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PubMed1976

In vitro correction of Hurler fibroblasts with bovine testicular hyaluronidase.

Bovine testicular hyaluronidase (endo-beta-N-acetyl hexosaminidase) has a significant corrective effect on cultured Hurler fibroblasts. Nonspecificity of this effect is indicated by its equally strong corrective effect on Hunter fibroblasts. Although all specimens of hyaluronidase also possessed iduronidase activity, a separate corrective effect could be attributed to the endo-N-acetyl hexosaminidase activity of at least one hyaluronidase (Wyeth M-151) for four reasons: (i) its very low content of iduronidase activity; (ii) a decrease in intracellular macromolecular mucopolysaccharides (believed to be largely dermatan sulfate) with a corresponding increase in intracellular and extracellular oligosaccharides; (iii) no measurable increase in iduronidase activity of hyaluronidase-treated cells despite near maximal correction; (iv) direct correlation between Hurler cell correction and hyaluronidase activity when enzymes of different strength were used at less than maximal correction.

Animals↗
PubMed1981

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors.

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.

Animals↗
PubMed1998

Hurler syndrome: II. Outcome of HLA-genotypically identical sibling and HLA-haploidentical related donor bone marrow transplantation in fifty-four children. The Storage Disease Collaborative Study Group.

Untreated patients with Hurler syndrome (MPSIH) experience progressive neurologic deterioration and early death. Allogeneic bone marrow transplantation (BMT) ameliorates or halts this course. The Storage Disease Collaborative Study Group was formed to evaluate the effectiveness and toxicity of BMT. Effectiveness was defined as engrafted survival with continuing cognitive development. Fifty-four patients deficient in leukocyte alpha-L-iduronidase enzyme activity (median age, 1.8 years; range, 0.4 to 7.9) received high-dose chemotherapy with or without irradiation and BMT from HLA-genotypically identical sibling (GIS) or HLA-haploidentical related (HIR) donors between September 16, 1983 and July 14, 1995; all children were included in this report. Thirty-nine of 54 patients (72%) engrafted following the first BMT. The probability of grade II to IV acute graft-versus-host disease (GVHD) at 100 days was 32% for GIS and 55% for HIR patients. The probability of extensive chronic GVHD was 0% for GIS and 24% for HIR patients. The actuarial probability of survival at 5 years was 64% for all patients, 75% for GIS patients, 53% for HIR patients, and 53% for patients with donor marrow engraftment. The baseline Mental Developmental Index (MDI) was examined both for children less than and greater than 24 months of age at BMT. Children transplanted before 24 months had a mean baseline MDI of 78, while those transplanted after 24 months had a mean baseline MDI of 63 (P = . 0002). Both baseline and post-BMT neuropsychologic data were available for 26 of 30 engrafted survivors. Of 14 patients transplanted before 24 months of age, nine demonstrated developmental trajectories that were normal or somewhat slower than normal. In contrast, of 12 patients transplanted after 24 months of age, only three showed developmental trajectories that were normal or somewhat slower than normal (P = .01). For children with a baseline MDI greater than 70, there was a significant correlation between the MDI at follow-up study and leukocyte alpha-L-iduronidase enzyme activity (P = .02). Children were more likely to maintain normal cognitive development if they were fully engrafted following BMT from a donor with homozygous normal leukocyte alpha-L-iduronidase enzyme activity. Children who developed acute GVHD of grade II or worse had significantly poorer cognitive outcomes (P < .009). No difference in the post-BMT MDI was observed between patients whose preparative therapies did (n = 10; radiation dose, 300 to 1,400 cGy) or did not (n = 16) include radiation. We conclude that MPSIH patients, particularly those less than 24 months of age with a baseline MDI greater than 70, can achieve a favorable long-term outcome with continuing cognitive development and prolonged survival after successful BMT from a related donor with homozygous normal enzyme activity.

Bone Marrow Transplantation↗
PubMed1993

Long-term outcome of Hurler syndrome following bone marrow transplantation.

