Patent Application
20070218469
This invention relates to the identification of mutations in the α chain of the lysosomal enzyme β-N-acetyl-hexosaminidase A. More particularly, the present invention concerns nucleic acid compositions and kits useful for screening, diagnosis and prognosis of a genetic defect which is associated with Tay Sachs disease.
Two major isozymes of the lysosomal enzyme β-N-acetyl-hexosaminidase (Hex) (EC 3.2.1.52) exist: Hex A, a heterodimer of α and β subunits, and Hex B, a homodimer of the β subunits. Hex A almost exclusively degrades the substrate GM2 ganglioside in the presence of an activator protein. Hex A deficiency, caused by mutations in the HEXA gene encoding the α subunit, results in accumulation of GM2 ganglioside, mainly in neuronal tissues and manifests in various progressive neurological syndromes termed GM2 gangliosidosis type B (Gravel et al 2001). The degree of GM2 ganglioside accumulation is directly correlated with the level of Hex A deficiency; thus, mutations that result in total absence of Hex A activity lead to Tay-Sachs disease (TSD), the severe-infantile form of GM2 gangliosidosis type B, whereas mutations that result in residual Hex A activity give rise to the milder juvenile and the late onset of TSD (LOTS) (MIM #272800). Possessing a residual level of activity more than 5% of the normal Hex A level was suggested as the “critical threshold” that allows one to escape the severe TSD, and exceeding a level of 10%, to escape the chronic LOTS form (Conzelmann et al 1983; Conzelmann and Sandhoff 1983-4).
To date, approximately 100 mutations, including neutral genetic polymorphisms, have been reported in the HEXA gene (Myerowitz, 1997; U.S. Pat. No. 5,217,865). Some of the mutations have been thoroughly characterized and a correlation between genotype and phenotype severity can be foreseen. However, there is no obvious correlation between genotypes and phenotypes of the neurological condition, especially in the non-infantile form of TSD. Although almost all LOTS patients carry the missense mutation G269S in compound heterozygosity with another missense mutation (Navon and Proia 1989; Paw et al 1989), they display different degrees of severity, and even patients with identical mutations manifest marked clinical heterogeneity (Argov and Navon 1984; Navon 1991).
U.S. Pat. No. 5,217,865 discloses methods for detecting mutations in the HEXA gene for diagnosis of Tay-Sachs disease patients and carriers. The patent describes a splice junction mutation and an insertion mutation, but does not disclose the novel mutations of the present invention.
The present invention describes newly identified point mutations in the α chain of beta-hexosaminidase (hereinafter HEXA) which are associated with Tay Sachs disease.
One such point mutation occurs at position 638 wherein Adenine is replaced with Cytosine (hereinafter 638A>C).
Another point mutation occurs at position 181, wherein Cytosine is replaced with Thymine (hereinafter 181C>T).
In a first of its aspects, the present invention provides a method to determine the presence or absence of an HEXA gene mutation in an individual comprising: isolating a nucleic acid sample being genomic DNA, cDNA, or RNA from an individual, and assessing the presence or absence of an allele carrying the mutation; wherein said mutation is either 638A>C or 181C>T.
The presence of two mutation-carrying alleles indicates that the individual is affected with Tay Sachs disease. The presence of one mutation-carrying allele indicates that the individual is either a heterozygote carrier of Tay Sachs disease or is affected with Tay Sachs disease depending on the identification of additional known mutations in the individual's second allele. Such additional mutations are well documented in the art, e.g. Myerowitz, 1997, U.S. Pat. No. 5,217,865.
In one embodiment the nucleotide probe specific for the 638A>C mutation is 5′AAAAGTGAAGCTCTCAGATGGGAAGGAAGGATC3′ (SEQ ID. No 11).
In another embodiment, the nucleotide probe specific for the 181C>T mutation is 5′CTCGTCGAAGACTGAGCA3′ (SEQ ID. No 12).
