Estimation of the frequency of hexosaminidase a variant alleles in the American Jewish population.

There appear to be several alleles of the hexosaminidase A (HEX A) gene that lead to different clinical syndromes. In addition to the infantile-onset Tay-Sachs disease (TSD), there is a juvenile-onset and an adult-onset form, which are also characterized by low HEX A levels. There are also apparently healthy adults with low HEX A activity. Based primarily on data from population screening for TSD carrier status, we estimate the allele frequency of the combined variant alleles for which data are available to be about 4.5 x 10(-4) and the frequency of adults showing zero HEX A levels (when tested using artificial substrate) to be about 1:67,000. The implications for population screening and prenatal diagnosis are discussed.     

Segregation of Tay-Sachs and Sandhoff alleles in a non-Jewish family.

A non-Jewish family is presented in which the genes for Tay-Sachs disease and Sandhoff disease are segregating. Individuals heterozygous for both alleles have low serum and white cell total hexosaminidase levels together with a proportion of heat-labile hexosaminidase A (HEX A) which falls in the normal range. The individuals would not be detected as carriers of Tay-Sachs disease or Sandhoff disease in a population screening program.     

Low levels of beta hexosaminidase A in healthy individuals with apparent deficiency of this enzyme.

Appreciable beta hexosaminidase A (hex A) activity has been detected in cultured skin fibroblasts and melanoma tissue from healthy individuals previously reported as having deficiency of hex A activity indistinguishable from that of patients with Tay-Sachs disease (TSD). Identification and quantitation of hex A, amounting to 3.5%-6.9% of total beta hexosaminidase activity, has been obtained by cellulose acetate gel electrophoresis, DEAE-cellulose ion-exchange chromatography, radial immunodiffusion, and radioimmunoassay. Previous family studies suggested that these individuals may be compound heterozygotes for the common mutant TSD gene and a rare (allelic) mutant gene. Thus, the postulated rate mutant gene appears to code for the expression of low amounts of hex A. Heterozygotes for the rare mutant may be indistinguishable from heterozygotes for the common TSD mutant. However, direct visualization and quantitation of hex A by the methods described may prevent false-positive prenatal diagnosis of TSD in fetuses having the incomplete hex A deficiency of the type described in the four healthy individuals.     

Characterization of a variant of beta-hexosaminidase: "hexosaminidase Paris".

A family (father and daughter) was found with a deficiency of hexosaminidase (HEX A and HEX B). Residual HEX A activity was about 30% of usual heterozygotes with very little HEX B activity. Thermostability of HEX A was decreased. No immunological cross reacting material was found for HEX A or B. The mechanism seems to be the production of abnormal, unstable beta subunits, which are still capable of combining with alpha subunits to form functional HEX A.     

Evidence for a hybrid hexosaminidase isoenzyme in heterozygotes for Sandhoff disease.

Patients with Sandhoff disease have less than 5% of normal levels of serum or tissue hexosaminidase activity. They are thought to have a defect in the structural gene for the beta chain of hexosaminidase (HEX). Heterozygotes for Sandhoff disease have approximately 50% of the total serum HEX activity of normals and more than 75% of the HEX is heat-labile. In normals, only 55%--65% of serum HEX is heat-labile. Serum HEX separates into three forms on DEAE cellulose chromatography: HEX A, a tetramer of 2 alpha and 2 beta chains, and HEX I and B composed solely of beta chains. The DEAE chromatograms from normals and Sandhoff heterozygotes did not differ in the relative distribution of HEX activity between peaks. In normals, the HEX A peak was heat-labile (60 degrees C for 9 min), but HEX I and B were heat-stable. In Sandhoff heterozygotes, however, HEX I and B were only 50%--53% heat-stable. This suggests the heterozygotes synthesized a hybrid enzyme containing both mutant and wild-type beta chains for HEX. The mutant beta chain renders the isoenzyme less stable to heating.     

Characterization of unusual hexosaminidase A (HEX A) deficient human mutants.

