Total Hexosaminidase Research Articles

Genetic variant reanalysis reveals a case of Sandhoff disease with onset of infantile epileptic spasm syndrome

BackgroundSandhoff disease (SD) i s an autosomal recessive lysosomal disease with clinical manifestations such as epilepsy, psychomotor retardation and developmental delay. However, infantile SD with onset of infantile epilepsy spasm syndrome (IESS) is extremely rare.Case presentationThe case presented here was a 22-month-old boy, who presented with IESS and psychomotor retardation/regression at 6 months of age. The patient showed progressive aggravation of seizures and excessive startle responses. The whole exome sequencing data, which initially revealed negative results, were reanalyzed and indicated a homozygous mutation at the c.1613 + 4del splice site of the HEXB gene. The activities of β-hexosaminidase A and total hexosaminidase were significantly decreased. The fundus examination showed cherry red spots at the macula.ConclusionsIESS can be an epileptic phenotype of infantile SD. Clinical phenotypes should be adequately collected in genetic testing. In the case of negative sequencing results, gene variant reanalysis can be performed when the patients show clinically suspicious indications.

GM2 gangliosidoses: evaluation of clinical, biochemical and genetic findings of patients with three novel mutations

Purpose: The aim of this study is to evaluate the diagnosis characteristics, clinic findings, phenotypical and genotypical features of children with GM2 gangliosidoses.
 Materials and Methods: The file records of 14 patients diagnosed with GM2 gangliosidoses in our clinic were retrospectively reviewed. The GM2 gangliosidoses diagnosis was confirmed by determining the levels of serum total hexosaminidase and β-hexosaminidase activity with genetic analysis. 
 Results: We identified a total of seven different mutations, three of which were novel (one in the HEXA gene and two in the HEXB gene) in 14 patients. We found a high frequency of c.1100_1111del (p.Gly367_Tyr370del) mutation in HEXA affected patients. The mean age at diagnosis was 13.46.3 months and 14.24.2 months for patients with Tay–Sachs disease (TSD) and Sandhoff disease (SD) respectively. Neuroregression was present in 92.9% of our patients. Of the 14 patients, 11 had epilepsy, 10 had developmental delay, 6 had hyperacusis, 6 had cherry-red spots and 6 had macrocephaly, but none of the patients had organomegaly. 
 Conclusion: GM2 gangliosidoses disease should be considered for children with developmental regression and/or delay. For early diagnosis, enzyme analysis and gene detection should be performed in children with suspected GM2 gangliosidoses in the presence of clinical findings.

Infantile onset Sandhoff disease: clinical manifestation and a novel common mutation in Thai patients

BackgroundSandhoff disease (SD) is an autosomal recessive lysosomal storage disorder, resulting in accumulation of GM2 ganglioside, particular in neuronal cells. The disorder is caused by deficiency of β-hexosaminidase B (HEX-B), due to pathogenic variant of human HEXB gene.MethodThis study describes clinical features, biochemical, and genetic defects among Thai patients with infantile SD during 2008–2019.ResultsFive unrelated Thai patients presenting with developmental regression, axial hypotonia, seizures, exaggerated startle response to noise, and macular cherry red spot were confirmed to have infantile SD based on deficient HEX enzyme activities and biallelic variants of the HEXB gene. In addition, an uncommon presenting feature, cardiac defect, was observed in one patient. All the patients died in their early childhood. Plasma total HEX and HEX-B activities were severely deficient. Sequencing analysis of HEXB gene identified two variants including c.1652G>A (p.Cys551Tyr) and a novel variant of c.761T>C (p.Leu254Ser), in 90 and 10% of the mutant alleles found, respectively. The results from in silico analysis using multiple bioinformatics tools were in agreement that the p.Cys551Tyr and the p.Leu254Ser are likely pathogenic variants. Molecular modelling suggested that the Cys551Tyr disrupt disulfide bond, leading to protein destabilization while the Leu254Ser resulted in change of secondary structure from helix to coil and disturbing conformation of the active site of the enzyme. Genome-wide SNP array analysis showed no significant relatedness between the five affected individuals. These two variants were not present in control individuals. The prevalence of infantile SD in Thai population is estimated 1 in 1,458,521 and carrier frequency at 1 in 604.ConclusionThe study suggests that SD likely represents the most common subtype of rare infantile GM2 gangliosidosis identified among Thai patients. We firstly described a potential common variant in HEXB in Thai patients with infantile onset SD. The data can aid a rapid molecular confirmation of infantile SD starting with the hotspot variant and the use of expanded carrier testing.