The invention further provides a method for screening or diagnosing patients or carriers of Tay Sachs disease comprising detecting said 181C>T or 638A>C mutation in said patients' genetic material.
In a preferred embodiment the invention allows for screening or diagnosing patients or carriers of Tay Sachs disease in the Ashkenazi Jewish population comprising detecting said 181C>T mutation in said patients' genome.
In another aspect the present invention provides a kit for detecting the 181C>T or 638A>C mutation, useful for screening the population or for diagnosing Tay Sachs disease or carrier status.
The invention further provides isolated nucleic acid sequences corresponding to the HEXA gene (SEQ ID NO. 1), wherein the Adenine nucleotide at position 638 is replaced with Cytosine, or portions thereof comprising the 638A>C mutation and its flanking regions.
The invention also provides isolated nucleic acid sequences corresponding to the HEXA gene (SEQ ID NO. 2), wherein the Cytosine nucleotide at position 181 is replaced with Thymine, or portions thereof comprising the 181C>T mutation and its flanking regions.
In accordance with another aspect, the present invention provides nucleic acid probes, at least 18 nucleotides ling, comprising a nucleotide sequence complementary to the nucleic acid sequences as set forth above.
The present invention further provides an expression vector comprising a coding sequence of a nucleic acid as set forth above operably linked with a promoter sequence capable of directing expression of the coding sequence in host cells for the vector.
In accordance with another aspect, the present invention provides host cells transformed with a vector as set forth above.
Another aspect of the present invention is a method of treating Tay-Sachs disease by gene therapy based on homologous recombination using corrective vectors. The method comprising: (a) providing corrective recombinant vectors comprising a nucleic acid sequence encompassing either position 638 of the HEXA gene, or position 181 of the HEXA gene and their respective flanking regions, wherein said nucleic acid sequence comprises the correct nucleic acid in said positions, e.g. adenine at position 638 and cytosine at position 181; (b) transfecting patients cells with said corrective vectors either ex vivo or in vivo under conditions allowing homologous recombination and expression of a correct, normal HEXA protein in sufficient quantities to reverse the disease condition.
The invention also concerns the corrective vectors described above, as well as their use for treating Tay-Sachs disease, and for preparing pharmaceutical compositions for the treatment of the disease. In another aspect the invention concerns pharmaceutical composition for the treatment of Tay-Sachs disease comprising the above-described corrective vectors.
The patent application file contains at least one drawing photograph executed in color. Copies of this patent application publication with color drawing photographs will be provided by the Office upon request and payment of the necessary fee.
In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Definitions:
“Screening” as used herein means examining a subject to identify the presence of a mutation associated with Tay-Sachs disease. Screening also pertains to mass examination of the population or of people at risk and is particularly useful for identifying carrier status to allow informed genetic counseling.
“Tay Sachs disease” and “GM2 gangliosidosis type B” are used interchangeably and mean an inherited autosomal recessive disorder caused by mutations in the α chain of the A form of β-hexosaminidase (HEXA). As used herein Tay-Sachs disease (TSD), includes the severe-infantile form of GM2 gangliosidosis type B, as well as the milder juvenile and the late onset forms of TSD (LOTS).
“Mutant hexosaminidase A gene” as used herein means the HEXA gene, wherein at least one nucleic acid substitution, and/or deletion and/or insertion occurs, which result in impairment of its essential function. As used herein the “normal” HEXA gene sequence refers to that sequence, which results in the normal phenotype and which is the most common haplotype found in the population.
“Genetic polymorphism” as used herein refers to the variation seen at a genetic locus wherein two or more different nucleotide sequences can coexist at the same genetic locus in the DNA. The different sequences may or may not result in disease.
“Tay-Sachs carrier” as used herein means a person whose chromosomes contain a mutant HEXA gene that may be transmitted to said person's offspring.