Two families with unusual hexosaminidase A (HEX A) mutations are described. In one, the proband had the Tay-Sachs disease phenotype with considerable HEX A activity. In the second, the proband was phenotypically normal with absent HEX A activity. Activities using ganglioside GM2 as substrate demonstrate markedly reduced activities in the first case and half-normal activities in the second. Pedigree analyses indicate the presence of two different mutations. In the first, the proband appears to be an allelic compound HEX A 2-4 where mutation HEX A 4 leads to a diminution of HEX A activity against GM2 but not for the synthetic substrate, 4MU-beta-D-N-acetyl-glucosaminide, with HEX A 2 being the Tay-Sachs disease (or similar) mutation. In the second family, the proband is an allelic compound HEX A 2-5 where mutation HEX A 5 leads to a diminution of HEX A activity against the synthetic substrate, 4MU-beta-D-N-acetyl-glucosaminide, but not for GM2. The presence of either mutation will lead to false-negative (HEX A 4) or false-positive (HEX A 5) assignments of heterozygosity or homozygosity for GM2 gangliosidosis when synthetic substrates are employed. In both families, DM2 N-acetyl-beta-D-galactosaminidase activity in fibroblasts was an accurate determinant of phenotype.     

The biochemical genetics of the hexosaminidase system in man.

Tay-Sachs disease and related GM2 ganglioside storage disorders result from the absence of one form of hexosaminidase, HEX A. The persistence of a second major hexosaminidase isozyme, HEX B, does not protect against the lethal accumulation of GM2 ganglioside in the central nervous system. Using immunologic and biochemical techniques, it has been demonstrated that the two major isozymes of hexosaminidase, HEX A and HEX B, share a common subunit, the structure of HEX A being designated (alpha beta)n and the structure of HEX B being designated as (beta2)n. The minor isozyme, HEX S, is an alpha chain homopolymer designated (alpha2)n, and HEX C seems unrelated to the HEX A, B, S system. The structures of other minor isozymes have not been totally resolved, but HEX I1, I2, and P (which may be identical to I2) appear to represent forms of HEX B.     

Ehlers-Danlos syndrome type IV: a multi-exon deletion in one of the two COL3A1 alleles affecting structure, stability, and processing of type III procollagen

We have studied a patient with severe, dominantly inherited Ehlers-Danlos syndrome type IV. The results indicate that this patient carries a deletion of 3.3 kilo-base pairs in the triple helical coding domain of one of the two alleles for the pro-alpha-chains of type III collagen (COL3A1). His cultured skin fibroblasts contain equal amounts of normal length mRNA and of mRNA shortened by approximately 600 bases, and synthesize both normal and shortened pro-alpha 1(III)-chains. In procollagen molecules containing one or more shortened chains, a triple helix is formed with a length of only about 780 amino acids. The mutant procollagen molecules have decreased thermal stability, are less efficiently secreted, and are not processed as their normal counterpart. The deletion in this family is the first mutation to be described in COL3A1.     

Juvenile GM2 gangliosidosis (AMB variant): inability to activate hexosaminidase A by activator protein.

Two sibling from a consanguineous Puerto Rican marriage were found to have a juvenile-onset type of lipidosis first noted at age 2 1/2 by expressing difficulties with motor function and developmental delay. They continued to deteriorate, showing muscle atrophy, spasticity, and loss of speech, and death occurred at ages 7 and 8. Examination of the brains from these patients revealed that the concentration of GM2 ganglioside was about 56% of the total gangliosides. Hexosaminidase and percent hexosaminidase A (HEX A) and other lysosomal enzymes were normal in cultured skin fibroblasts, liver, and brain. The concentration of the activator protein required for the enzymatic hydrolysis of GM2 ganglioside was in high normal levels in the brain of the patient available. However, the HEX A from the patient's brain and liver as well as from skin fibroblast lysates could not be activated to hydrolyze GM2 ganglioside by the activator protein from a control or himself. The HEX A from a control could be activated by the activator protein from controls or this patient. These patients appear to have a defect in HEX A, which does not affect it heat stability, electrophoretic migration, and activity toward fluorogenic substrates, but may affect the binding of the activator protein required for GM2 ganglioside hydrolysis. We propose to call these patients the AMB variant of GM2 gangliosidosis to denote the mutation in HEX A but with normal levels of HEX A and B with synthetic substrates. This is to distinguish these patients from those missing the activator protein and normal HEX A and B levels.     

Hereditary heat-labile hexosaminidase B: a variant whose homozygotes synthesize a functional HEX A.