Clinical and radiological findings in Brazilian patients with mucolipidosis types II/III.

The present study aims to provide orientation for clinicians and radiologists to recognize the most prevalent findings leading to diagnosis in mucolipidosis from a description of the natural history of five Brazilian cases. We conducted an observational and retrospective study of five patients with clinical and radiological diagnosis of mucolipidosis. Clinical evaluation consisted of information obtained from records and including physical, neurologic, and dysmorphic evaluations. Radiologic studies consisted of complete skeletal radiographs of all patients. Enzyme assessment was performed for confirmation of the diagnosis. The five patients were referred for genetic evaluation due to disproportionate short stature with short trunk accompanied by waddling gait. Age at referral varied from 11months to 28years. The most prevalent findings were joint restriction (4/5 patients), neuropsychomotor developmental delay (3/5), coarse facies (2/5), hypertrophic cardiomyopathy (2/5), and mental retardation (1/4 patients). The most common radiological findings were anterior beaking of the vertebral bodies (5/5), shallow acetabular fossae (5/5), epiphyseal dysplasia (5/5), platyspondyly (4/5), pelvic dysplasia (4/5), decreased bone mineralization (4/5), scoliosis (3/5), wide and oar-shaped ribs (3/5), generalized epiphyseal ossification delay (3/5), and hypoplasia of basilar portions of ilea (3/5). Enzyme assessment showed α-iduronidase, α-mannosidase, β-glucuronidase, hexosaminidase A, and total hexosaminidase increased in plasma and normal glycosaminoglycans concentration. One patient was clinically classified as ML II and four patients as ML III. The follow-up of five patients showed the typical clinical and radiological findings allowing the diagnosis, thus improving clinical management and providing adequate genetic counseling. Clinicians and radiologists can take advantage of the information from this work, enhancing their differential diagnosis ability.

CLINICAL AND GENETIC CHARACTERISTICS OF MUCOLIPIDOSIS II AND IIIA TYPES IN CHILDREN

The article is devoted to a rare pathology from a group of accumulation diseases with an autosomal recessive type of inheritance – mucolipidosis II and IIIA types. The disease is characterized by a greater phenotypic similarity to mucopolysaccharidosis. Objective: analysis of genophenotypic parameters in Russian patients with mucolipidosis II and IIIA types. The activity of lysosomal enzymes in plasma (β-glucuronidase, total hexosaminidase and N-acetyl-α-D-glucosaminidase) was measured using a standard technique using chromogenic and fluorogenic substrates. Genomic DNA of peripheral blood leukocytes was isolated using a set of reagents Preb 100 (DIAtomTM). Amplification of all exons of the GNPTAB gene was carried out by polymerase chain reaction (PCR) followed by direct non-radioactive sequencing by Sanger. 50 patients aged from 1.5 to 10 years were examined. The clinical symptoms of the disease included: a Hurler-like phenotype, growth retardation, skeletal, cardiac and vascular damage, and CNS. Mucolipidosis type II (I-cell disease) was characterized by a more severe course. The clinical diagnosis was confirmed by the results of laboratory methods of investigation: normal parameters of renal excretion of glycosiminoglycans (GAG), high (5-15 times higher than normal) activity of lysosomal hydrolases in blood plasma and detection of mutations in the GNPTAB gene. 35 probands are completely genotyped. In 8 patients only 8 mutant alleles were detected; 7 mutations were not detected. Six new mutations in exons 1 (p.I31N; p.Q36P), 10 (p.L398P), 11 (p.W446X) and 13 (p.S738X; c.2250delT) were found, including a frequent mutation for Russian patients P.S738X (21% alleles). The most common (31.4% alleles) in the Russian cohort of patients was a known small deletion c. 3503_3504delTC, leading to a reading frameshift. A clinical observation of a child with type 2 mucolipidosis (I-cell disease) with typical symptomatology of the disease caused by two nonsense mutations of the GNPTAB gene is presented: p.S738X / p.R375X. In conclusion, it is emphasized that the identification of mutations of the GNPTAB gene provides prediction of the severity of the disease course, an understanding of the mechanisms of its development that will contribute to the development of pathogenetic treatment methods, improving the quality of life of patients and effective medical and genetic counseling.