“Tay-Sachs patient” as used herein means a person who carries a mutant HEXA gene on both chromosomes on which the gene is carried, such that the clinical symptoms of Tay-Sachs disease are exhibited.
“Allele” as used herein means an alternative form of a gene. A single allele for each gene is inherited separately from each parent.
“Oligonucleotide primers” as used herein means a molecule comprised of more than three deoxyribonucleotides or ribonucleotides. Its exact length will depend on many factors relating to the ultimate function or use of the oligonucleotide primer including temperature, source of the primer and use of the method. The oligonucleotide primer can occur naturally as in a purified restriction digest or be produced synthetically. The oligonucleotide primer is capable of acting as a point of initiation of synthesis when placed under conditions which induce synthesis of a primer extension product complementary to a nucleic acid strand. The conditions can include the presence of nucleotides and an inducing agent such as DNA polymerase at a suitable temperature and pH. Although the primer preferably is single stranded, it may alternatively be double stranded. If it is double stranded, the primer must first be treated to separate its strands before it is used to produce extension products. In the preferred embodiment, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent.
“Complementary” as used herein means a nucleic acid molecule having a sequence which complements a reference template sequence, whereby the two sequences can specifically hybridize.
“Variant” as used herein means a nucleic acid molecule encoding a mutant HEXA gene having at least 95% homology (identity) in the nucleic acid level to the mutant HEXA gene sequence of SEQ ID Nos. 1 or 2.
“Flanking” as used herein means the sequence of nucleotides adjacent, on both sides of the mutated nucleic acid (e.g. adjacent to position 638 or adjacent to position 181).
Most of the late onset Tay Sachs (LOTS) disease Ashkenazi and non-Ashkenazi patients have a common molecular scheme, harboring the 805G>A mutation responsible for the less severe form of GM2 gangliosidosis type B in compound heterozygosity with an infantile mutation.
This invention relates to newly identified mutations in the HEXA gene which are associated with LOTS disease. More specifically, one mutation is an A to C change at base pair position 638 of the HEXA gene, and another mutation is C to T change at base pair position 181 of said gene.
Without being limited by theory, it is assumed that both mutations alter the secondary structure of the α-precursor peptide resulting in its retention in the ER/cis Golgi Network and its subsequent proteosomal degradation.
Mis-sense mutations in the HEXA gene which affect the tertiary structure of the early α precursor in the ER/cis Golgi Network and disrupt its folding or dimerization have been previously reported. Such mutant proteins are retained in the ER/cis Golgi Network by the quality control system and are subsequently directed to proteosomal degradation and thus their routing to the lysosomes is prevented (Mahuran 1999; Hiller et al 1996). Indeed, both 638A>C and 181C>T result in non-conservative amino-acid substitutions: the 638A>C mutation results in substitution of Y to S and 181C>T results in substitution of L to S. These substitutions are likely to result in alteration of the secondary structure of the α-precursor peptide. It can be concluded that the two identified mutations are disease-causing mutations.
With knowledge of the novel mutations of the HEXA gene as disclosed herein, screening for presymptomatic homozygotes and heterozygous carriers, including prenatal diagnosis and screening can be readily carried out.
Individuals carrying mutations in the HEXA gene may be detected at either the DNA or RNA level using a variety of techniques that are well known in the art. The mutation analysis may be performed on samples of RNA by reverse transcription into cDNA.
The genomic DNA or RNA used for the diagnosis may be obtained from an individual's cells, such as those present in peripheral blood, urine, saliva, surgical specimen, and autopsy specimens. The DNA may be used directly or may be amplified in vitro through use of methods well known in the art e.g. PCR, prior to mutation analysis. Genomic HEXA may be amplified using any set of primer pairs as previously disclosed (Triggs-Raine et al 1991). As a non limiting example, cDNA may be amplified using oligonucleotide primers designated SEQ ID. Nos. 3-10, as detailed in Table 2. For the identification of HEXA 638A>C exon 6 should be amplified e.g. using primers of SEQ. ID. Nos. 5 and 6. For the amplification of 181C>T exon 1 should be amplified e.g. using primers of SEQ ID. Nos. 3 and 4.
