A genetic testing code of practice for insurers.

The Association of British Insurers has issued a Genetic Testing Code of Practice. The document consists of a forward and six parts, namely; Code of Practice, Statement of Duties of our Insurance Company's Chief Medical Director, Statement of the ABI Genetics advisors responsibilities, Principals of the adjudication system, the legal and ethical framework and confidentiality guidelines.

Underwriting lethal genetic diseases.

There are thousands of single gene deposits that cause increased morbidity or mortality risks. Few have complete penetrance leading to certain death. Most can be underwritten with affordable increases in premium; many at standard rates. As we learn more about penetrance for specific mutations we can learn to be more aggressive in underwriting inherited risks. I have described approaches to underwriting untested applicants to Huntington disease, and tested applicants who carry dominant mutations leading to breast, ovarian and colon cancer.

Proposal: a position for the insurance industry on genetic testing.

Proposed legislation to limit the use of genetic test results in insurance underwriting has appeared with increasing frequency at the state and federal level. The proponents seek to protect consumers from unfair discrimination by insurers. Can we not develop a proactive approach, acknowledging that we do not need to do prospective testing while we assert that we must retain the current status of equivalency of information between the underwriter and the applicant?

GM2 ganglioside activator occurs in multiple forms.

The protein which activates the hydrolysis of GM2 ganglioside by hexosaminidase A was purified from human kidney. The GM2 activator had a molecular mass of 28 kDa by gel filtration and was resolved into three major bands using polyacrylamide gel electrophoresis in the presence of SDS with molecular masses of 23, 22 and 21 kDa. These three bands corresponded respectively to strongly binding, weakly binding and non-binding fractions of GM2 activator chromatographed through concanavalin A-Sepharose, indicating that GM2 activator exists in multiple glycosylated forms.

Classification of disorders of GM2 ganglioside hydrolysis using 3H-GM2 as substrate.

Rates of GM2 ganglioside hydrolysis by fibroblasts from normal controls and patients with GM2 gangliosidosis were measured in situ, with cells growing in tissue culture by assaying the decrease in cell-incorporated 3H-GM2 over time, and in vitro by assaying the rate of 3H-GM2 hydrolysis using fibroblast extracts in the presence of no additives, sodium taurocholate, and GM2 activator protein. In tissue culture, normal cells hydrolyzed cell-incorporated GM2 while fibroblasts from patients with GM2 gangliosidosis did not. The half life of GM2 in normal fibroblasts was 78 hours. In vitro, only normal fibroblast extracts hydrolyzed GM2 in the absence of additives. In the presence of 10 mM sodium taurocholate, rates of GM2 hydrolysis by normal fibroblast extracts were increased 5-16-fold, fibroblast extracts from AB and B1 variant patients hydrolyzed GM2 at normal rates, cell extracts from patients with Tay-Sachs disease hydrolyzed GM2 at nearly normal rates, and cell extracts from Sandhoff disease patients hydrolyzed GM2 at about 10% of normal rates. In the presence of 1 microgram of GM2 activator, rates of GM2 hydrolysis by normal fibroblast extracts were increased 8-25-fold, fibroblast extracts from a patient with the AB variant hydrolyzed GM2 at normal rates, and cell extracts from other variants of GM2 gangliosidosis did not hydrolyze GM2. The results suggest that measuring the persistence of 3H-GM2 in tissue culture over time will detect any variant of GM2 gangliosidosis and may be the ideal way to test for the presence of this disease. Variants can be distinguished by assaying the hydrolysis of 3H-GM2 using cell extracts in the absence of additives, with sodium taurocholate, and with activator.

Molecular heterogeneity in the infantile and juvenile forms of Sandhoff disease (O-variant GM2 gangliosidosis).

