1/84. Enzyme studies in GM2 gangliosidiosis, and their application in prenatal diagnosis. Assay of hexosaminidase a and B enzymes in four cases with developmental regression and cherry red spot on fundus examination confirmed that three cases had tay-sachs disease, and one case had sandhoff disease. prenatal diagnosis was carried out by hexosaminidase enzyme assay in amniotic fluid and cells in one family, and chorionic villus sample in the second family. The fetus was diagnosed to be unaffected in one, and affected in the other family. Assay of hexosaminidase a and B is useful for specific diagnosis of GM2 gangliosidosis, and for prenatal diagnosis to reduce the burden of these disorders. ( info) |
| tay-sachs disease (TSD) is caused by mutation of the HEXA gene, which results in a deficiency of the alpha-subunit of hexosaminidase a. The major mutation in Japanese TSD is a G-to-T transversion at the 3'-splice site of intron 5. We established a fluorescent competitive allele-specific polymerase chain reaction (FCAS-PCR) method for detection of the mutation and applied it to prenatal diagnosis of a Japanese TSD family. FCAS-PCR distinguished the wild and mutant alleles clearly, with broad ranges in the amount of template dna, the dNTP concentration, the MgCl2 concentration and the number of PCR cycles. After obtaining ethics committee approval and informed consent from the parents in the index family, chorionic villus sampling was performed. FCAS-PCR analysis using chorionic villus dna disclosed that the fetus was homozygous for the mutation. To confirm the diagnosis, direct sequencing analysis of the genomic PCR fragment was performed, and showed the same results as those of the FCAS-PCR analysis. FCAS-PCR proved to be helpful for carrier screening and prenatal diagnosis in TSD families in the Japanese population. It would also be a useful dna-diagnostic method for many other inherited disorders. ( info) |
| The late-onset form of GM2 gangliosidosis (tay-sachs disease) is an autosomal-recessive disorder with progressive neurologic disease, mainly characterized by motor neuron and spinocerebellar dysfunction. The majority of patients are of Ashkenazi Jewish origin. 31Phosphorus magnetic resonance spectroscopy of the brain was performed to study the metabolic changes of a 16-year-old patient with late-onset tay-sachs disease who had a heterozygous Gly269-->Ser mutation in the hexosaminidase a encoding gene in compound heterozygosity with another, yet unidentified mutation. Severe changes in phosphorus metabolism with a decreased amount of phosphodiesters and membrane-bound phosphates were demonstrated, suggesting an activation of phosphodiesterases by accumulating gangliosides. The clinical findings were well related to the changes in spectroscopically determined metabolites. ( info) |
| The brief communication describes a 2-year-old child who presented with delayed achievement and regression of milestones, seizures of multiple types, exaggerated response to sound, inability to see and bilateral cherry red spots. In addition to these typical manifestations of the late infantile variety of tay-sachs disease, unilateral ptosis was present. The magnetic resonance imaging of brain revealed abnormalities consistent with an advanced stage of the disease. ( info) |
5/84. GM2 gangliosidosis variant B1 neuroradiological findings. Variant B1 is a rare type of GM2 gangliosidosis. Clinically, it shows a wide spectrum of forms ranging from infantile to juvenile. We report the first magnetic resonance imaging (MRI) findings from three patients affected by GM2 gangliosidosis variant B1, two presenting with the infantile form and one with the juvenile form. The MRI appearances of the two patients with the infantile form disease are congruent with those reported for the early-onset type of both Tay-Sachs and Sandhoff diseases, and are characterized by early involvement of the basal ganglia and thalamus with cortical atrophy appearing later. In contrast, the patient with the juvenile form of variant B1 showed progressive cortical and white-matter atrophy of the supratentorial structures and, to a lesser extent, the infratentorial structures. No basal ganglia or thalamic anomalies were observed. Because in the adult forms of both Tay-Sachs and Sandhoff diseases a progressive cerebellar atrophy represents the only abnormality detectable, it appears that an MRI pattern peculiar to GM2 gangliosidosis can be defined. This pattern ranges from the basal ganglia injury associated with the early and severe demyelination process noted in the infantile form of the disease, to cerebellar atrophy with no supratentorial anomalies in the adult form. An "intermediate" MRI picture, with cortical atrophy and mild cerebellar atrophy, but without basal ganglia impairment, can be observed in the juvenile form. In addition, our investigations suggest that MRI abnormalities in GM2 gangliosidosis correlate with the clinical form of the disease rather than with the biochemical variant of the enzymatic defect. ( info) |
6/84. Biochemical and molecular characterization of mutant hexosaminidase a in a Turkish family. BACKGROUND: tay-sachs disease is a form of monosialoganglioside triaose (GM2) gangliosidosis that results from the mutations in the alpha-subunit gene of hexosaminidase a. In the B1 variant, the active site of the alpha-subunit of the enzyme is thought to be affected. In the present study, a patient who had previously been diagnosed as a B1 variant is further analyzed. The patient's parents and brother were also analyzed. methods: Single-stranded conformational polymorphism (SSCP) and dna sequencing analysis were conducted in all cases. In addition, hexosaminidase a (Hex A) was isolated from leukocyte homogenates of the patient's parents and brother using DE 52 ion-exchange chromatography, and thermostability analyses of the isolated enzymes were performed. RESULTS: hexosaminidase a of the parents was found to be more thermostable than normal Hex A. dna sequencing analysis revealed a 12-bp deletion mutation in exon 10 of the Hex A gene. The patient was a homozygote and the parents were heterozygotes for the mutation, which could also be observed at the dna double strands by SSCP analysis. These deleted bases are located within the catalytic domain of the alpha-subunit. CONCLUSIONS:The 12-bp deletion mutation in exon 10 of Hex A is responsible for the increased thermostability of the enzyme. Considering this mutation has previously been found in a Turkish Tay-Sachs patient, the patient in the present study may have another mutation on the Hex B gene that causes decreased thermostability of the enzyme. Thermal inactivation assay may not be sufficient for a correct diagnosis in such unusual cases. ( info) |