In situ hybridization may also be used to detect the HEXA mutations of the invention.
The methodology for preparing nucleic acids in a form that is suitable for mutation detection is well known in the art. For example, suitable probes for detecting a given mutation include the nucleotide sequence at the mutation site and encompass a sufficient number of nucleotides to provide a means of differentiating a normal from a mutant allele. Any probe or combination of probes capable of detecting any one of the HEXA mutations herein described are suitable for use in this invention. Examples of suitable probes include those complementary to either the coding or non coding strand of the DNA. Similarly, suitable PCR primers are complementary to sequences flanking the mutation site. Production of these primers and probes can be carried our in accordance with anyone of the many routine methods, e.g. as disclosed in Sambrook et al. sup 45.
Probes for use with this invention should be long enough to specifically identify or amplify the relevant HEXA mutations with sufficient accuracy to be useful in evaluating the risk of an individual to be a carrier or having Tay-Sachs disease. In general, suitable probes and primers will comprise, preferably at a minimum, an oligomer of at least 16 nucleotides in length. Since calculations for mammalian genomes indicate that for an oligonucleotide 16 nucleotides in length, there is only one chance in ten that a typical cDNA library will fortuitously contain a sequence that exactly matches the sequence of a nucleotide. Therefore, suitable probes and primers are preferably 18 nucleotides long, which is the next larger oligonucleotide fully encoding an amino acid sequence (i.e., 6 amino acids in length).
By use of nucleotide sequences provided by this invention, effective and accurate testing procedures are made available to identify carriers of mutant 638A>C and 181C>T alleles of HEXA, as well as pre- and postnatal diagnosis of fetuses, children and adults carrying either one or two mutant alleles. These testing procedures should be combined with assays directed towards the identification of other Tay-Sachs mutations known in the art and together will generate a more accurate screening tool that allows the identification of a broad array of carriers and patients which were previously not identified.
Many versions of conventional genetic screening tests are known in the art. Several examples are U.S. Pat. No. 5,217,865 describing screening for Tay-Sachs disease, WO 91/02796 for cystic fibrosis, U.S. Pat. No. 5,227,292 for neurofibromatosis, WO 93/06244 for Gaucher disease and WO 02/059381 for familial dysautonomia. Thus, in accordance with the state of the art regarding assays for such genetic disorders, several types of assays are conventionally employed for mutation detection. For example, the detection of the 638A>C or 181C>T mutations in the HEXA gene sequence can be accomplished by a variety of methods including but not limited to, DNA sequencing, restriction-fragment-length-polymorphism (RFLP) analysis based on allele-specific restriction-endonuclease cleavage, hybridization with allele-specific oligonucleotide probes including immobilized oligonucleotides, oligonucleotide or GeneChip arrays, peptide nucleic acid (PNA) probes, methylation-specific PCR, pyrosequencing analysis, acycloprime analysis, reverse dot blot, TaqMan, Molecular Beacons, Intercalating dye, FRET primers, AlphaScreen, SNPstream, genetic bit analysis, Multiplex minisequencing, SNaPsht, MassEXTEND, MassArray, GOOD assay, Microarray miniseq, arrayed primer extension, Microarray primer extension, Tag arrays, Coded microspheres, template-directed incorporation (TDI), fluorescence polarization, Colorimetric oligonucleotide ligation assay (OLA), Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlock probes, Rolling circle amplification, Invader assay, MLPA and MS-MLPA.
The identification of the novel HEXA mutations of the invention also has therapeutic implications. Indeed, one aspect of the present invention is a therapy which circumvents or overcomes the defect in the HEXA gene caused by the 638A>C or 181C>T mutations via a gene therapy approach based on site-directed mutagenesis.
