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PubMed1994

Hepatitis C virus polyprotein processing: kinetics and mutagenic analysis of serine proteinase-dependent cleavage.

Hepatitis C virus (HCV) serine proteinase (Cpro-2) is responsible for the processing of HCV nonstructural (NS) protein processing. To clarify the mechanism of Cpro-2-dependent processing, pulse-chase and mutation analyses were performed by using a transient protein production system in cultured cells. Pulse-chase study revealed the sequential production of HCV-NS proteins. Production of p70(NS3) and p66(NS5B) were rapid. An 89-kDa processing intermediate protein (p89) was observed during the early part of the chase. p89 seemed to be cleaved first into a 31-kDa protein (p31) and a p58/56(NS5A). p31 was further processed into p4(NS4A) and p27(NS4B). Mutation analysis of cleavage sites of NS4A/4B, NS4B/5A, and NS5A/5B revealed that cleavage at each site is essentially independent from cleavage occurring at the other site.

Animals↗
PubMed2005

Determinants of simulated RNA evolution.

Models of RNA secondary structure folding are widely used to study evolution in theory and simulation. However, systematic studies of the parameters involved are rare. In this paper, we study by simulation how RNA evolution is influenced by three different factors, namely the mutation rate, scaling of the fitness function, and distance measure. We found that for low mutation rates the qualitative evolutionary behavior is robust with respect to the scaling of the fitness function. For efficient mutation rates, which are close to the error threshold, scaling and distance measure have a strong influence on the evolutionary behavior. A global distance measure that takes sequence information additively into account lowers the error threshold. When using a local sequence-structure alignment for the distance, we observed a smoother evolution of the fitness over time. Finally, in addition to the well known error threshold, we identify another threshold of the mutation rate, called divergence threshold, where the qualitative transient behavior changes from a localized to an exploratory search.

Animals↗
PubMed2006

Severe episodic neurological deficits and permanent mental retardation in a child with a novel FHM2 ATP1A2 mutation.

OBJECTIVE: Attacks of familial hemiplegic migraine (FHM) are usually associated with transient, completely reversible symptoms. Here, we studied the ATP1A2 FHM2 gene in a young girl with episodes of both very severe and transient neurological symptoms that were triggered by mild head trauma as well as permanent mental retardation. Her family members suffered from hemiplegic and confusional migraine attacks. METHODS: Mutation analysis of the ATP1A2 gene was performed by direct sequencing of all exons and flanking intronic regions, using genomic DNA of the proband. Functional consequences of the mutation were analyzed by cellular survival assays. RESULTS: We identified a novel G615R ATP1A2 mutation in the proband and several of her family members. Functional analysis of mutant Na,K-ATPase in cellular survival assays showed a complete loss-of-function effect. INTERPRETATION: Permanent mental retardation in children may be caused by ATP1A2 mutations.

Arginine↗
PubMed1994

Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase.

An early step in GH action involves tyrosine phosphorylation of various cellular proteins. Recently, it has been shown in murine preadipocytes that GH promotes the association of its receptor (the GHR) with and the activation of the JAK2 tyrosine kinase. In this study, we confirmed the human (h) GH-induced association of JAK2 with hGHR in IM-9 cells by coimmunoprecipitation experiments using anti-hGHR serum. We further examined the interaction of JAK2 with the GHR cytoplasmic domain by two lines of investigation. For in vitro studies, we assayed by immunoblotting the ability of cell-derived JAK2 to interact with glutathione S-transferase fusion proteins containing elements of the hGHR cytoplasmic domain. A fusion protein containing the entire hGHR cytoplasmic domain (residues 271-620) specifically associated with JAK2 independent of prior stimulation of cells with hGH. This interaction was not dependent on tyrosine phosphorylation of either partner. Mutational analysis of the hGHR cytoplasmic domain component of the fusions indicated that a membrane-proximal 20-residue region that includes the proline-rich box 1 was necessary for the interaction. This region appeared to cooperate with another region(s), largely in the N-terminal one third of the cytoplasmic domain, to promote full interaction with JAK2. For in vivo reconstitution experiments, wild-type (WT) and mutant rabbit GHRs (rGHRs) along with murine JAK2 were expressed by transient transfection in COS-7 cells. rGHR mutations were confined to the cytoplasmic domain and included C-terminal truncations as well as internal deletions of residues 297-406 and 278-292 (the latter contains box 1). All mutant rGHRs were expressed at the cell surface and bound hGH to a degree similar to the WT rGHR. Receptors were tested for their ability to mediate the hGH-induced immunoprecipitability of JAK2 with phosphotyrosine (APT) antibodies. A rGHR truncated to residue 275 [rGHR-(1-275)], which contains only five cytoplasmic residues, failed to mediate JAK2 APT precipitability in response to hGH. In contrast, WT rGHR; the C-terminal truncations rGHR-(1-542), rGHR-(1-390), and rGHR-(1-317); and the rGHR-(d297-406) deletion mutant maintained this ability. Deletion of the 278-292 box 1-containing region in the context of either rGHR-(d297-406) or WT rGHR eliminated detectable hGH-induced JAK2 APT precipitability. Interestingly, rGHR-(1-292), which includes box 1, was not able to mediate significant hGH-induced JAK2 APT precipitability.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Migraine-like disorder segregating with mtDNA 14484 Leber hereditary optic neuropathy mutation.

