Biochemical and Molecular Investigation of Hexosaminidase A Deficiency in GM2 Gangliosidosis Genotypes

Note:The reliable diagnosis of Tay-Sachs disease (TSD) and detection of TS heterozygotes is dependent upon an accurate assessment of the catalytic activity of hexosaminidase A (Hex A). 4-methylumbelliferyl-.6-D-N -acetylglucosamine-6-sulfate (4MUGS) was synthesized in high yield by the sulfation of 4-methylumbelliferyl-S-DN- acetylglucosamine (4MUG) with chlorosulfonic acid, producing a substrate preferentially hydrolyzed by Hex A. By application of an empirical formula, 4MUGS assay values are transformed to allow the calculation of Hex A and Hex B activities without the requirement of thermal fractionation. The usefulness of this substrate for the detection of TS heterozygotes, by both manual and automated assay procedures, is demonstrated. The diagnosis of TSD genotypes, particularly the B1 TSD and Hex A-deficient healthy adult variants, is significantly improved with clear implications for screening and prenatal diagnosis. Chromatographic separation of Hex isozymes and analysis of the profiles obtained by 4MUG and 4MUGS hydrolysis allows for the differentiation of later-onset forms of TSD. The kinetic characterization of the B1 mutation using 4MUGS and the investigation of the TSD mutation in French Canadians by a DNA-based procedure demonstrates the heteroallelism within populations.Le diagnostic sur de la maladie de Tay-Sachs et le dépistage des hétérozygotes de la maladie de Tay-Sachs sont dépendant de la mesure précise de l'activité de l'hexosaminidase A. J'ai synthétise le substrat 4-methylumbelliferyl-B-DN- acetylglucosamine-6-sulfate (4MUGS) par sulfation de la 4-methylumbelliferyl-BD- N-acetylglucosamine (4MUG) avec l'acide chlorosulfonique. La sulfation de 4MUG produit un substrat spécifique pour l'hexosaminidase A. Par l'emploi d'une formule empirique, les montants de l'essai peuvent être transformer en activité des hexosaminidases A et B sans !'inactivation thermale. L'utilité du substrat 4MUGS pour le dépistage des porteurs de tare par les essai manuels et automatiques est démontré. Le diagnostic de la maladie de Tay-Sachs, en particulier les variante B1 et les adulte en bonne santé sans l'activité de l'hexosaminidase A est amélioré, avec implication pour le dépistage et le diagnostic prénatal. Utilisant la chromatographie, les hexosaminidases sont isolées. Les maladies de Tay-Sachs avec une présentation tardive peuvent être distingues par l'examen des profils obtenus à l'hydrolyse enzymatique des 4MUG et 4MUGS. La caractérisation cinétique de la mutation B1 par 4MUGS et l'investigation de la mutation Tay-Sachs chez les Canadiens français démontrent la variation dans les populations

Frequencies of cystic fibrosis mutations in the Maine population: high proportion of unknown alleles in individuals of French-Canadian ancestry.

Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders in Caucasian populations. A mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes this disorder. Reported here is the first analysis of CF mutations in the Maine population. We have screened 263 CF chromosomes for 16 previously reported mutations. Analysis of DNA from 124 apparently unrelated CF patients and 15 obligate carrier parents (whose partner and affected child were unavailable for study) resulted in the identification of 91% of the CF alleles and complete genotyping of 85% of the patients. The frequencies (%) of these mutations in the Maine population are delta F508 (75% of the chromosomes), G85E (0.76), R117H (0.76), I148T (1.1), 621 + 1G --\u3e T (1.1), 711 + 1G --\u3e T (3.0), A455E (1.1), 1717-1G --\u3e A (1.1), G542X (1.9), G551D (1.9), R560T (0.76), Y1092X (0.38), W1282X (0.38), and N1303K (1.5). The exon 10 mutation, delta I507, and the exon 11 mutation, R553X, were not observed. Surprisingly, whereas only 5% of the alleles remain unidentified in the non-French population, the unidentified proportion in the French population is 19%. CF testing for the Maine population will be further improved as the as yet unidentified CF mutations in this population are characterized

Intron/exon structure confirms that mouse Zfy1 and Zfy2 are members of the ZFY gene family.

Zfy1 and Zfy2 are homologous zinc finger genes on the mouse Y Chromosome. To ask whether these genes are properly classified as members of the ZFY family, we have characterized and compared their genomic organization to that of mouse Zfx, human ZFX, and human ZFY. We show that Zfy1 has 11 exons distributed across at least 56 kb, and Zfy2 has a minimum of 9 exons distributed across at least 52 kb. The Zfy2 locus contains regions similar in size and sequence to all 11 exons of Zfy1, plus an additional 5\u27 UTR exon. All splice sites conform to the GT-AG rule. There are two instances of additional AG dinucleotides immediately 5\u27 of 3\u27 splice sites. Zfy1 and Zfy2 are homologous to other ZFY family members within the coding region, but the untranslated regions show no sequence similarity. Within the coding region, there is conservation of exon length and splice sites, with each splice preceding the second nucleotide of a codon. We conclude that Zfy1 and Zfy2 are indeed members of the ZFY family, which has evolved from a single common ancestral gene