Tandem mass spectrometric enzyme assay for simultaneous detection of Tay-Sachs and Sandhoff diseases in dried blood spots for newborn screening.

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 ß-hexosaminidase A (HexA) or both ß-hexosaminidase A and ß-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.

Thymic involution and corticosterone level in Sandhoff disease model mice: new aspects the pathogenesis of GM2 gangliosidosis.

Sandhoff disease (SD) is a lysosomal disease caused by a mutation of the HEXB gene associated with excessive accumulation of GM2 ganglioside (GM2) in lysosomes and neurological manifestations. Production of autoantibodies against the accumulated gangliosides has been reported to be involved in the progressive pathogenesis of GM2 gangliosidosis, although the underlying mechanism has not been fully elucidated. The thymus is the key organ in the acquired immune system including the development of autoantibodies. We showed here that thymic involution and an increase in cell death in the organ occur in SD model mice at a late stage of the pathogenesis. Dramatic increases in the populations of Annexin-V(+) cells and terminal deoxynucletidyl transferase dUTP nick end labeling (TUNEL) (+) cells were observed throughout the thymuses of 15-week old SD mice. Enhanced caspase-3/7 activation, but not that of caspase-1/4, -6 ,-8, or -9, was also demonstrated. Furthermore, the serum level of corticosterone, a potent inducer of apoptosis of thymocytes, was elevated during the same period of apoptosis. Our studies suggested that an increase in endocrine corticosterone may be one of the causes that accelerate the apoptosis of thymocytes leading to thymic involution in GM2 gangliosidosis, and thus can be used as a disease marker for evaluation of the thymic condition and disease progression.

New cases of adult-onset Sandhoff disease with a cerebellar or lower motor neuron phenotype.

Sandhoff disease is a lipid-storage disorder caused by a defect in ganglioside metabolism. It is caused by a lack of functional N-acetyl-beta-d-glucosaminidase A and B due to mutations in the HEXB gene. Typical, early-onset Sandhoff disease presents before 9 months of age with progressive psychomotor retardation and early death. A late-onset form of Sandhoff disease is rare, and its symptoms are heterogeneous. As drug trials that aim to intervene in the disease mechanism are emerging, the recognition and identification of Sandhoff disease patients-particularly those with atypical phenotypes-are becoming more important. The authors describe six new late-onset Sandhoff cases demonstrating cerebellar ataxia or lower motor neuron (LMN) involvement combined with, mostly subclinical, neuropathy. Two different mutations were found: IVS 12-26 G/A and c.1514G-->A. In patients with either progressive cerebellar ataxia or LMN disease in the setting of a possibly recessive disorder, Sandhoff disease should be suspected, even when the onset age is over 45 years.

Identification of Sandhoff disease in a Thai family: clinical and biochemical characterization.

Sandhoff disease is a GM2 gangliosidosis that is rare in Thailand. The authors report a Thai family with two children known to have infantile form of Sandhoff disease. The index case exhibited mitral valve prolapse with mitral regurgitation as an early sign, which is a rare presentation in Sandhoff disease. Thereafter the patient had developmental regression, startle reaction, and cherry red spots. The diagnosis was confirmed by biochemical analysis.

Biomarkers for disease progression and AAV therapeutic efficacy in feline Sandhoff disease.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme ß-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were largely normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated SD patients and those in future clinical trials.

Tay-Sachs and Sandhoff diseases: enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards.