The authors report neurologic features in a large family harboring the mitochondrial DNA (mtDNA) mutation T14484C associated with Leber hereditary optic neuropathy (LHON). In the maternal line the mtDNA mutation was associated with optic neuropathy or migraine with aura or without aura and transient neurologic/visual disturbances. The segregation of familiar cases of migraine and LHON mutation broadens the clinical phenotype associated with a primary LHON mutation.

Adolescent↗
PubMed1992

The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function.

The UL5 protein of herpes simplex virus type 1, one component of the viral helicase-primase complex, contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases. Although this superfamily contains more than 20 members ranging from bacteria to mammalian cells and their viruses, the importance of these motifs has not been addressed experimentally for any one of them. In this study, we have examined the functional significance of these six motifs for the UL5 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. A transient replication complementation assay was used to test the effect of each mutation on the function of the UL5 protein in viral DNA replication. In this assay, a mutant UL5 protein expressed from an expression clone is used to complement a replication-deficient null mutant with a mutation in the UL5 gene for the amplification of herpes simplex virus origin-containing plasmids. Eight mutations in conserved regions and three similar mutations in nonconserved regions of the UL5 gene were analyzed, and the results indicate that all six conserved motifs are essential to the function of UL5 protein in viral DNA replication; on the other hand, mutations in nonconserved regions are tolerated. These data provide the first direct evidence for the importance of these conserved regions in any member of the superfamily of DNA and RNA helicases. In addition, three motif mutations were introduced into the viral genome, and the phenotypic analyses of these mutants are consistent with results from the transient replication complementation assay. The ability of these three mutant UL5 proteins to form specific interactions with other members of the helicase-primase complex, UL8 and UL52, indicates that the functional domains required for replication activity of UL5 are separable from domains responsible for protein-protein interactions. It is anticipated that this type of structure-function analysis will lead to the identification of protein domains that contribute not only to the enzymatic activities of helicase or primase but also to protein-protein interactions within members of the complex.

Amino Acid Sequence↗
PubMed1994

Cleavage at the N-terminal site of Alzheimer amyloid beta/A4 protein is essential for its secretion.

To characterize the secretory pathway of Alzheimer amyloid beta/A4 protein (beta/A4), mutated cDNAs of amyloid precursor protein (APP) were expressed transiently in COS cells. Although the expression of C100 (Met671-Asn770) resulted in the secretion of beta/A4-like peptide, the cells expressing the mutated APPs with the longer N-terminal domain beyond beta/A4 sequence secreted little beta/A4-like peptide. The C-terminal domain truncated APPs, with a stop codon at the end of beta/A4 sequence or the predicted membrane spanning domain produced little beta/A4-like peptide. When beta/A4 was expressed with direct addition of the signal sequence, beta/A4-like peptide was found in the cell lysate but little of it was secreted. Hence, the secretion of beta/A4 was started initially by cleavage at the N-terminal site of beta/A4, although cleavage at its C-terminal site was also necessary for its secretion.

Alzheimer Disease↗
PubMed1980

Ribosomal precursor RNA metabolism and cell division in the yeast Saccharomyces cerevisiae.