Previous reports suggested a therapeutic response of lysosomal storage diseases such as Hurler syndrome following bone marrow transplantation. However, a clearer understanding of outcome has awaited long-term follow-up. We evaluated prospectively 11 consecutive patients with Hurler syndrome receiving marrow from an HLA-identical sib donor between September 1983-October 1988. Follow-up evaluations included assessment of donor engraftment by restriction fragment polymorphism analysis, determination of leukocyte alpha-L-iduronidase level, measurement of lumbar cerebrospinal fluid (CSF) pressure, computerized tomography (CT) of the brain, and psychometric testing. In this series there was a survival rate of 9/11 (82%) with all survivors showing complete (7 patients) or partial (2 patients) donor engraftment. Prospective longitudinal evaluation of the 9 surviving children, now 3.8-8.9 years posttransplantation (median 5.5) demonstrated persistence of previously deficient leukocyte alpha-L-iduronidase at levels reflecting the donor genotype and degree of donor engraftment. Urinary glycosaminoglycan excretion declined to near-normal within 5 months of donor engraftment. Prior to treatment, 7 of 8 children studied were found to have occult intracranial hypertension (lumbar CSF pressure > 20 cm CSF); however, all surviving children attained normal or near-normal pressure within 18 months of donor engraftment. Longterm follow-up CT imaging of the brain did not show progressive volume loss (cerebral atrophy) after donor engraftment. Of 9 survivors, 4 children having a developmental quotient (DQ, Mental Development Index on Bayley Scales of Infant Development) above 80 prior to transplantation subsequently maintained IQ scores above this level. However, 5 patients with lower pretransplant DQ scores now have significant cognitive deficits and attention deficit hyperactivity disorder. Progressive brain damage resulting from communicating hydrocephalus may be prevented by successful engraftment. Early transplantation of children with Hurler syndrome who have normal intelligence is likely to have the clearest benefit because long-term intellectual outcome will be limited by brain damage which has occurred prior to treatment.

Bone Marrow Transplantation↗
PubMed1995

Molecular genetics of mucopolysaccharidosis type I: diagnostic, clinical, and biological implications.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive disease caused by mutations in the alpha-L-iduronidase (IDUA) gene. These mutations lead to a deficiency of the glycosidase alpha-L-iduronidase (IDUA), which is required for the degradation of heparan sulphate and dermatan sulphate and thus the storage of these glycosaminoglycans in the lysosome. There is a wide range of clinical phenotypes in MPS-I (eponyms: Hurler syndrome, severe; Hurler/Scheie syndrome, intermediate; Scheie syndrome, mild), which makes prediction of disease severity and genetic counselling difficult. However, since cloning of the IDUA gene, mutation analysis has provided some molecular explanations for the range of MPS-I phenotypes, in turn facilitating the selection and evaluation of patients undergoing experimental treatment protocols such as bone marrow transplantation. A total of 46 mutations now have been defined for MPS-I consisting of 8 nonsense mutations, 21 missense mutations, 3 splice site mutations, and 14 minor deletions and/or insertions. Furthermore, 30 polymorphisms or nonpathogenic sequence variants have been defined, including 7 amino acid substitutions. Among patients of European origin, there are two major MPS-I mutations and a number of less frequent mutations. It is possible to follow mutation analysis of 292 patients, which can be divided into eight main patient groups of different ethnic and/or geographic origin with significant variation in mutant allele frequencies. A complex picture of molecular heterogeneity is emerging, building a valuable database for genotype/phenotype correlation. Mutation analysis is also providing some of the first clues into the structure and function of IDUA.

Codon, Nonsense↗
PubMed1983

Prenatal diagnosis of the Hurler syndrome: report on 40 pregnancies at risk.

In 40 pregnancies at risk for the Hurler syndrome 13 affected fetuses were detected by the demonstration of an alpha-L-iduronidase deficiency and an increased level of 35S-sulphate incorporation. The diagnosis were confirmed by the analysis of fetal tissues and/or cultured fetal skin fibroblasts. Microassays for alpha-L-iduronidase, using phenyl alpha-L-iduronide and more recently 4-methyl-umbelliferyl alpha-L-iduronide, enabled a reliable diagnosis to be made within 15 to 18 days after amniocentesis. 35S-sulphate incorporation has been a valuable adjunct in cases with a low (heterozygote) enzyme activity.

Amniocentesis↗
PubMed2001

Enzyme replacement therapy in feline mucopolysaccharidosis I.