Polycystic kidneys and GM2 gangliosidosis-like disease in neonatal springboks (Antidorcas marsupialis).

Clinical, gross, histopathologic, electron microscopic findings and enzymatic analysis of 4 captive, juvenile springboks (Antidorcas marsupialis) showing both polycystic kidneys and a storage disease are described. Springbok offspring (4 of 34; 12%) were affected by either one or both disorders in a German zoo within a period of 5 years (2008-2013). Macroscopic findings included bilaterally severely enlarged kidneys displaying numerous cysts in 4 animals and superior brachygnathism in 2 animals. Histopathologically, kidneys of 4 animals displayed cystic dilation of the renal tubules. In addition, abundant cytoplasmic vacuoles with a diameter ranging from 2 to 10 μm in neurons of the central and peripheral nervous system, hepatocytes, thyroid follicular epithelial cells, pancreatic islets of Langerhans and renal tubular cells were found in 2 springbok neonates indicative of an additional storage disease. Ultrastructurally, round electron-lucent vacuoles, up to 4 μm in diameter, were present in neurons. Enzymatic analysis of liver and kidney tissue of 1 affected springbok revealed a reduced activity of total hexosaminidase (Hex) with relatively increased HexA activity at the same level of total Hex, suggesting a hexosaminidase defect. Pedigree analysis suggested a monogenic autosomal recessive inheritance for both diseases. In summary, related springboks showed 2 different changes resembling both polycystic kidney and a GM2 gangliosidosis similar to the human Sandhoff disease. Whether the simultaneous occurrence of these 2 entities represents an incidental finding or has a genetic link needs to be investigated in future studies.

Incidence and carrier frequency of Sandhoff disease in Saskatchewan determined using a novel substrate with detection by tandem mass spectrometry and molecular genetic analysis

Sandhoff disease is a rare progressive neurodegenerative genetic disorder with a high incidence among certain isolated communities and ethnic groups around the world. Previous reports have shown a high occurrence of Sandhoff disease in northern Saskatchewan. Newborn screening cards from northern Saskatchewan were retrospectively screened in order to investigate the incidence and determine the carrier frequency of Sandhoff disease in these communities. PCR-based screening was conducted for the c.115delG (p.(Val39fs)) variant in the HEXB gene that was previously found in 4 Sandhoff disease patients from this area. The carrier frequency for this allele was estimated to be ~1:27. MS/MS-based screening of hexosaminidase activity along with genetic sequencing allowed for the identification of additional variants based on low total hexosaminidase activity and high % hexosaminidase A activity relative to c.115delG carriers. In total 4 pathogenic variants were discovered in the population (c.115delG, c.619A>G, c.1601G>T, and c.1652G>A) of which two are previously unreported (c.1601G>T and c.1652G>A). The combined carrier frequency of these alleles in the study area was estimated at ~1:15. Based on the number of cases of Sandhoff disease from this area we estimate the incidence to be ~1:390 corresponding to a child being born with the disease every 1–2years on average. The results from our study were then compared with variants in the HEXB gene from the genomes available from the 1000 Genomes project. A total of 19 HEXB variants were found in the 1092 genomes of which 5 are suspected of having a deleterious effect on hexosaminidase activity. The estimated carrier frequency of Sandhoff disease in Saskatchewan at 1:15 is more than 3 times higher than the carrier frequency in the global sample provided by the 1000 Genomes project at 1:57.