Gene therapy, utilizing recombinant DNA technology to deliver specifically designed corrective vectors into patient cells, will supply the patient with a normal, corrected gene product in vivo. Accordingly, a recombinant vector is designed comprising a nucleic acid sequence encompassing position 638 of the HEXA gene, or position 181 of the HEXA gene and their respective flanking regions, wherein said nucleic acid sequence comprises the correct nucleic acid, e.g. adenine at position 638 and cytosine at position 181. In gene therapy of Tay-Sachs disease, the corrective vector is delivered to the affected individual in a form and amount such that homologous recombination occurs and the correct gene is expressed and will translate into sufficient quantities of HEXA protein to reverse the effects of the mutated HEXA gene. Current approaches to gene therapy include viral vectors, cell-based delivery systems and delivery agents. Further, ex vivo gene therapy could also be useful. In ex vivo gene therapy, cells (either autologous or otherwise) are transfected with the corrective vector and implanted or otherwise delivered into the patient. Such cells thereafter express the normal HEXA gene product in vivo and would be expected to assists a patient with Tay-Sachs disease. Ex vivo gene therapy is described in U.S. Pat. No. 5,399,346.
Appropriate vectors include retroviruses, adenovirus, adeno associated virus (AAV), vaccinia virus, bovine papilloma virus or members of the herpes virus group such as Epstein-Barr virus. The viral vectors are preferably replication deficient. Non viral methods of in vivo delivery are also suitable, e.g. liposomes.
Materials and Methods
Description of Patients which Participated in the Present Study
Patient I: A 30-year-old woman, born in Russia to unrelated parents and with no illnesses worthy of note in her family. She was in good health until age 15, when she started having difficulties in jumping, walking and coordination. Her symptoms worsened rapidly within three years. She had emotional lability and was treated by a psychiatrist under the diagnosis of hysterical bursts. At age 24 she was diagnosed as having ‘ALS’. Since then she had more difficulties walking and her speech has become unclear. On examination she had dysarthric speech with nasal quality. Cranial nerves were intact. Upper limbs showed normal strength and muscle mass. There was severe atrophy of thigh muscles. Strength of iliopsoas, quadriceps, and hamstrings was 2-3/5 (MRC scale) bilaterally. Other lower limbs muscles were normal. Deep tendon reflexes were normal. She had pronounced dysmetria on finger-nose test. Her gait was waddling, and she used Gower's maneuver to get up from sitting. MRI showed severe cerebellar atrophy with normal structure of the cerebral hemispheres. EMG revealed severe, chronic nonactive neurogenic changes in the lower limbs muscles.
To summarize, the clinical features were of cerebellar ataxia and lower motor neuron signs of the lower limbs.
Patient II: This 67-year-old Ashkenazi Jew was a mother of two children who were diagnosed as LOTS: the son had an unusually slow ALS-like syndrome and the daughter had a diffuse neurodegenerative disease affecting mentation, cerebellum, motor neurons, and pyramidal system. She noticed tremor of the hands (more on the left) after the delivery of her first daughter at age 30. The tremor worsened with years and she needed someone to assist her with eating and dressing and lost her ability to write. She did not report any weakness of the hands and was still walking six km every day. There were no sensory or cognitive symptoms. Examination revealed mild, slow rhythmic tremor of the head and tongue at rest. A marked coarse tremor of both hands (more on the left) was noted at rest and this tremor was aggravated upon action and became flapping-like. There was no weakness but the muscle tone was markedly reduced in the upper limbs and slightly reduced in the lower. Tendon reflexes were elicited and there were no pyramidal signs. The patient could not perform repeated changing movements of her hands as the tremor became so marked that she needed to hold one hand with the other. The tremor was also aggravated at walking but her gait was otherwise normal. She did have minimal impairment of the shin to heal test. She did not show any cognitive or psychiatric decline and did not have any signs of neurological involvement of other systems until death at age 83.