Enzyme replacement therapy (ERT) has long been considered an approach to treating lysosomal storage disorders caused by deficiency of lysosomal enzymes. ERT is currently used to treat Gaucher disease and is being developed for several lysosomal storage disorders now that recombinant sources of the enzymes have become available. We have continued development of ERT for mucopolysaccharidosis I (MPS I) using the feline model. Recombinant alpha-L-iduronidase was administered intravenously at low dose (approximately 0.1 mg/kg or 25,000 units/kg) to four cats and high dose (0.5 mg/kg or 125,000 units/kg) to two cats on a weekly basis for 3- or 6-month terms. Clinical examinations showed distinct clearing of corneal clouding in one cat although clinical effects in the others were not evident. Biochemical studies of the cats showed that the enzyme was distributed to a variety of tissues although the liver and spleen contained the highest enzyme activities. Glycosaminoglycan storage was decreased in liver and spleen, and the histologic appearance improved in liver, spleen, and renal cortex. Enzyme was not consistently detected in cerebral cortex, brainstem, or cerebellum and the histologic appearance and ganglioside profiles did not improve. A variety of other tissues showed low variable uptake of enzyme and no distinct improvement. IgG antibodies to alpha-L-iduronidase were observed in five cats with higher titers noted when higher doses were administered. Mild complement activation occurred in three cats. Enzyme replacement therapy was effective in reversing storage in some tissues at the biochemical and histologic level in MPS I cats but an improved tissue distribution and prevention of a significant immune response could make the therapy more effective.

Animals↗
PubMed1980

Hybridization studies of fibroblasts from Hurler, Scheie, and Hurler/Scheie compound patients: support for the hypothesis of allelic mutants.

Heterokaryons were made by fusion of alpha-L-iduronidase-deficient fibroblasts from patients with the Hurler, Scheie, or Hurler/Scheie compound syndrome. The fused cell populations remained alpha-L-iduronidase deficient and accumulated 35S-labeled glycosaminoglycans (35S-GAG) to the same extent as the parental cells strains. Also, when 35S-GAG accumulation was studied by autoradiography at the level of single bi- and multinuclear hybrid cells, no evidence was found for genetic complementation. The results support the hypothesis that the mutations in the Hurler and Scheie syndromes are allelic, and they are compatible with the view that patients with intermediate phenotypes represent genetic compounds.

Alleles↗
PubMed1979

Hurler-Scheie phenotype: a report of two pairs of inbred sibs.

Four cases from two families with dermatan sulfate mucopolysacchariduria who lack alpha-L-iduronidase in peripheral leukocytes are described. The clinical and roentgenographic features of these cases represent an intermediate phenotype between Hurler's syndrome and Scheie's syndrome, and both parents in each family are first cousins. In the presence of parental consanguinity, a phenotypic variation or a third mutant allele at the iduronidase locus seems to be a more reasonable explanation for these cases than a genetic compound.

Adult↗
PubMed1981

Prenatal diagnosis of Hurler's syndrome--biochemical studies on the affected fetus.

A prenatal diagnosis of Hurler's syndrome was made in a pregnancy at risk in a family with two affected children. The fetus was diagnosed as having Hurler's syndrome on the basis of a deficiency of alpha-L-iduronidase in the cultured amniotic cells. The glycosaminoglycans (GAG) content in the supernatant of the amniotic fluid was increased about 1.5 fold compared with that in the control, and increases of heparan sulfate and dermatan sulfate were observed on electrophoresis. The diagnosis could be confirmed by the deficiencies of alpha-L-iduronidase in the liver and brain from the affected fetus. GAG content in the liver from the affected fetus was increased approximately 10 fold as compared with that in the control fetal liver, and most of the GAG were degraded. The GAG content was observed to be increased two fold in the brain, and dermatan sulfate, which was not detected in normal fetal brain, was identified. beta-Galactosidase activities in the affected liver and brain were decreased to 30-50% of the control, and an altered hexosaminidase A was also observed in the liver.

Amniotic Fluid↗
PubMed1993

Long-term clinical progress in bone marrow transplanted mucopolysaccharidosis type I patients with a defined genotype.

Two mucopolysaccharidosis type I (MPS-I) patients, subjected to bone marrow transplantation (BMT) more than 10 years ago, have recently had their alpha-L-iduronidase genotypes defined. Both patients, homozygous for the relatively common W402X mutation, received BMT when they were 14 and 11 months of age, and are now 12 and 14 years old, respectively. Untreated MPS-I patients, homozygous for W402X, have an extremely severe clinical phenotype with rapid clinical deterioration and death before 6 years of age. The 12-year-old patient, with limited mobility, is coping well at school, while the other patient is wheelchair-bound with severe disability in his lower limbs, and attends a school for the physically handicapped. Both patients have less than normal intelligence with slowly continuing losses. A third MPS-I patients, diagnosed at the age of 6 months, was felt, prior to BMT at 14 months, to have a severe phenotype. Twelve years post-BMT, he is ambulatory, albeit with restricted movement, and has normal intelligence. This patient did not have a defined MPS-I genotype and had alpha-L-iduronidase protein and activity consistent with a less severe outcome than the first two patients. We conclude that BMT has significantly slowed down the clinical regression of the W402X phenotype. We propose that if further gains are to be made, BMT should be performed within the first few months of life. Early diagnosis is therefore essential.