There are two major beta-hexosaminidase, EC 3.2.1.52, isozymes in normal human tissues. They exist as active dimers of alpha- and/or beta-subunits. A defect of their beta-subunit results in Sandhoff disease (O-variant GM2 gangliosidosis), an inherited, clinically heterogeneous, lysosomal storage disease. The status of the HEXB gene, pre beta-polypeptide chain mRNA, and residual beta-hexosaminidase activities were examined in a clinically and ethnically diverse collection of 16 fibroblast cell lines from patients with Sandhoff disease. Differentiation of the two major clinical types, infantile and juvenile onset, could be made by the determination of the activity of the residual beta-hexosaminidase eluting in the same pH range as hexosaminidase A. All the juvenile lines were found to have normal or reduced levels of pre beta-chain mRNA and no gross abnormalities in the HEXB gene. Of the 11 infantile type cell lines examined, four were found to contain no detectable pre beta-chain mRNA. Two cell lines in this group contained partial gene deletions localized to the 5' end of the HEXB gene. One of these cell lines has previously been assigned to the single complementation group in Sandhoff disease, conclusively demonstrating that the primary gene defect in the majority of Sandhoff cases is in the HEXB gene itself. These data suggest that each clinical group is made up of a collection of different HEXB mutations.

Isolation of cDNA clones coding for the alpha-subunit of human beta-hexosaminidase. Extensive homology between the alpha- and beta-subunits and studies on Tay-Sachs disease.

The lysosomal beta-hexosaminidases (N-acetyl-beta-glucosaminidase, EC 3.2.1.30) occur as two major isozymes, hexosaminidase A (alpha beta a beta b) and hexosaminidase B (2(beta a beta b)). To facilitate the investigations of the biosynthesis and structure of the enzymes and the nature of mutation in Tay-Sachs disease, we have isolated cDNA clones coding for the alpha-subunit. The polypeptide chains of hexosaminidase A (30 mg) were digested with trypsin, and peptides were isolated by reverse phase high pressure liquid chromatography and their amino acid sequences determined. One of alpha-chain peptides contained a string of seven amino acids from which two sets of oligonucleotides were specified. They were used to screen the SV40-transformed human fibroblast cDNA library of Okayama and Berg. Three cDNA clones, designated pHexA, identified from among 5 X 10(5) clones screened, contained the deduced amino-acid sequences of five alpha-chain peptides. Genomic DNA homologous to pHexA cDNA mapped to human chromosome 15 in somatic cell hybrids, as expected for the pre-alpha-polypeptide. Two of the clones contained identical polyadenylation sites, while the third was polyadenylated about 450 base pairs downstream. The two types of clones were found to correspond to a major 2.0-kilobase pair and a minor 2.3-kilobase pair mRNA species. Blot hybridizations of mRNA and DNA from Tay-Sachs variant fibroblasts revealed absence or reduction of levels of both mRNA species among infantile and juvenile variants, but no observable DNA alterations. Alignment of the pre-alpha- and pre-beta-polypeptides revealed 55% nucleotide and 57% amino acid homology. These data suggest a common origin of the HEXA and HEXB genes and account for the similar substrate specificities of the alpha-dimer subunit, hexosaminidase S, and hexosaminidase B.

Characterization by nuclear magnetic resonance of the concanavalin A binding oligosaccharide on the beta b chain of placental beta-hexosaminidase B: lectin binding to the separated polypeptide chains of hexosaminidases A and B.

The type and distribution of the oligosaccharides on each polypeptide of human placental beta-hexosaminidases A (alpha (beta a beta b)) and B (2(beta a beta b)) were examined. The denatured polypeptides were separated by isoelectric focussing in a polyacrylamide slab gel and each gel was then overlaid with 125I-labelled lectins. The study indicated that the beta a chain contains negligible carbohydrate, the beta b chain contains both the high-mannose and a complex type oligosaccharide, and the alpha chain contains predominantly high-mannose or hybrid type moieties. Two asparagine-linked high-mannose type oligosaccharides present on the beta b polypeptide of beta-hexosaminidase B were isolated by concanavalin A chromatography and by reverse-phase high pressure liquid chromatography. Proton nuclear magnetic resonance characterization of the oligosaccharides revealed an equimolar glycan mixture of the high-mannose type structure Man5 and Man6.

HPLC analysis of urinary sulfatide: an aid in the diagnosis of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) presents as six separate variant forms, four allelic and two non-allelic. It is diagnosed in the laboratory by a decrease in the fibroblast or leukocyte arylsulfatase A activity, generally against an artificial substrate. Since residual enzyme activity is not always an indicator of presence or absence of disease, it may be helpful to supplement this information with that of the presence or absence of sulfatide storage in the body. We have improved the HPLC analysis of sulfatide by the use of a sulfated internal standard, sulfatoxymonoalkylmonoacylgalactosylglycerol. Normal urines contain approximately 0 to 0.2 nmol sulfatide/mg creatinine, whereas MLD urines may contain 5 to 7.5 nmol/mg. There is no increase in plasma sulfatide compared to controls in the age group of MLD patients which we studied (up to 4 years).