When shifted from 23 degrees C to 36 degrees C, cells of a non-temperature-sensitive strain of yeast arrest transiently in G1 before continuation of the cell division cycle. When shifted to 36 degrees C, cells harboring a temperature-sensitive rna mutation behave similarly. Others have shown that temperature shift transiently decreases the rates of production and processing of ribosomal precursor RNA (rpreRNA). Production of rpreRNA is soon restored to normal levels in these strains, but normal processing of these repreRNA transcripts is restored only in non-temperature-sensitive strains. Therefore these experiments serve to eliminate from cell cycle considerations the involvement of processing of rpreRNA, while maintaining the established correlation between cell cycle behavior and rpreRNA production.

Cell Division↗
PubMed2002

Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes.

BACKGROUND: Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR subunit are located in various functional domains, whereas fully described mutations in AChR non- subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two -subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern. METHODS: These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells. RESULTS: The authors identified two SCCMS mutations in the AChR subunit, L78P and L221F. Both mutations prolonged ACh-induced ion channel activations. L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and L221F shows variable penetrance in one of the two families that were identified harboring this mutation. CONCLUSION: SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.

Adolescent↗
PubMed2005

Structure, function, and evolution of transient and obligate protein-protein interactions.

Recent analyses of high-throughput protein interaction data coupled with large-scale investigations of evolutionary properties of interaction networks have left some unanswered questions. To what extent do protein interactions act as constraints during evolution of the protein sequence? How does the type of interaction, specifically transient or obligate, play into these constraints? Are the mutations in the binding site of an interacting protein correlated with mutations in the binding site of its partner? We address these and other questions by relying on a carefully curated dataset of protein complex structures. Results point to the importance of distinguishing between transient and obligate interactions. We conclude that residues in the interfaces of obligate complexes tend to evolve at a relatively slower rate, allowing them to coevolve with their interacting partners. In contrast, the plasticity inherent in transient interactions leads to an increased rate of substitution for the interface residues and leaves little or no evidence of correlated mutations across the interface.

Amino Acid Substitution↗
PubMed2001

The arginine-552-cysteine (R1315C) mutation within the A1 loop of von Willebrand factor induces an abnormal folding with a loss of function resulting in type 2A-like phenotype of von Willebrand disease: study of 10 patients and mutated recombinant von Willebrand factor.

The study identified 10 patients from 6 families with prolonged bleeding time, decreased von Willebrand factor (vWF) ristocetin cofactor activity (RCoF) to vWF:Ag (antigen) ratio, and reduced ristocetin-induced platelet agglutination as well as ristocetin- or botrocetin-induced binding of plasma vWF to platelet glycoprotein Ib (GpIb). In addition, all patients showed a decrease of intermediate-molecular-weight (intermediate-MW) and high-molecular-weight (HMW) multimers of vWF. In the heterozygous state, a cysteine-to-threonine (C --> T) transversion was detected at nucleotide 4193 of the VWF gene of all patients and lead to the arginine (R)522C substitution in the A1 loop of vWF mature subunit (R1315C in the preprovWF). By in vitro mutagenesis of full-length complementary DNA (cDNA) of vWF and transient expression in COS-7 cells, the mutated C552 recombinant vWF (C552rvWF) was found to exhibit decreased expression, abnormal folding, and lack of intermediate-MW and HMW multimers. In addition, direct binding of botrocetin to C552rvWF, as well as ristocetin- and botrocetin-induced binding of C552rvWF to GpIb, was markedly decreased. Although being localized in an area of the A1 loop of vWF where most of the type 2B mutations that induce a gain-of-function have been identified, the R552C mutation induces a 2A-like phenotype with a decrease of intermediate-MW and HMW multimers as well as a loss-of-function of vWF in the presence of either ristocetin or botrocetin. (Blood. 2001;97:952-959)

Adult↗
PubMed2005

Identification of a novel cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5'-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber.

