Genotyping an immunodeficiency causing c.1624-11G>A ZAP70 mutation in Canadian Mennonites.

BACKGROUND: Primary immunodeficiency is a life-threatening genetic disease that appeared to have an increased incidence in Manitoba Mennonites. Determining the genetic basis of this immunodeficiency was an essential step for providing early and appropriate medical intervention. METHODS: Initially, DNA from probands affected with primary immunodeficiency and their family members was assessed for linkage to genes previously associated with immunodeficiency. Candidate genes were sequenced to identify the causative mutation. The frequency of the mutation among first and second degree relatives, as well as apparently unrelated community members was analyzed using a PCR-based assay. RESULTS: A previously described c.1624-11G>A mutation in ZAP70 was identified as the causative mutation in all affected probands that were analyzed. Among 125 study participants of Mennonite descent, 79 genotyped as normal, 39 were carriers and seven were affected. None of 115 non-Mennonite random individuals carried the mutation, whereas one of ten random DNA samples from individuals who self-identified as Mennonite was a carrier. CONCLUSIONS: In collaboration with the target community, we have developed a robust screening test for determining ZAP70 genotype. Early identification of affected individuals has provided an opportunity for timely clinical intervention, while carrier identification has allowed for genetic counselling of at risk couples.

Molecular basis of succinylcholine sensitivity in a prairie Hutterite kindred and genetic characterization of the region containing the BCHE gene.

The tetrameric glycoprotein butyrylcholinesterase (BChE; EC 3.1.1.8) is one of two enzymes that hydrolyze choline esters. The controlling gene (BCHE) is comprised of four coding exons and is located on chromosome 3q26. Based on BChE activity measurements in the presence and absence of dibucaine, usual (designated U) and atypical (designated A) gene products have been distinguished. Homozygotes for the A gene product are at risk for prolonged apnea following exposure to the surgical anesthetics succinylcholine or mivacurium. In this report, we detail biochemical and molecular investigations of succinylcholine sensitivity in a prairie Hutterite kindred. Our results establish that BChE activities in the family members are impacted by two distinct BCHE mutations, namely, c.209A>G p. D70G and c.1615G>A p. A539T. However, homozygotes for the c.209A>G mutation (i.e., atypical or A) are the only individuals whose BChE activity could lead to adverse reactions to succinylcholine. Interestingly, haplotype analysis of the chromosomal region containing BCHE indicates that the c.209A>G mutation is carried on a unique haplotype, suggesting that it was likely introduced into the population only once. Conversely, the c.1615G>A mutation is carried on various haplotypes and was likely introduced into the population more than once.

Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins.

To identify proteins interacting in the insulin-signaling pathway that might define new pathways or regulate existing ones, we have employed the yeast two-hybrid system. In a two-hybrid screen of a human liver cDNA library, we identified the human growth factor receptor bound 14 (hGrb14) adaptor protein as a partner of the activated insulin receptor. Additional analysis of the insulin receptor--hGrb14 interaction in the yeast two-hybrid system revealed that the SH2 domain of hGrb14 was not the sole region involved in binding the activated insulin receptor. The insulin-stimulated interaction between hGrb14 and the insulin receptor was also observed in different mammalian cultured cell lines. This association was detected at 1 min of insulin stimulation and was maximal at 10 nM and greater concentrations of insulin. Chinese hamster ovary cells stably expressing the insulin receptor (CHO-IR) and hGrb14 were used to examine the effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation of proteins; in general, increasing levels of hGrb14 expression resulted in a reduction in tyrosine phosphorylation. This decrease was demonstrated for the specific proteins src homology-containing and collagen-related protein (Shc), insulin receptor substrate-1 (IRS-1), and Downstream of tyrosine Kinase (Dok). The broad effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation suggest that it acts early in the insulin-signaling pathway.

Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase.

beta-Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of beta-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. Heritable deficiency of this enzyme results in various forms of GalNAc-beta(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-beta(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs disease. We have determined the x-ray crystal structure of a beta-hexosaminidase from Streptomyces plicatus to 2.2 A resolution (Protein Data Bank code ). beta-Hexosaminidases are believed to use a substrate-assisted catalytic mechanism that generates a cyclic oxazolinium ion intermediate. We have solved and refined a complex between the cyclic intermediate analogue N-acetylglucosamine-thiazoline and beta-hexosaminidase from S. plicatus to 2.1 A resolution (Protein Data Bank code ). Difference Fourier analysis revealed the pyranose ring of N-acetylglucosamine-thiazoline bound in the enzyme active site with a conformation close to that of a (4)C(1) chair. A tryptophan-lined hydrophobic pocket envelopes the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr(393) and Asp(313) appear important for positioning the 2-acetamido group of the substrate for nucleophilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization. This complex provides decisive structural evidence for substrate-assisted catalysis and the formation of a covalent, cyclic intermediate in family 20 beta-hexosaminidases.

Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX.

Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysaccharidosis IX, was recently described. This patient had a surprisingly mild clinical phenotype, including notable periarticular soft tissue masses, mild short stature, an absence of neurological or visceral involvement, and histological and ultrastructural evidence of a lysosomal storage disease. To determine the molecular basis of mucopolysaccharidosis IX, we analyzed two candidate genes tandemly distributed on human chromosome 3p21.3 and encoding proteins with homology to a sperm protein with hyaluronidase activity. These genes, HYAL1 and HYAL2, encode two distinct lysosomal hyaluronidases with different substrate specificities. We identified two mutations in the HYAL1 alleles of the patient, a 1412G --> A mutation that introduces a nonconservative amino acid substitution (Glu268Lys) in a putative active site residue and a complex intragenic rearrangement, 1361del37ins14, that results in a premature termination codon. We further show that these two hyaluronidase genes, as well as a third recently discovered adjacent hyaluronidase gene, HYAL3, have markedly different tissue expression patterns, consistent with differing roles in HA metabolism. These data provide an explanation for the unexpectedly mild phenotype in mucopolysaccharidosis IX and predict the existence of other hyaluronidase deficiency disorders.

Structural and functional characterization of Streptomyces plicatus beta-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis.

We have sequenced the Streptomyces plicatus beta-N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human beta-N-acetylhexosaminidases whose deficiency results in various forms of GM2 gangliosidosis. Based upon the x-ray structure of Serratia marcescens chitobiase (SmChb), we generated a three-dimensional model of SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human beta-N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selected and mutated putative SpHex active site residues. Arg162 --> His mutation increased Km 40-fold and reduced Vmax 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human beta-N-acetylhexosaminidase A (HexA) and Arg349 in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex. Glu314 --> Gln reduced Vmax 296-fold, reduced Km 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246 --> Asn reduced Vmax 2-fold and increased Km only 1.2-fold, suggesting that Asp246 may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the x-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.

W474C amino acid substitution affects early processing of the alpha-subunit of beta-hexosaminidase A and is associated with subacute G(M2) gangliosidosis.

Mutations in the HEXA gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), that abolish Hex A enzyme activity cause Tay-Sachs disease (TSD), the fatal infantile form of G(M2) gangliosidosis, Type 1. Less severe, subacute (juvenile-onset) and chronic (adult-onset) variants are characterized by a broad spectrum of clinical manifestations and are associated with residual levels of Hex A enzyme activity. We identified a 1422 G-->C (amino acid W474C) substitution in the first position of exon 13 of HEXA of a non-Jewish proband who manifested a subacute variant of G(M2) gangliosidosis. On the second maternally inherited allele, we identified the common infantile disease-causing 4-bp insertion, +TATC 1278, in exon 11. Pulse-chase analysis using proband fibroblasts revealed that the W474C-containing alpha-subunit precursor was normally synthesized, but not phosphorylated or secreted, and the mature lysosomal alpha-subunit was not detected. When the W474C-containing alpha-subunit was transiently co-expressed with the beta-subunit to produce Hex A (alphabeta) in COS-7 cells, the mature alpha-subunit was present, but its level was much lower than that from normal alpha-subunit transfections, although higher than in those cells transfected with an alpha-subunit associated with infantile TSD. Furthermore, the precursor level of the W474C alpha-subunit was found to accumulate in comparison to the normal alpha-subunit precursor levels. We conclude that the 1422 G-->C mutation is the cause of Hex A enzyme deficiency in the proband. The resulting W474C substitution clearly interferes with alpha-subunit processing, but because the base substitution falls at the first position of exon 13, aberrant splicing may also contribute to Hex A deficiency in this proband.

Limb girdle muscular dystrophy in Manitoba Hutterites does not map to any of the known LGMD loci.

Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of disorders affecting primarily the shoulder and pelvic girdles. Autosomal dominant and recessive forms have been identified; 8 have been mapped and 1 more has been postulated on the basis of exclusion of linkage. An autosomal recessive muscular dystrophy was first described in 1976 in the Hutterite Brethren, a North American genetic and religious isolate [Shokeir and Kobrinsky, 1976; Clin Genet 9:197-202]. In this report, we discuss the results of linkage analysis in 4 related Manitoba Hutterite sibships with 21 patients affected with a mild autosomal recessive form of LGMD. Because of the difficulties in assigning a phenotype in some asymptomatic individuals, stringent criteria for the affected phenotype were employed. As a result, 7 asymptomatic relatives with only mildly elevated CK levels were assigned an unknown phenotype to prevent their possible misclassification. Two-point linkage analysis of the disease locus against markers linked to 7 of the known LGMD loci and 3 other candidate genes yielded lod scores of < or = -2 at theta = 0.01 in all cases and in most cases at theta = 0.05. This suggests that there is at least 1 additional locus for LGMD.

Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system.

The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.

Benign HEXA mutations, C739T(R247W) and C745T(R249W), cause beta-hexosaminidase A pseudodeficiency by reducing the alpha-subunit protein levels.