Hypermethylation contributes to down-regulation of lysosomal β-hexosaminidase α subunit in prostate cancer cells

β-Hexosaminidase, involved in degradation of glycoproteins and glycosphingolipids, is altered in several tumours leading to enhanced migration capacity. To date, the expression of the β-hexosaminidase isoenzymes in prostate cancer cells has not been elucidated. By using PC3, LNCaP, DUCaP, MDAPCa 2b, and hyperplasic prostate (BPH-1) cell lines, we analysed the β-hexosaminidase activity in each cell line and determined β-hexosaminidase α subunit gene expression in PC3, LNCaP, and BPH-1. We then investigated the methylation status of the gene promoter and determined the cellular responses of PC3 and LNCaP after transfection with β-hexosaminidase α subunit. We found that each prostate cancer cell line had a decrease in total hexosaminidase activity and that the lack of hexosaminidase A activity, observed in PC3 and LNCaP cells, was associated with mRNA disappearance. The HEXA promoter region in LNCaP and PC3 cell lines had methylated CpG islands, as confirmed by 5'-Aza-2'-deoxycitidine treatment, in PC3 cells, used as cell cancer model. We also tested, the involvement of hexosaminidase A in the migration capacity by migration assay using Hex α subunit-transfected PC3. Finally, we found that, after Hex α subunit transfection, both PC3 and LNCaP were less susceptible to exogenous ceramide treatment. Results indicate a likely contribution of the lysosomal enzyme to the acquisition of cancerous features.

Expanding the spectrum of HEXA mutations in Indian patients with Tay–Sachs disease

Tay–Sachs disease is an autosomal recessive neurodegenerative disorder occurring due to impaired activity of β-hexosaminidase-A (EC 3.2.1.52), resulting from the mutation in HEXA gene. Very little is known about the molecular pathology of TSD in Indian children except for a few mutations identified by us. The present study is aimed to determine additional mutations leading to Tay–Sachs disease in nine patients confirmed by the deficiency of β-hexosaminidase-A (<2% of total hexosaminidase activity for infantile patients) in leucocytes. The enzyme activity was assessed by using substrates 4-methylumbelliferyl-N-acetyl-β-d-glucosamine and 4-methylumbelliferyl-N-acetyl-β-d-glucosamine-6-sulfate for total-hexosaminidase and hexosaminidase-A respectively, and heat inactivation method for carrier detection. The exons and exon–intron boundaries of the HEXA gene were bi-directionally sequenced on an automated sequencer. ‘In silico’ analyses for novel mutations were carried out using SIFT, Polyphen2 and MutationT@ster software programs. The structural study was carried out by UCSF Chimera software using the crystallographic structure of β-hexosaminidase-A (PDB-ID: 2GJX) as the template. Our study identified four novel mutations in three cases. These include a compound heterozygous missense mutation c.524A>C (D175A) and c.805G>C (p.G269R) in one case; and one small 1bp deletion c.426delT (p.F142LfsX57) and one splice site mutation c.459+4A>C in the other two cases respectively. None of these mutations were detected in 100 chromosomes from healthy individuals of the same ethnic group. Three previously reported missense mutations, (i) c.532C>T (p.R178C), (ii) c.964G>T (p.D322Y), and (iii) c.1385A>T (p.E462V); two nonsense mutations (i) c.709C>T (p.Q237X) and (ii) c.1528C>T (p.R510X), one 4bp insertion c.1277_1278insTATC (p.Y427IfsX5) and one splice site mutation c.459+5G>A were also identified in six cases. We observe from this study that novel mutations are more frequently observed in Indian patients with Tay–Sachs disease with clustering of ~73% of disease causing mutations in exons 5 to 12. This database can be used for a carrier rate screening in the larger population of the country.