Cytokinins and light activate the transcription of the cucumber NADPH-protochlorophyllide reductase (POR) gene. We have previously reported that 2.3 kb of the 5'-region of this gene contains a cis-element that is responsive to cytokinin. In this study, to identify the cytokinin-responsive cis-element corresponding to chlorophyll biosynthesis and chloroplast development, we performed transient expression assays in etiolated cucumber cotyledons. A 5'-deletional analysis indicated that a 411-bp fragment (-451 to -40 bp) contained at least one of the cis-elements related to cytokinin-responsiveness. Gel mobility shift assays also detected cytokinin-enhanced binding in this region. DNase I footprinting analysis, using a 150-bp fragment (-490 to -340 bp) as the probe, identified the cytokinin-enhanced protected sequence as 5'-ATATTAGTGATAT-3'. More detailed gel mobility shift and competition analyses identified 5'-TATTAG-3' as the sequence critical for cytokinin-enhanced binding. Mutations in the identified sequence in the transient expression assay caused a reduced but retained cytokinin-responsiveness, as well as low reporter activity of untreated control. These results suggest that the identified sequence is a novel cis-element exhibiting cytokinin-dependent protein binding in vitro, which may function effectively when interacting with other cytokinin-related elements. The effects of this element on the chloroplast development are discussed in relation to other cytokinin-related elements.

Base Sequence↗
PubMed1999

Estrogen-dependent and independent activation of the P1 promoter of the p53 gene in transiently transfected breast cancer cells.

Loss of p53 function by mutational inactivation is the most common marker of the cancerous phenotype. Previous studies from our laboratory have demonstrated 17 beta estradiol (E2) induction of p53 protein expression in breast cancer cells. Although direct effects of E2 on the expression of p53 gene are not known, the steroid is a potent regulator of c-Myc transcription. In the present studies, we have examined the ability of E2 and antiestrogens to regulate the P1 promoter of the p53 gene which contains a c-Myc responsive element. Estrogen receptor (ER)-positive T47D and MCF-7 cells were transiently transfected with the P1CAT reporter plasmid and levels of CAT activity in response to serum, E2 and antiestrogens were monitored. Factors in serum were noted to be the dominant inducers of chloramphenicol acetyltransferase (CAT) expression in MCF-7 cells. The levels of CAT were drastically reduced when cells were maintained in serum free medium (SFM). However, a subtle ER-mediated induction of CAT expression was detectable when MCF-7 cells, cultured in SFM, were treated with E2. In serum-stimulated T47D cells, the CAT expression was minimal. The full ER antagonist, ICI 182 780 (ICI) had no effect. Treatment with E2 or 4-hydroxy tamoxifen (OHT) resulted in P1CAT induction; OHT was more effective than E2. Consistent with c-Myc regulation of the P1 promoter, E2 stimulated endogenous c-Myc in both cell lines. Two forms of c-Myc were expressed independent of E2 stimuli. The expression of a third more rapidly migrating form was E2-dependent and ER-mediated since it was blocked by the full ER antagonist, ICI, but not by the ER agonist/antagonist OHT. These data demonstrate both ER-mediated and ER-independent regulation of c-Myc and the P1 promoter of the p53 gene, and show differential effects of the two classes of antiestrogens in their ability to induce the P1 promoter of the p53 gene in breast cancer cells.

Breast Neoplasms↗
PubMed1999

Genetic and nongenetic factors influencing plasma homocysteine levels in patients with ischemic cerebrovascular disease and in healthy control subjects.