Homozygosity for a mutant allele at the beta-chain locus of hexosaminidase (HEX), resulting in a variant of heat-labile HEX B, is reported for the first time in two healthy children. HEX activity in their sera, leukocytes, and cultured skin fibroblasts is severely deficient when measured on the synthetic substrate 4-MU-GLcNAc. However, their cultured skin fibroblasts synthesize and process both alpha and beta chains of HEX, and their lymphoid cells hydrolyze normally the natural ganglioside GM2. This mutation is, therefore, different from at least one of the beta-chain mutations found in previously published families with heat-labile HEX B.     

Sequence-based evidence for major histocompatibility complex-disassortative mating in a colonial seabird

The major histocompatibility complex (MHC) is a polymorphic gene family associated with immune defence, and it can play a role in mate choice. Under the genetic compatibility hypothesis, females choose mates that differ genetically from their own MHC genotypes, avoiding inbreeding and/or enhancing the immunocompetence of their offspring. We tested this hypothesis of disassortative mating based on MHC genotypes in a population of great frigatebirds (Fregata minor) by sequencing the second exon of MHC class II B. Extensive haploid cloning yielded two to four alleles per individual, suggesting the amplification of two genes. MHC similarity between mates was not significantly different between pairs that did (n = 4) or did not (n = 42) exhibit extra-pair paternity. Comparing all 46 mated pairs to a distribution based on randomized re-pairings, we observed the following (i): no evidence for mate choice based on maximal or intermediate levels of MHC allele sharing (ii), significantly disassortative mating based on similarity of MHC amino acid sequences, and (iii) no evidence for mate choice based on microsatellite alleles, as measured by either allele sharing or similarity in allele size. This suggests that females choose mates that differ genetically from themselves at MHC loci, but not as an inbreeding-avoidance mechanism.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

More than one mutant allele causes infantile Tay-Sachs disease in French-Canadians

Two Tay-Sachs disease (TSD) patients of French-Canadian origin were shown by Myerowitz and Hogikyan to be homozygous for a 7.6-kb deletion mutation at the 5' end of the hexosaminidase A α-subunit gene. In order to determine whether all French-Canadian TSD patients were homozygotes for the deletion allele and to assess the geographic origins of TSD in this population, we ascertained 12 TSD families of French-Canadian origin and screened for occurrence of mutations associated with infantile TSD. DNA samples were obtained from 12 French-Canadian TSD families. Samples were analyzed using polymerase-chain-reaction (PCR) amplification followed by hybridization to allele-specific oligonucleotides (ASO) or by restriction analysis of PCR products. In some cases Southern analysis of genomic DNA was performed. Eighteen of the 22 independently segregating mutant chromosomes in this sample carried the 7.6-kb deletion mutation at the 5' end of the gene. One chromosome carried the 4-nucleotide insertion in exon 11 (a “Jewish” mutation). In this population no individuals were detected who had the substitution at the splice junction of exon 12 previously identified in Ashkenazi Jews. One chromosome carried an undescribed B¹ mutation; this allele came from a parent of non-French-Canadian origin. Patients in three families carried TSD alleles different from any of the above mutations. The 5' deletion mutation clusters in persons originating in southeastern Quebec (Gaspé) and adjacent counties of northern New Brunswick.     

The use of microsatellites for detecting DNA polymorphism, genotype identification and genetic diversity in wheat

A set of 20 wheat microsatellite markers was used with 55 elite wheat genotypes to examine their utility (1) in detecting DNA polymorphism, (2)in the identifying genotypes and (3) in estimating genetic diversity among wheat genotypes. The 55 elite genotypes of wheat used in this study originated in 29 countries representing six continents. A total of 155 alleles were detected at 21 loci using the above microsatellite primer pairs (only 1 primer amplified 2 loci; all other primers amplified 1 locus each). Of the 20 primers amplifying 21 loci, 17 primers and their corresponding 18 loci were assigned to 13 different chromosomes (6 chromosomes of the A genome, 5 chromosomes of the B genome and 2 chromosomes of the D genome). The number of alleles per locus ranged from 1 to 13, with an average of 7.4 alleles per locus. The values of average polymorphic information content (PIC) and the marker index (MI) for these markers were estimated to be 0.71 and 0.70, respectively. The (GT)_n microsatellites were found to be the most polymorphic. The genetic similarity (GS) coefficient for all possible 1485 pairs of genotypes ranged from 0.05 to 0.88 with an average of 0.23. The dendrogram, prepared on the basis of similarity matrix using the UPGMA algorithm, delineated the above genotypes into two major clusters (I and II), each with two subclusters (Ia, Ib and IIa, IIb). One of these subclusters (Ib) consisted of a solitary genotype (E3111) from Portugal, so that it was unique and diverse with respect to all other genotypes belonging to cluster I and placed in subcluster Ia. Using a set of only 12 primer pairs, we were able to distinguish a maximum of 48 of the above 55 wheat genotypes. The results demonstrate the utility of microsatellite markers for detecting polymorphism leading to genotype identification and for estimating genetic diversity. Received: 15 May 1999 / Accepted: 27 July 1999     