7/84. A new point mutation (G412 to A) at the last nucleotide of exon 3 of hexosaminidase alpha-subunit gene affects splicing. We report the sixth mutation associated with the infantile form of tay-sachs disease in the Turkish population. The mutation is a single nucleotide transition (G to A) at the last nucleotide of exon 3 of hexosaminidase a (HEX A) alpha-subunit gene. The 14 exons and their flanking sequences of the HEX A gene were amplified and analyzed by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Sequencing of exon 3 showed a homozygous mutation. Cultured patient's fibroblasts produced no detectable mRNA for HEX A alpha-subunit gene by Northern blot analysis. We speculate that abnormal mRNA was rapidly degraded following transcription. Our data are consistent with the idea that the severe infantile form of tay-sachs disease is associated with a total lack of Hex A activity in the patient. A similar mutation (G to T) had been observed at the 5'-donor splice site of exon 3. It resulted in abnormal splicing and skipping of exon 3. The other acceptor and donor splice site mutations described in the HEX A gene ablate normal mRNA splicing. Identification of multiple mutant HEX A alleles shows molecular heterogeneity of infantile tay-sachs disease in our population. ( info) |
8/84. A glycine250--> aspartate substitution in the alpha-subunit of hexosaminidase a causes juvenile-onset tay-sachs disease in a Lebanese-Canadian family. The mutation causing juvenile tay-sachs disease (TSD) in two sibs of Lebanese-Maronite origin is described. An mRNA-containing extract of cultured fibroblasts obtained from one of the probands was used as a template to amplify the coding sequence of the hexosaminidase a (Hex A) alpha-subunit. Sequencing of amplified cDNA fragments revealed a single alteration, guanine to adenine at nt 749 creating a G250D mutation. The mutation introduces a new recognition site for the restriction enzyme Eco RV, permitting identification of heterozygotes for this allele following PCR amplification and Eco RV digestion of exon 7 sequences from genomic dna templates. In order to test the effect of this substitution, an in vitro mutagenized cDNA construct was introduced into a mammalian expression vector and transfected into monkey Cos-1 cells separately or along with a beta-cDNA expression vector. When the mutant alpha-cDNA was the only gene introduced into cos cells no enzymatic activity above endogenous COS cell activity was detected. Cotransfection of normal alpha-cDNA and beta-cDNA followed by immunoprecipitation of human Hex A resulted in 20-fold increase in the ratio between positive and negative (mock transfection) control values. This allowed the detection of some residual activity (12% of the positive control) when the mutant alpha-cDNA replaced its wild-type counterpart. The predicted protein environment in which the mutation occurs is compared to that of the adult-onset tay-sachs disease mutation caused by a Gly269-->Ser substitution in exon 7.(ABSTRACT TRUNCATED AT 250 WORDS) ( info) |
9/84. A new tay-sachs disease B1 allele in exon 7 in two compound heterozygotes each with a second novel mutation. Three novel Tay--Sachs Disease (TSD) mutations have been identified in two unrelated, non-Jewish compound heterozygous patients. A G772C transversion mutation causing an Asp258His substitution is shared by both patients. The mutant enzyme had been characterized, on the basis of previous kinetic studies (1) as a B1, or alpha-subunit active site mutation. This is the first B1 mutation not found in codon 178 (exon 5). A C508T transition causing an Arg170Trp substitution also occurred in one of the patients. The third mutation is a two base deletion occurring in exon 8 involving the loss of either nts 927-928 or 929-930 in codon 310. The deletion creates an inframe termination codon 35 bases downstream. The Arg170Trp mutation was also detected in a third unrelated TSD patient. In both families this allele was traced to French Canadian ancestors originating in the Estrie region of the province of quebec. This mutation is the third TSD allele unique to the French Canadian population and the ancestral origins of the carrier parents are distant from the center of diffusion of the more common 7.6 kb deletion mutation which is in the eastern part of the province. ( info) |
10/84. Heterozygosity for the "DN allele" (G533-greater than A) of the beta-hexosaminidase alpha subunit gene identified by direct dna sequencing in a family with the B1 variant of GM2-gangliosidosis. A new patient having clinical, pathologic and biochemical features of the exceedingly rate B1 variant of GM2-gangliosidosis is described. This patient, of northern European (non-Ashkenazi) ancestry, is the first affected child of this ethnic background available for molecular genetic analysis; thus, she represented an opportunity to identify a new mutation associated with this phenotype or, conversely, to further characterize the DN allele of the hexosaminidase alpha gene (G533-greater than A) as a more widely distributed mutation not previously observed in this gene pool. As a means of rapid analysis, we report a strategy for PCR amplification and direct dna sequencing of exon 5 of the hexosaminidase alpha gene, the most frequent site of mutations in this condition. With this technique, the father of the affected child was determined to be heterozygous for the DN allele, while the mother was found to have only the normal sequence in this region. This observation further extends the known geographic and ethnic distribution of this mutation, and suggests the likelihood that the DN allele has been derived by multiple independent mutational events. ( info) |