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha-subunit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alphaalpha) and Hex A (alphabeta) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta-N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha-subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.

Heterozygosity for Tay-Sachs and Sandhoff diseases among Massachusetts residents with French Canadian background.

OBJECTIVES: The frequency of Tay-Sachs disease (TSD) heterozygosity is increased among French Canadians in eastern Quebec. A large proportion of the New England population has French Canadian heritage; thus, it is important to determine if they too are at increased risk for TSD heterozygosity. This prospective study was designed to assess the TSD heterozygote frequency among people with French Canadian background living in Massachusetts. A simultaneous screen for heterozygosity for Sandhoff disease, a related genetic disorder, was also undertaken. METHODS: 1260 non-pregnant subjects of French Canadian background were included in the study. beta hexosaminidase activity was measured in blood samples, and results were evaluated for TSD and Sandhoff disease heterozygosity. Samples from the TSD heterozygotes were also subjected to mutation analysis. RESULTS: Of the 1260 samples studied, 22 (1 in 57; CI 1 in 41, 1 in 98) were identified as TSD heterozygotes by enzymatic analyses and 11 subjects (1 in 114; CI 1 in 72, 1 in 280) were identified as Sandhoff disease heterozygotes. Three of the 22 TSD heterozygotes were found to have benign pseudodeficiency mutations, resulting in a maximum TSD heterozygote frequency of 19 in 1260 (1 in 66; CI 1 in 46, 1 in 120). Together, these data provide a maximum frequency of heterozygosity for TSD or Sandhoff disease of 30 in 1260 (1 in 42; CI 1 in 31, 1 in 64) in this population. CONCLUSIONS: Simultaneous screening for TSD and Sandhoff disease heterozygosity by assay of beta hexosaminidases A and B activities provides a possible method for use with subjects of French Canadian background. The relevance of some of the novel mutations identified in this group needs further study. However, the comparatively high combined frequency of TSD and Sandhoff disease heterozygosity indicates a need for discussion regarding the appropriateness of carrier testing for these disorders for persons of French Canadian background in Massachusetts.

An A-to-G mutation at the +3 position of intron 8 of the HEXA gene is associated with exon 8 skipping and Tay-Sachs disease.

Tay-Sachs disease (TSD) results from a deficiency of beta-hexosaminidase A (EC 3.2.1.52) activity. A child with late-infantile TSD was found to have two HEXA mutations, 986 + 3A-->G (A-->G at the +3 position of intron 8) and 533G-->A, associated with the variant B1 form of TSD. We were able to detect exon 8-deleted, but no correctly spliced HEXA mRNA, from the non-533G-->A allele in this patient. This suggests that 986 + 3A-->G results in missplicing and, together with 533G-->A, TSD.

Mutational analyses of Tay-Sachs disease: studies on Tay-Sachs carriers of French Canadian background living in New England.

Tay-Sachs disease (TSD) results from mutations in HEXA that cause Hex A deficiency. Heterozygote-screening programs have been applied in groups with an increased TSD incidence, such as Ashkenazi Jews and French Canadians in Quebec. These programs are complicated by benign mutations that cause apparent Hex A deficiency but not TSD. Benign mutations account for only approximately 2% of Jewish and approximately 36% of non-Jewish enzyme-defined carriers. A carrier frequency of 1/53 (n = 1,434) was found in an ongoing prospective analysis of persons of French Canadian background living in New England by using an enzyme-based assay. DNA from enzyme-defined carriers from this population was analyzed to determine the molecular basis of Hex A deficiency. Samples (36) were tested for common mutations, and samples that were negative for these were screened for uncommon or novel mutations by using SSCP analysis. Exons showing mobility shifts were sequenced, and most mutations were confirmed by restriction enzyme digestion. Known disease-causing mutations were found in nine samples (four had a 7.6-kb deletion found in 80% of French Canadian TSD alleles), and known benign mutations were found in four samples. Seven novel changes were identified, including G748A in four samples. The molecular basis of Hex A deficiency in this carrier population differs from that of French Canadian TSD patients. Screening centers should be aware of the presence of benign mutations among U.S. French Canadians or Franco-Americans.(ABSTRACT TRUNCATED AT 250 WORDS)

PCR-based analysis of voltage-gated K+ channels in vascular smooth muscle.

Irregularities in K+ currents form the basis of several cardiovascular dysfunctions, among which are arrhythmias and vasospasms. The developmental regulation of voltage-gated K+ channels, however, has been difficult to study. A novel approach was therefore employed to examine these channels in muscle tissue. Primers for a PCR-based analysis were designed using published nucleic acid sequences for voltage-gated K+ channels. Final selection of the primer pairs was based on the homology present in the S4 and H5 transmembrane domains. A specific band was amplified with these primers using RNA isolated from both rat A10 vascular smooth muscle cells and rat heart tissue.