Adult Sandhoff Disease With 2 Mutations in the HEXB Gene Presenting as Brachial Amyotrophic Diplegia

Sandhoff disease is a rare autosomal recessive metabolic disorder of GM2 gangliosides. It is caused by a lack of functional N-acetyl-β-D-glucosaminidase A and B because of mutations in the HEXB gene. We describe a 55-year-old woman with adult Sandhoff disease presenting as brachial amyotrophic diplegia. The assay of total hexosaminidase involving A and B showed decreased level of these activities. Hex-A was 4.6 nmol·min·mL (normal: 7.0-20.0 nmol·min·mL) and Hex-B was 0.1 nmol·min·mL (normal: 1.0-10.0 nmol·min·mL), respectively. Analysis of HEXB gene demonstrated 2 point mutations that were located at the exon 5 (c.619A>G) and exon 11 (c.1250C>T). Compound heterozygosity of these 2 mutations may trigger the development of distinct adult Sandhoff disease phenotype. Sandhoff disease should be considered in the differential diagnosis of lower motor neuron disease, such as brachial amyotrophic diplegia, even if the age at onset is more than 50 years.

Salivary exoglycosidases in gestational diabetes

As exoglycosidases have been described as potential markers of salivary gland pathology, we decided to check the possibility of the use of these enzymes in the detection of salivary gland involvement in gestational diabetes. For this purpose diabetic pregnant women were compared to pregnant and non-pregnant healthy women. The activities of total HEX as well as GLU in the saliva were determined in duplicate according to Marciniak et al. The activities of GAL, FUC, and MAN in the saliva were determined in duplicate according to Zwierz et al. It was found that the specific activities of exoglycosidases in the saliva of diabetic pregnant women significantly increased in comparison to healthy pregnant and non-pregnant women. Increased specific activity of exoglycosidases suggests that gestational diabetes provokes structural/functional alterations in salivary glands and changes in the salivary glycoconjugates metabolism.

Salivary hexosaminidase in smoking alcoholics with bad periodontal and dental states

BackgroundA sensitive alcohol marker, β-hexosaminidase (HEX), in the saliva of alcoholics, is investigated for the first time. MethodsThe activity, specific-activity and output of total HEX and its isoenzymes HEX A and HEX B were measured in the saliva of healthy controls (C), alcohol-dependent non-smokers (ANS), and alcohol-dependent smokers (AS). ResultsWe observed a significantly increased activity/specific-activity and output of HEX A in the ANS and AS groups, due to the inflammatory state of the oral-cavity/salivary-glands. Significantly increased activity of HEX A contributed to an increase in the salivary activity of the total HEX in the ANS group. A significant decrease in the activity/specific-activity of HEX B in AS seemed to be due to HEX B inactivation by cigarette smoke. We noticed a tendency for deteriorated dental state (lower decayed-missing-filled-teeth index – DMFT), worse periodontal state (higher gingival index – GI and papilla-bleeding index – PBI) in AS, and worse periodontal state (higher GI) in ANS, as compared to the controls. We found no differences in the salivary protein concentrations between all groups and decreased salivary flow in both alcoholic groups as compared to the controls. In alcoholics, the area under the curve (AUC) for HEX A activity/specific-activity was significantly greater than for HEX and HEX B. The salivary HEX A activity/specific-activity had good/excellent sensitivity and specificity in smoking and non-smoking alcoholics, whereas salivary HEX and HEX B had poor/fair sensitivity and specificity. ConclusionsSalivary HEX A may be helpful in the diagnosis of chronic alcohol intoxication, even in smokers.

Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India.

Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

Effect of sample collection, temperature and time of storage on β-galactosidase and total hexosaminidase activities in dried blood collected on filter paper

Dried blood spots (DBS) on filter paper is a valuable sampling technique in clinical chemistry, but the stability of enzymes used in the diagnosis of lysosomal storage diseases (LSDs) needs to be evaluated. In a first experiment, blood from 20 subjects was collected using a syringe without additives and distributed into EDTA tubes, heparin tubes, and spotted on filter paper for the comparison of sampling effects. In a second experiment, blood from 30 healthy subjects was spotted on filter paper and analyzed for β-galactosidase and total hexosaminidase activities after storage of the samples at different temperatures for up to 180 days. Initially, we observed that enzyme activities were the same, independent of the collection method. When DBS was stored at 37°C the activity of β-galactosidase dropped to 85% of the initial value after 180 days (p<0.05). At all other temperatures (-20°C, 4°C and 25°C), the results were within the methodological error. Total hexosaminidase activity did not change significantly during the entire study period and at different storage temperatures. The two enzymes investigated in the present study may be stored for up to 17 days (β-galactosidase) or 180 days (total hexosaminidase) until analysis without loss of activity.

