. Biochemical diagnosis relies on the measurement of β-hexosaminidases A and B activities, which is able to detect lysosomal enzyme deficiency but fails to identify defects in GM2AP. We developed a rapid, specific and sensitive liquid chromatography-mass spectrometry-based method to measure simultaneously GM1, GM2, GM3 and GD3 molecular species. Gangliosides were analysed in plasma from 19 patients with GM2-Gangliosidosis: Tay-Sachs (n = 9), Sandhoff (n = 9) and AB variant of GM2-Gangliosidosis (n = 1) and compared to 20 age-matched controls. Among patients, 12 have a late adult-juvenile-onset and 7 have an infantile early-onset of the disease. Plasma GM2 molecular species were increased in all GM2-Gangliosidosis patients (19/19), including the patient with GM2A mutation, compared to control individuals and compared to patients with different other lysosomal storage diseases. GM234:1 and GM234:1/GM334:1 ratio discriminated patients from controls with 100% sensitivity and specificity. GM234:1 and GM234:1/GM334:1 were higher in patients with early-onset compared to those with late-onset of the disease, suggesting a relationship with severity. Longitudinal analysis in one adult with Tay-Sachs disease over 9 years showed a positive correlation of GM234:1 and GM234:1/GM334:1 ratio with age at sampling. We propose that plasma GM2 34:1 and its ratio to GM3 34:1 could be sensitive and specific biochemical diagnostic biomarkers for GM2-Gangliosidosis including AB variant and could be useful as a first line diagnostic test and potential biomarkers for monitoring upcoming therapeutic efficacy.

Nonalcoholic fatty liver disease (NAFLD) and impaired glycemic control are closely linked; however, the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. The high secretory capacity of the liver and impairments in protein secretion in NAFLD suggest that endocrine changes in the liver are likely to contribute to glycemic defects. We identify hexosaminidase A (HEXA) as an NAFLD-induced hepatokine in both mice and humans. HEXA regulates sphingolipid metabolism, converting GM2 to GM3 gangliosides-sphingolipids that are primarily localized to cell-surface lipid rafts. Using recombinant murine HEXA protein, an enzymatically inactive HEXA(R178H) mutant, or adeno-associated virus vectors to induce hepatocyte-specific overexpression of HEXA, we show that HEXA improves blood glucose control by increasing skeletal muscle glucose uptake in mouse models of insulin resistance and type 2 diabetes, with these effects being dependent on HEXA\'s enzymatic action. Mechanistically, HEXA remodels muscle lipid raft ganglioside composition, thereby increasing IGF-1 signaling and GLUT4 localization to the cell surface. Disrupting lipid rafts reverses these HEXA-mediated effects. In this study, we identify a pathway for intertissue communication between liver and skeletal muscle in the regulation of systemic glycemic control.

OBJECTIVE: To explore the genetic basis for a girl featuring epilepsy, developmental delay and regression.
METHODS: Clinical data of the patient was collected. Activities of hexosaminidase A (Hex A) and hexosaminidase A&B (Hex A&B) in blood leukocytes were determined by using a fluorometric assay. Peripheral blood samples were collected from the proband and six members from her pedigree. Following extraction of genomic DNA, whole exome sequencing was carried out. Candidate variants were verified by Sanger sequencing.
RESULTS: Enzymatic studies of the proband have shown reduced plasma Hex A and Hex A&B activities. Genetic testing revealed that she has carried c.1260_1263del and c.1601G>C heterozygous compound variants of the HEXB gene. Her mother, brother and sister were heterozygous carriers of c.1260_1263del, while her father, mother, three brothers and sister did not carry the c.1601G>C variant, suggesting that it has a de novo origin. Increased eosinophils were discovered upon cytological examination of peripheral blood and bone marrow samples.
CONCLUSIONS: The compound heterozygous variants of c.1260_1263del and c.1601G>C of the HEXB gene probably underlay the Sandhoff disease in this child. Eosinophilia may be noted in infantile Sandhoff disease.