Moderately elevated plasma homocysteine levels have been established as an independent risk factor for atherosclerosis and its complications, including cerebrovascular disease. A common mutation (C677T) in the gene encoding for the enzyme methylenetetrahydrofolate reductase (MTHFR) has been linked to increased plasma homocysteine levels in homozygous carriers, particularly in the presence of low folate levels. However, the results of most of the previous studies suggest that the C677T MTHFR mutation is not a significant risk factor for arterial disease. This discrepancy might, at least partly, be due to the fact that plasma homocysteine levels are influenced by several other factors, including age, gender, renal function, and vitamin status. We investigated the relation between plasma homocysteine levels, the C677T MTHFR mutation, and these other factors in a population of 96 patients with transient ischemic attacks or minor strokes and in 96 age- and sex-matched healthy control subjects. We further tested the value of a multivariate model for the prediction of plasma homocysteine levels under particular consideration of the MTHFR mutation status. In the patients, plasma homocysteine levels were significantly higher than in the healthy control subjects. With regard to the MTHFR mutation, the distribution of the C/C, C/T, and T/T genotypes was not significantly different between patients and healthy control subjects. Univariate (linear regression) analysis revealed significant (positive) correlations between plasma homocysteine levels on the one hand and age and creatinine on the other, the latter particularly in subjects with creatinine levels in the upper quartile. Significant (negative) correlations were found between plasma homocysteine levels, vitamin B12, and folate levels. However, these relations could much better be expressed by means of a multiplicative regression model. T/T subjects exhibited slightly higher homocysteine levels than C/C and C/T subjects; however, the differences between the 3 genotypes were not significant. Multivariate (stepwise regression) analysis revealed age, vitamin B12 levels, folate levels, and creatinine levels as significant independent variables influencing plasma homocysteine levels, whereas the MTHFR mutation status and gender were removed from the model. Considering all 192 subjects, only 28.8% of the variance of plasma homocysteine levels could be accounted for by the model. However, in homozygous carriers of the MTHFR mutation, the predictive power of the model is very high, explaining 76.1% of the variance of plasma homocysteine levels. According to our results, the C677T mutation does not constitute a major risk factor for transient ischemic attack or minor stroke, even under consideration of other possibly confounding factors that are known to affect plasma homocysteine levels. However, it is possible to predict plasma homocysteine levels in homozygous carriers of the mutation with high accuracy. The knowledge of the MTHFR mutation status may therefore help to identify subjects at high risk for hyperhomocysteinemia.

Adult↗
PubMed2002

Beyond the proton abstracting role of Glu-376 in medium-chain acyl-CoA dehydrogenase: influence of Glu-376-->Gln substitution on ligand binding and catalysis.

The active site residue, Glu-376, of medium-chain acyl-CoA dehydrogenase (MCAD) has been known to abstract the alpha-proton from acyl-CoA substrates during the course of the reductive half-reaction. The site-specific mutation of Glu-376-->Gln(E376Q) slows down the octanoyl-CoA-dependent reductive half-reaction of the enzyme by about 5 orders of magnitude due to impairment in the proton-transfer step. To test whether the carboxyl group of Glu-376 exclusively serves as the active site base (for abstracting the alpha-proton) during the enzyme catalysis, we undertook a detailed kinetic investigation of the enzyme-ligand interaction and enzyme catalysis, utilizing octanoyl-CoA/octenoyl-CoA as a physiological substrate/product pair and the wild-type and E376Q mutant enzymes as the catalysts. The transient kinetic data revealed that the E376Q mutation not only impaired the rate of octanoyl-CoA-dependent reduction of the enzyme-bound FAD, but also impaired the association and dissociation rates for the binding of the reaction product, octenoyl-CoA. Besides, the E376Q mutation correspondingly impaired the kinetic profiles for the quenching of the intrinsic protein fluorescence during the course of the above diverse (i.e., "chemistry" versus "physical interaction") processes. A cumulative account of the experimental data led to the suggestion that the carboxyl group of Glu-376 of MCAD is intimately involved in modulating the microscopic environment (protein conformation) of the enzyme's active site during the course of ligand binding and catalysis. Arguments are presented that the electrostatic interactions among Glu-376, FAD, and CoA-ligands are responsible for structuring the enzyme's active site cavity in the ground and transition states of the enzyme during the above physicochemical processes.

Acyl Coenzyme A↗
PubMed2001

A common mutation in the gene for coagulation factor XIII-A (VAL34Leu): a risk factor for primary intracerebral hemorrhage is protective against atherothrombotic diseases.