Adolescent↗
PubMed1986

Neuropathological and clinical correlations in Hurler disease.

We report studies on two patients (1 and 2) with Hurler disease. They both had all of the non-neurological features of Hurler disease to a similar and extreme degree and similar signs of brain damage on computed tomography. However, intellectual function was unusually well-preserved in patient 1, but seriously and typically impaired in patient 2. The reason for this discrepancy has been investigated by reference to the neuropathological findings, the results of alpha-L-iduronidase assays using different substrates and comparisons to other cases (patients 3 and 4). We suggest that patient 1 is an unusual variant of the disease who may have had a very low residual alpha-L-iduronidase activity in neuronal cells only, and that this could not be demonstrated by either enzyme assays on whole brain using the 4-methylumbelliferyliduronide substrate (Crow et al., 1983) or in studies on fibroblast lysates using a radioactive disaccharide substrate.

Brain↗
PubMed2001

Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations.

The lysosomal storage disorder, mucopolysaccharidosis type I (MPS I), is caused by a deficiency of the enzyme alpha-L-iduronidase, which is involved in the breakdown of dermatan and heparan sulphates. There are three clinical phenotypes, ranging from the Hurler form characterised by skeletal abnormalities, hepatosplenomegaly and severe mental retardation, to the milder Scheie phenotype where there is aortic valve disease, corneal clouding, limited skeletal problems, but no mental retardation. In this study, 85 MPS I families (73 Hurler, 5 Hurler/Scheie, 7 Scheie) were screened for 9 known mutations (Q70X, A75T, 474-2a>g, L218P, A327P, W402X, P533R, R89Q, 678-7g>a). W402X was the most frequent mutation in our population (45.3%) and Q70X was the second most frequent (15.9%). In 30 families, either one or both of the mutations were not identified, which accounted for 25.9% of the total alleles. Therefore, all 14 exons of the alpha-L-iduronidase gene were screened in these patients and 23 different sequence changes were found, 17 of which were previously unknown. The novel sequence changes include 4 deletions (153delC, 628del5, 740delC, 747delG), 5 nonsense mutations (Q60X, Y167X, Q400X, R619X, R628X), 6 missense mutations (C205Y, G208V, H240R, A319V, P496R, S633L), a splice site mutation (IVS12+5g>a), and a rare polymorphism (A591T). The polymorphism and novel missense mutations were transiently expressed in COS-7 cells and all of them except the polymorphism showed complete loss of enzyme activity. In total, 165 of the 170 mutant alleles were identified in this study and despite the high frequency of W402X and Q70X, the identification of many novel mutations unique to individual families further highlights the genetic heterogeneity of MPS I.

Animals↗
PubMed2005

Marrow stromal cells from patients affected by MPS I differentially support haematopoietic progenitor cell development.

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

Adolescent↗
PubMed2006

Cardiac findings after enzyme replacement therapy for mucopolysaccharidosis type I.

Mucopolysaccharidosis type I is a lethal autosomal recessive storage disease caused by a deficiency of lysosomal alpha-L-iduronidase and the consequent systemic accumulation of glycosaminoglycan. Cardiomyopathy and valvar insufficiency occur as glycosaminoglycan accumulates in the myocardium, expands the spongiosa of cardiac valves, and proliferates within the myointima of the epicardial coronary arteries. Congestive heart failure and death occur within the first decade of life in the most severe cases. Allogeneic hematopoietic stem cell transplantation, used in severe forms of the disease, markedly prolongs survival, alleviates ventricular hypertrophy, and preserves cardiac function, but cardiac valves continue to thicken and valvular insufficiency progresses. Enzyme replacement therapy with human recombinant alpha-L-iduronidase has been proposed as an alternativee therapy for patients with mucopolysaccharidosis type I in whom the risk/benefit ratio of hematopoietic stem cell transplantation seems unfavorable. The investigators report the cardiac findings in a small series of 5 children with mucopolysaccharidosis type I who received enzyme replacement therapy for as long as 7 years. No deaths occurred during treatment. Left ventricular hypertrophy, which was present before therapy, resolved in all cases, and myocardial function remained normal. In contrast, the mitral and aortic valves remained thickened and, in some instances, developed progressive thickening and regurgitation. In conclusion, long-term enzyme replacement therapy has some clear benefits for the myocardium, but the cardiac valves appear unresponsive, and the ultimate effect on the coronary vasculature is unknown.