The Effect of the Binge Drinking Session on the Activity of Salivary, Serum and Urinary -Hexosaminidase: Preliminary Data

Our report is the first to show that an acute ingestion (6 h) of a relatively large, yet tolerable dose of alcohol (120-160 g), significantly increases activity of total serum beta-hexosaminidase (total beta-HEX), beta-HEX A and beta-HEX B isoenzymes, as well as salivary total beta-HEX and urinary beta-HEX A, in eight infrequent binge drinkers. An increase in the activity of serum and urinary total HEX is mainly due to its secretory isoenzyme beta-HEX A.

Connective tissue alterations in women with pelvic organ prolapse and urinary incontinence

Alterations in collagen synthesis and metabolism have previously been reported in patients with pelvic organ prolapse (POP) and/or urodynamic stress incontinence (USI). Since urinary incontinence does not always associate with POP, the objective of this study was to examine connective tissues from patients with USI plus POP, and patients with prolapse only. Biopsies from the uterosacral ligaments were obtained during the operation from POP patients (n =28), and from continent women (control group, n =12) who underwent surgery for other benign reasons. POP patients were classified following urodynamic tests and symptom questionnaire with respect to the presence (n =14) or absence (n =14) of USI. N-terminal propeptides of collagen (PINP and PIIINP), TGF-beta and leptin were measured in plasma. Hydroxyproline and glycosaminoglycan (GAGs) concentrations and total hexosaminidase activity were measured in tissue samples. Histological sections were prepared using Masson's trichrome technique, and digitised solutions were used for imaging provided by Soft Imaging System GmBh. Statistical evaluations were made by the Kruskal-Wallis test. A significant decrease in hydroxyproline content was found in USI+POP women in comparison to controls (p<0.05). In contrast, histopathological examination revealed an increased density of collagen in USI+POP patients. Hexosaminidase activity was decreased in both groups with POP, but no change in the amount of GAGs was observed. Markers of collagen synthesis (PINP, PIIINP), and factors related to the collagen synthesis (TGF-beta, leptin) remained unaltered. Our biochemical and morphological findings suggest a different organisation of collagen fibres in tissues of patients with USI+POP, when compared with both the controls and the POP patients.

Comparison of the measurement of lysosomal hydrolase activity in mycoplasma-contaminated and non-contaminated human fibroblast cultures treated with mycoplasma removal agent

Objectives: To investigate the effect of both mycoplasma contamination and of its remover (MRA), through human fibroblasts culture over the activity of some lysosomal hydrolases. Design and methods: Activity was measured in contaminated fibroblasts before and after the addition of MRA. Results were compared with the enzymatic activity in control fibroblasts with and without MRA. Results: Only β-glucosidase showed no significant alteration in the presence of either mycoplasma or MRA. Total hexosaminidase and β-galactosidase underwent significant interference in the presence of the mycoplasma and the MRA. The % of hexosaminidase A and arylsulphatase A altered their activity only in the presence of MRA. β-Glucuronidase changed its activity only in the presence of mycoplasma. Conclusions: The fibroblast enzymes behaved differently in the presence of MRA and/or mycoplasma, demonstrating the sensitivity of these hydrolases. Our work suggests that mycoplasma and MRA alter the activity of some lysosomal hydrolases.