The role of a common polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recently investigated as a protective genetic factor against arterial and venous thrombosis. In addition, the less frequent Leu34 allele has been described as a risk factor for intracerebral hemorrhage. We evaluated the prevalence of this polymorphism by PCR in three case-control studies of patients diagnosed as having primary intracerebral hemorrhage (PCH, n = 130), coronary heart diseases (CHD, n = 240; myocardial infarction/no myocardial infarction, 120/120), and cerebrovascular diseases (CVD, n = 240; cerebral infarction/transient ischaemic attack, 120/120). The matched control groups consisted of patients admitted to the hospital without history of vascular disease. In addition, 200 healthy subjects were investigated. The frequency of the mutated allele (Leu34) was higher in patients with PCH than in controls (33.8% vs. 23.1%, P = 0.009) and lower in CHD and CVD patients compared to controls (18.1% vs. 25.2%, P = 0.010 and 17.3% vs. 24.2%, P = 0.011, respectively). Moreover, among the patients with CHD, the Leu34 allele was underrepresented in cases with myocardial infarction than without (12.9% vs. 23.3%, P = 0.004) and than in controls (12.9% vs. 25.2%, P < 0.001). Similar findings were obtained in patients with CVD comparing the cases with cerebral infarction versus cases with transient ischaemic attack (12.5% vs. 22.1%, P = 0.008) and versus controls (12.5% vs. 24.2%, P < 0.001). Finally, considering altogether the groups of ischaemic patients (CHD and CVD, n = 480), it was noted a trend towards a higher mean age of the clinical onset in homozygotes for the Leu allele than in the wild types (P = 0.078). This study indicates that in our population possession of the FXIII Val34Leu mutation predisposes to the occurrence of primary intracerebral hemorrhage and protects against cerebral and myocardial infarction. A wider modulatory role in the progression and onset of atherothrombotic diseases could be ascribed to FXIII Val34Leu.

Age of Onset↗
PubMed2002

Ikaros, a lymphoid-cell-specific transcription factor, contributes to the leukemogenic phenotype of a mink cell focus-inducing murine leukemia virus.

Mink cell focus-inducing (MCF) viruses induce T-cell lymphomas in AKR/J strain mice. MCF 247, the prototype of this group of nonacute murine leukemia viruses, transforms thymocytes, in part, by insertional mutagenesis and enhancer-mediated dysregulation of cellular proto-oncogenes. The unique 3' (U3) regions in the long terminal repeats of other murine leukemia viruses contain transcription factor binding sites known to be important for enhancer function and for the induction of T-cell lymphomas. Although transcription factor binding sites important for the biological properties of MCF 247 have not been identified, pathogenesis studies from our laboratory suggested to us that binding sites for Ikaros, a lymphoid-cell-restricted transcriptional regulator, affect the biological properties of MCF 247. In this report, we demonstrate that Ikaros binds to predicted sites in U3 sequences of MCF 247 and that site-directed mutations in these sites greatly diminish this binding in vitro. Consistent with these findings, ectopic expression of Ikaros in murine cells that do not normally express this protein significantly increases transcription from the viral promoter in transient gene expression assays. Moreover, site-directed mutations in specific Ikaros-binding sites reduce this activity in T-cell lines that express Ikaros endogenously. To determine whether the Ikaros-binding sites are functional in vivo, we inoculated newborn mice with a variant MCF virus containing a mutant Ikaros-binding site. The variant virus replicated in thymocytes less efficiently and induced lymphomas with a delayed onset compared to the wild-type virus. These data are consistent with the hypothesis that the Ikaros-binding sites in the U3 region of MCF 247 are functional and cooperate with other DNA elements for optimal enhancer function in vivo.

3' Untranslated Regions↗
PubMed2002

Contribution of arylsulfatase A mutations located on the same allele to enzyme activity reduction and metachromatic leukodystrophy severity.

The occurrence of different mutations on the same arylsulfatase A allele is not uncommon, due to the high frequency of several variants, among which the pseudodeficiency mutations are particularly important. We identified a late infantile metachromatic leukodystrophy patient carrying on one allele the new E253K mutation and the known T391S polymorphism, and on the other allele the common P426L mutation, usually associated with the adult or juvenile form of the disease, and the N350S and *96A>G pseudodeficiency mutations. To analyze the contribution of mutations located on the same allele to enzyme activity reduction, as well as the possible phenotype implications, we performed transient expression experiments using arylsulfatase A cDNAs carrying the identified mutations separately and in combination. Our results indicate that mutants containing multiple mutations cause a greater reduction of ARSA activity than do the corresponding single mutants, the total deficiency likely corresponding to the sum of the reductions attributed to each mutation. Consequently, each mutation may contribute to ARSA activity reduction, and, therefore, to the degree of disease severity. This is particularly important for the alleles containing a disease-causing mutation and the pseudodeficiency mutations: in these alleles pseudodeficiency could play a role in affecting the clinical phenotype.

Alleles↗