Adolescent↗
PubMed2003

Delivery of recombinant gene product to canine brain with the use of microencapsulation.

An alternative approach to somatic gene therapy is to deliver a therapeutic protein by implanting "universal" recombinant cells that are immunologically protected from graft rejection with alginate microcapsules. This strategy has proved successful in reversing pathologic conditions in several rodent models of human disease (dwarfism, lysosomal storage disease, hemophilia, cancer). In particular, neurologic disease and behavioral deficit in the mouse model of a neurodegenerative disease (mucopolysaccharidosis [MPS] VII) were significantly improved through the intraventricular implantation of the recombinant encapsulated cells. Here we report the feasibility of delivering recombinant gene products to the central nervous systems (CNSs) of dogs, first using human growth hormone as a marker for delivery in normal dogs and then using alpha-iduronidase as a therapeutic product for delivery in the MPS I dog that is genetically deficient in this lysosomal enzyme. Madin-Darby canine kidney cells were genetically modified to express either human growth hormone or canine alpha-iduronidase, then enclosed in alginate-poly-l-lysine-alginate microcapsules of about 500 microm in diameter. The encapsulated cells were implanted into the brain under steoreotaxic guidance. The brains were monitored with computed tomographic scans before and after surgery and examined biochemically and histologically. Delivery of gene products, as measured in the plasma and cerebrospinal fluid sampled periodically through 21 days or in various regions of the brain after death showed that the delivery of both gene products was extremely low but detectable. However, we noted extensive inflammatory reactions, both at the sites of implantation and in the immediate vicinity of the implanted microcapsules. Hence for this technology to be applicable to the CNSs of larger animals and human beings, a more accurate and less invasive neurosurgical procedure, more biocompatible microcapsule-recombinant cell combinations, and higher output of recombinant products must be developed.

Animals↗
PubMedDate not supplied

Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I.

Enzyme replacement therapy (ERT) has been developed for several lysosomal storage disorders, including mucopolysaccharidosis I (MPS I), and is effective at reducing lysosomal storage in many tissues and in ameliorating clinical disease. However, intravenous ERT does not adequately treat storage disease in the central nervous system (CNS), presumably due to effects of the blood-brain barrier on enzyme distribution. To circumvent this barrier, we studied whether intrathecal (IT) recombinant human alpha-L-iduronidase (rhIDU) could penetrate and treat the brain and meninges. An initial dose-response study showed that doses of 0.46-4.14 mg of IT rhIDU successfully penetrated the brain of normal dogs and reached tissue levels 5.6 to 18.9-fold normal overall and 2.7 to 5.9-fold normal in deep brain sections lacking CSF contact. To assess the efficacy and safety in treating lysosomal storage disease, four weekly doses of approximately 1 mg of IT rhIDU were administered to MPS I-affected dogs resulting in a mean 23- and 300-fold normal levels of iduronidase in total brain and meninges, respectively. Quantitative glycosaminoglycan (GAG) analysis showed that the IT treatment reduced mean total brain GAG to normal levels and achieved a 57% reduction in meningeal GAG levels accompanied by histologic improvement in lysosomal storage in all cell types. The dogs did develop a dose-dependent immune response against the recombinant human protein and a meningeal lymphocytic/plasmacytic infiltrate. The IT route of ERT administration may be an effective way to treat the CNS disease in MPS I and could be applicable to other lysosomal storage disorders.

Animals↗
PubMed1992

Architecture of the canine IDUA gene and mutation underlying canine mucopolysaccharidosis I.

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease caused by deficiency of alpha-L-iduronidase. In addition to the well-known human forms (Hurler, Hurler/Scheie, and Scheie syndromes), there exists a canine model of the disease. By using previously described canine cDNA encoding alpha-L-iduronidase as a probe, the canine IDUA gene has been cloned and characterized. It contains 14 exons spread over 13 kb. An unusual GC dinucleotide was found at the donor splice site of intron 11. A transcriptional start site was identified by primer extension 177 bp upstream of the initiator AUG codon. The upstream region was found to be similar to the promoter region of many housekeeping genes: it is GC rich and has seven potential Sp1 binding sites but no TATA box or CAAT motif. The mutation in canine MPS I was localized to the area of intron 1 by RT-PCR, identified by sequence analysis of amplified genomic DNA, and confirmed by restriction analysis; it is a G-->A transition in the donor splice site of intron 1. The mutation causes retention of intron 1 in the RNA and creates a premature termination codon at the exon-intron junction.

Animals↗