A thermodynamic study of β-N-acetylhexosaminidase enzyme heterogeneity in cerebrospinal fluid from patients with multiple sclerosis

Background. ß-N-acetylhexosaminidase (Hex) is a lysosomal hydrolase, whose determination in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) has provided discordant results.Methods. Total Hex and its isoenzymes Hex A and Hex B were determined using a thermodynamic procedure in the CSF of 27 patients with definitive MS, 8 with possible MS, 9 with meningitis, 14 with other neurological diseases, and in 10 controls without any neurological disease.Results. In the group of patients with definitive MS, the total Hex and Hex A were significantly higher than in the control group (p<0.001), with a possible association of greater enzymatic activities with the presence of oligoclonal bands and recent relapse; however, an overlap was detected for the activities of total Hex and its isoenzymes between the groups of patients with different neurological diseases. A significant correlation was obtained for neuron-specific enolase (NSE) with total Hex and Hex A and Hex B isoenzymes (p<0.001); however, in the partial correlation statistical significance was only obtained between NSE and Hex A (p<0.001) which is the most abundant Hex isoenzyme in the brain.Conclusions. Although the inflammatory process in MS mainly takes place in the perivascular zone, with little activity in the cerebral parenchyma, the significant increase of NSE and Hex A isoenzyme in CSF reveals a neuronal damage. The disease status may have effect on the CSF Hex activity.

Common HEXB polymorphisms reduce serum HexA and HexB enzymatic activities, potentially masking Tay-Sachs disease carrier identification

A DNA-proven Tay-Sachs disease (TSD) carrier and his brother were found to have serum percent Hexosaminidase A (%HexA) enzymatic activities in the non-carrier range, while the leukocyte %HexA profiles clearly identified them as TSD heterozygotes. Both their serum HexA and HexB enzymatic activities were below reference range, suggesting inheritance of mutations in both the HEXA (α-subunit) and HEXB (β-subunit) genes. DNA sequencing revealed that both individuals, carried the common HEXA 1277_1278insTATC mutation, and two common HEXB polymorphisms: [619A>G (+) delTG]. To determine if these HEXB polymorphisms reduce HexA and HexB enzymatic activities, 69 DNA samples from subjects previously screened enzymatically in both serum and leukocytes for TSD carrier status were selected for either high, mid-range or low serum Total Hex (defined as the sum of HexA and HexB) activities and were tested for the HEXB mutations. Further, three additional TSD carriers ascertained by the atypical pattern of normal serum %HexA but carrier leukocyte %HexA, were found to have the [delTG (+) 619A>G] genotype. In addition, the frequency of the [delTG (+) 619A>G] genotype was significantly higher ( P < 0.01) in subjects with low serum HexB enzymatic activities. Given the high frequency of the [delTG (+) 619A>G] haplotype in the Ashkenazi Jewish population (approximately 10%), up to 10% of TSD carriers may have normal serum %HexA values with low total Hex. Accordingly, serum %HexA should not be the sole criterion used for carrier status determination. Where total Hex activity is reduced, further testing with leukocyte Hex profiles is indicated.

β-Hexosaminidase isoenzyme profiles in serum, plasma, platelets and mononuclear, polymorphonuclear and unfractionated total leukocytes

Objectives: The relative proportion in percentage of the isoenzyme A of β-hexosaminidase (Hex) is the single discriminatory function most frequently used for the biochemical screening of heterozygote Tay–Sachs disease carriers. It has been suggested that the assay of the Hex isoenzymes in homogeneous cell preparations is preferable to that in mixed total leukocytes which present greater interindividual variation. The major aim of our study was the evaluation of this hypothesis. Design and methods: Total Hex and its Hex A and Hex B isoenzymes were determined in different samples of serum and plasma ( n = 81) as well as in lysates of platelets ( n = 75), and mononuclear ( n = 81), polymorphonuclear ( n = 81) and mixed total leukocytes ( n = 33). Results: The interindividual variations found for % Hex A in the different biological samples were: plasma (CV = 23.4%), platelets (CV = 10.2%), mononuclear (CV = 5.7%), polymorphonuclear (CV = 5.3%) and total leukocytes (CV = 7.1%). Although the relative proportion of Hex A was significantly greater in polymorphonuclear than in mononuclear leukocytes ( P < 0.001), a statistical significance was not attained for the correlation between the relative proportions of blood polymorphonuclear cells and Hex A in mixed total leukocytes ( r = 0.220). Conclusions: The use of total leukocyte lysates does not appear to introduce a significant increase for the interindividual variation of the Hex A isoenzyme relative proportion in relation to the use of homogeneous cell preparations.