BACKGROUND: Tay-Sachs disease (TSD), Sandhoff disease (SD) and variants are caused by deficient activity of the lysosomal enzymes hexosaminidase A (HA) and total hexosaminidase (TH) (hexosaminidase A plus B), respectively. For diagnosis, these enzymes are usually measured in plasma or extracts of leukocytes. We describe methods for the assay of hexosaminidase A and total hexosaminidase activities in dried blood spots (DBSs) on filter paper. MATERIALS AND METHODS: We studied 163 healthy controls, 9 Tay-Sachs patients, 4 Sandhoff patients, 18 obligate carriers and the newborn-screening cards from two patients with Tay-Sachs and one patient with Sandhoff disease. To tubes containing a 3-mm-diameter blood spot, we added elution liquid and substrate solution. After incubation at 37 degrees C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. RESULTS AND CONCLUSIONS: The described methodology is useful to distinguish patients with Tay-Sachs disease or Sandhoff disease from carriers and controls using samples that are sufficiently stable to be transported to the testing laboratory by mail. The diagnosis of both diseases from a newborn-screening card (NSC) was clearly demonstrated, even after storage for up to 38 months at room temperature. The newborn-screening card has been added to the biological materials that allow the identification of patients with Tay-Sachs disease and Sandhoff disease.

Ultrastructure of the conjunctiva, skin, and gingiva: a case of Sandhoff's disease in a Jewish patient.

Pleomorphic membranous cytoplasmic bodies that indicated glycolipid storage were found in the conjunctiva, skin, and gingiva of a Jewish patient with Sandhoff's disease. The clinical symptoms were typical of GM2 gangliosidosis. Both hexosaminidase A and hexosaminidase B activities were deficient in the leukocytes and serum. Glycosaminoglycan levels in cultured fibroblasts were elevated. Membranous cytoplasmic bodies were observed in high concentrations in a large proportion of the vascular endothelial cells, pericytes, and Schwann cells and to a somewhat lesser extent in the fibrocytes of all tissues studied. Ultrastructural analysis of the conjunctiva, skin, and gingiva as an aid for the diagnosis of Sandhoff's disease is suggested.

[Sandhoff's and Tay-Sachs disease--based on our own cases]. / Choroba Sandhoffa oraz Tay Sachsa--w oparciu o przypadki wlasne.

The authors described two infant with Sandhoff's and Tay-Sachs disease. Tay-Sachs disease is well-known inherited disease leading to an accumulation of gangliosides in the brain and retina. Sandhoffs disease (GM2 gangliosidosis type 0) was diagnosed in an infant, in whom a progressive neurological disorder and cherry-red foveal spots were developed. In addition, to the general clinical examination, indirect ophthalnoscopy, blood white cells enzymatic examination are used to make definitive diagnosis. In this cases, the early eye fundus examination allowed us to make the proper diagnosis. The fundus change is characterized by the "cherry-red spot" in the central area.

Lyso-GM2 ganglioside: a possible biomarker of Tay-Sachs disease and Sandhoff disease.

To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.

Sandhoff disease: a prevalent form of infantile GM2 gangliosidosis in Lebanon.

All cases clinically diagnosed as Tay-Sachs disease at the American University Hospital, Beirut, during a period of 22 years (1957--1979) were reviewed. Of a total of 15 cases, seven had serum hexosaminidase tested and proved to have Sandhoff disease. In two other cases, parents were tested and found to be Sandhoff carriers. These results indicate that Sandhoff disease is relatively prevalent in Lebanon and that it may represent the more common form of infantile GM2 gangliosidosis in this country.

Usefulness of 4-methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyrano sid e for the diagnosis of GM2 gangliosidoses in leukocytes.

4-Methylumbelliferyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized and tested as a substrate for the diagnosis of GM2 gangliosidoses using leukocytes. Less than 2% of normal activity was measured in homogenates from patients with typical Tay-Sachs disease and from a patient with a variant form having 37% Hexosaminidase A by heat denaturation using the usual fluorogenic substrate. An adult patient had 8.5% of normal activity. Three patients with Sandhoff disease were found to have values ranging from 17% to 37% of normal. These values overlap the range found for carriers of Tay-Sachs disease, and suggest that the usefulness of this substrate, while excellent for diagnosing B variants of GM2 gangliosidosis, requires further study. Perhaps when used together with 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside excellent discriminations of patients, carriers and controls will be realized.