Novel polymorphisms of goat growth hormone and growth hormone receptor genes and their effects on growth traits

In this study, the polymorphisms of growth hormone (GH) gene 5' promoter region and intron 8, exons 4 and 10 of growth hormone receptor (GHR) gene were analyzed in Xinong Saanen goats (SG) and Boer goats (BG). Two alleles (A and B) and three genotypes (AA, AB and BB) were detected at P1 locus of GH gene, and two alleles (G and T) and two genotypes (GG and GT) were detected at P4 locus of GHR gene by PCR-SSCP analysis. In addition, two single nucleotide polymorphisms (SNPs)-A73C (P1 locus) and G114T (P4 locus), were identified by DNA sequencing. The frequencies of alleles A and B in the two goat breeds were 0.61-0.62, and 0.39-0.38, respectively, and the frequencies of alleles G and T in the two goat breeds were 0.82-0.86, and 0.18-0.14, respectively. The SNP loci were in Hardy-Weinberg disequilibrium in both goat breeds (P<0.05). Polymorphisms of GH and GHR genes were shown to be associated with growth traits in BG breed. AA and GG genotypes were associated with superior growth traits in 1-, 2- and 3-month old individuals. Hence, AA and GG genotypes are suggested to be a molecular marker for superior growth traits in BG breed.     

A naturally occurring mutation of insulin receptor alanine 1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance

We have identified a previously undescribed genetic variant of the insulin receptor (Ala1134----Thr1134) in a family with the Type A syndrome of insulin resistance. Using the polymerase chain reaction to amplify insulin receptor cDNA and genomic DNA (exon 19), this mutation was detected in 1/2 alleles in the proband, her two affected sisters, and her affected father. Two normal alleles were present in the unaffected mother. No additional structural changes were encoded by the remainder of the proband's receptor cDNA. The Ala1134 mutant receptor was expressed in Chinese hamster ovary cells. The expressed mutant receptors were processed normally and displayed normal affinity of insulin binding but were markedly deficient in insulin-stimulated autophosphorylation. The mutant receptor was unable to catalyze the phosphorylation of the endogenous substrate, pp185, and insulin-stimulated kinase activity toward an exogenous substrate in vitro also was markedly impaired. Ala1134 is a highly conserved residue located in a consensus sequence found in most tyrosine kinases. It is likely that this previously uncharacterized residue and/or the immediate region surrounding it are important for normal kinase function in other members of this receptor family. This study also demonstrates that severe insulin resistance with dominant inheritance may be caused by a missense mutation in one allele of the insulin receptor gene.     

Mutation screening of 17 Japanese patients with neuropathic Gaucher disease

Using PCR and PCR-single strand conformation polymorphism (SSCP) we have identified gene mutations in 17 Japanese patients with neuropathic Gaucher disease (type 2, 9 cases; type 3, 8 cases). The L444P, F213I, D409H, and 1447 del 20 and 1447 ins TG mutations accounted for eight (type 2, 6; type 3, 2), seven (type 2, 2; type 3, 5), three (type 3), and three (type 2) alleles, respectively. Three alleles were unique. Ten alleles (type 2, 5; type 3, 5) could not be identified. The genotypes, D409H/?, L444P/?, L444P/F213I, and F213I/?, were identified in three, three, two, and two patients, respectively. Six patients had a unique genotype and none of the mutant alleles could be identified in one patient. The data indicate that the genotypes in Japanese patients with neuropathic Gaucher disease are found to be heterogeneous and the genotype prevalence and mutated alleles are unique.     