Discrimination of the isozymes of human placental hexosaminidase by kinetic parameter estimation.

Human placental beta-N-acetylhexosaminidase (EC 3.2.1.52) (HEX) is a lysosomal glycosyl hydrolase with an acidic pH optimum. Four isozymes (HEX B, HEX I1, HEX I2, and HEX A) have been isolated from human placenta. HEX BA derived from the subunit rearrangement of HEX A was also prepared. To determine if the isozymes of hexosaminidase differ in their kinetic parameters, the conditions for 4-methylumbelliferyl-beta-D-N-acetylglucosaminide hydrolysis were optimized for each isozyme. The Km values were essentially similar and varied from 0.64 +/- 0.06 for HEX BA to 0.85 +/- 0.13 for HEX I1. The Vmax values were similar only for HEX I1 (3.90 +/- 0.28 kat kg-1) and HEX I2 (4.40 +/- 0.17). Vmax values varied significantly between HEX A (9.68 +/- 0.52), HEX B (8.00 +/- 0.75), HEX BA (4.81 +/- 0.17), and the HEX I values.

A statistical comparison of parameter estimation for the Michaelis-Menten kinetics of human placental hexosaminidase.

To enable the most effective method of kinetic discrimination between a group of isozymes such as those of human placental hexosaminidases (HEX), three methods estimating the parameters of the Michaelis-Menten equation were evaluated. Computer-simulated experiments were performed under various conditions. They indicated that, in the presence of constant absolute or relative errors, the method of unweighted nonlinear least squares yielded slightly more precise and accurate parameters than the method of the direct linear plot. Parameters calculated from the Lineweaver-Burk plot were very imprecise and inaccurate. The direct linear plot was comparatively resistant to outlier observations; however, only when outliers were substantial did the method become superior to nonlinear least squares. The calculation of a confidence limit is necessary for the evaluation of any resulting differences in the kinetic parameters for a set of isozymes. This can easily be calculated from either the Lineweaver-Burk plot or the nonlinear least-squares method. However, those calculated from the Lineweaver-Burk plot are biased, especially at higher levels of experimental errors. Therefore, the nonlinear least-squares method is the one most suited for the discrimination of a group of enzymes based on their kinetic parameters.

Isolation of cDNA clones coding for the beta subunit of human beta-hexosaminidase.

The major forms of beta-hexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) occur as multimers of alpha and beta chains--hexosaminidase A (alpha beta a beta b) and hexosaminidase B 2(beta a beta b). To facilitate the investigation of beta-chain biosynthesis and the nature of mutation in Sandhoff disease, a human hexosaminidase beta-chain cDNA clone was isolated. Hexosaminidase B (10 mg) was treated with CNBr, five peptide fragments were isolated by reverse-phase HPLC, and their amino acid sequences were determined. One of these contained a string of six amino acids from which an oligonucleotide probe was defined. The simian virus 40-transformed human fibroblast cDNA library of Okayama and Berg was screened by colony hybridization with the radiolabeled probe. Thirteen probe-binding clones were selected out of 50,000 clones screened. Four of these designated pHex were shown to be identical at their 3' ends by restriction enzyme mapping, differing only in their 5' extensions (1.4-1.7 kilobases). The nucleotide sequence of a 174-base-pair segment contained the deduced amino acid sequence of two of the five CNBr peptides, indicating that the pHex clones encode the beta subunit of hexosaminidase. In addition, pHex cDNA was found homologous to multiple bands in digests of genomic human DNA totaling 43 kilobases (kb), all of which were mapped to chromosome 5 in somatic cell hybrids, as expected of the HEXB gene. The pHex cDNA also hybridized to a 2.2-kilobase RNA that apparently codes for the pre-beta-polypeptide of hexosaminidase. This RNA species was absent in the fibroblasts of one of three patients with Sandhoff disease examined. We anticipate that these clones will be of value to diagnosis and carrier detection of Sandhoff disease in affected families.