Restriction fragment-length polymorphisms of class II gene sequences in mice expressing minor structural variants of I-Ak and I-Ap

Serologic and structural analyses of the I-A molecules expressed among a large collection of wild mouse-derived H-2 haplotypes has led to the definition of "families" of I-A alleles which encode antigenically similar molecules that are identical in more than 90% of their tryptic peptides. Two of these families, denoted the I-Ak and I-Ap families, consist of 10 I-A alleles which encode I-A molecules whose structures are closely related to either I-Ap or I-Ak. The evolutionary relationships of the I-A alleles in these families were assessed by a molecular analysis of their genomic structures. The A alpha and A beta alleles within these I-A families were compared by analysis of restriction fragment-length polymorphisms (RFLP) detected at high stringency by Southern blot hybridization with DNA probes specific for either A alpha or A beta. The polymorphic restriction enzyme sites detected in this survey were distributed over more than 7 kb of genomic DNA surrounding each gene. Because both A alpha and A beta are encoded by about 700 bp of exon DNA, the majority of the restriction enzyme sites assayed by this RFLP analysis reflect polymorphisms in noncoding regions. The DNA sequence homologies of these alleles were estimated from the RFLP results with seven restriction endonucleases by calculating the fraction homologous value as defined previously. The results indicate that evolutionarily dissimilar I-A alleles can encode I-A molecules with very similar structures. The five I-A alleles in the I-Ak family could be divided into two discrete groups, denoted K1 and K2, on the basis of their restriction fragment (RF) genotypes. The RF genotypes of alleles within each group shared more than 80% of the restriction fragments for both A and A beta. In contrast, the RF genotypes of alleles in group K1 differed extensively from those in group K2, indicating that alleles in these separate groups may not be evolutionarily closely related. These observations suggest that gene conversion or intragenic recombinational events may have been involved in the evolution of groups K1 and K2 in the I-Ak family. The RF genotypes of alleles in the I-Ap family demonstrated a close evolutionary relationship among all but two of the alleles. These two alleles encoded I-A molecules whose structures were the least related to I-Ap of any of the alleles in the I-Ap family.(ABSTRACT TRUNCATED AT 400 WORDS)     

中国人胃癌和正常组织基因组DNA中癌基因Ha-ras的限制性片段长度多态性研究

本文报道以PHS-49为探针,分析了中国人群中36例胃癌患者癌组织和25位正常人体组织基因组DNA中Ha-ras基因的BamHI限制性片段长度多态性(RFLPs)。发现了10种不同长度的片段和18种基因型。其中4种小于6kb的BamHI片段是迄今国外未见报道的,这可能是中国人群遗传多态性的一个特征。此外,在胃癌组织中发现Ha-ras的一些稀有等位基因和基因型的频率明显高于正常人群。对两名胃癌患者家系中的11名成员也作了RFLPs分析,发现有些成员出现3条和4条限制性片段的杂合个体,表明这些个体的染色体上含有的Ha-ras基因不只一份拷贝。对上述现象的可能原因作了分析和讨论。     

Chromosome-specific desynapsis in the n = 2 race of Haplopappus gracilis (Compositae)

During cytological screening for pollen sterility in a wild population of Haplopappus gracilis (n = 2), several partially sterile plants were found that had good pachytene pairing but varying numbers of univalents. Some plants had chromosome A bivalents or A univalents, while in the same cells chromosome B had only bivalents. In other plants the reverse condition occurred; the B chromosome had B bivalents or B univalents and only A bivalents. This demonstrates a chromosome-specific effect for the desynapsis genes. Hybridization between the two homozygous mutant genotypes produced only normal bivalents; this indicates the two mutants are not alleles and each is recessive. An F2 generation showed independent assortment of the desynaptic mutations. The chromosome A bivalent is the larger of the two and normally has one or two chiasmata; the B bivalent normally has a single chiasma. Chiasmata distribution was tested in the desynaptic mutant A bivalents and showed an acceptable fit to a binomial distribution. This occurs also in heterozygous, asynaptic pairing control gene mutations. Analysis of the NOR bivalent in two hologenomic desynaptic mutations in tomato also showed a good fit to a binomial distribution of chiasmata. This indicates the same methods are applicable to diverse species.