Thus, this patient had a very atypical syndrome of rubro-cerebellar tremor without other neurological phenomena.
The patients were tested for Hex A activities in serum and leukocytes either by the heat inactivation method using 4-methylumbelliferyl 2-acetoamido-2-deoxy-β D-glucopyranoside (4-MUG) or by 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide-6-sulfo (4-MUGS) as a substrate (Fuchs et al 1983; Inui and Wenger 1984). All patients were shown to have severe Hex A deficiency in the range known for patients suffering from GM2 gangliosidosis type B.
Nucleic Acid Analysis
Genomic DNA was isolated from whole blood and from cultured fibroblasts using the Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, Minn.).
Total RNA was isolated from fibroblasts using TriPure™ reagent (Roche Diagnostics, Basel, Switzerland) and mRNA was reverse transcribed using synthetic oligo-d(T) and Superscript II RNAse H-Reverse Transcriptase (Gibco Invitrogen Corporation, Carlsbad, Calif.).
PCR reactions were performed with Taq Polymerase (Bioline, Boston, Mass.).
Sequencing of PCR products from patients' cDNA and genomic DNA was performed using 373A DNA sequencing system, Applied Biosystems, Foster City, Calif.
Expression in COS-7 Cell Line
The different identified mutations were introduced into wild-type pcDNA3α vector (the α cDNA in pBluescript II KS vector was kindly provided by Dr. R. L. Proia and subcloned in pcDNA3 vector using Xhol/Notl sites) using either the QuickChange™ Site-Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) (638A>C) or the Transformer™ Site Directed Mutagenesis Kit, 2nd version (Clontech, Palo Alto, Calif.) (181C>T).
Briefly, the COS-7 cell-line was grown in Dulbecco's modified Eagle's medium, supplemented with 20% fetal calf serum (FCS), antibiotics and L-glutamin (Biological Industries, Beit Haemek, Israel). Next, the cells were transiently transfected with the different cDNA constructs in Opti-MEM I Reduced Medium (Gibco Invitrogen Corporation, Carlsbad, Calif.) using FuGene6 reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Finally, the cells were harvested 48 h post transfection.
Hex S Enzymatic Activities and Protein Levels
Protein concentrations were determined by the Bradford method (BioRad Laboratories, Inc., Hercules, Calif.).
For the enzyme assay, the harvested cells were re-suspended in 0.05M sodium citrate buffer, pH 4.2, and subjected to five rounds of freezing and thawing. The cell extracts were assayed for Hex S activity using 4-MUGS (Sigma, Ronkonkoma, N.Y.) specific for the catalytic activity of the α subunit (Fuchs et al 1983; Inui and Wenger 1984). All transfection experiments were performed in duplicate and enzymatic reactions in triplicate to ensure reliability of the results.
For Western blotting, the harvested cells were lysed for 1 h in a lysis buffer containing NP-40 and Complete Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland). Equal amounts of total proteins from transfected COS-7 cells extracts (2.5 μg) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Laemmli 1970). The membranes were blocked in 5% BSA and then incubated overnight at 4° C. with 1:800 dilution of rabbit anti human Hex A IgG (kindly provided by Dr. R. A. Gravel) and 1 h with 1:25,000 dilution of Horseradish Peroxidase conjugated donkey anti-rabbit IgG (Jackson Immunoresearch). Finally, the membranes were developed using Chemiluminescent Detection System (ECL, Santa Cruz Biotechnology).
Immunolocalization in Intracellular Compartments
COS-7 cells were seeded onto cover slips at low-density 24 h post transfection. Following 24 h incubation, the cells were fixed and gently permeabilized with cold methanol at −20° C. for 30 min, blocked with 1% BSA, and incubated overnight with a 1:700 dilution of rabbit anti-human Hex A IgG at 4° C. The cells were then incubated with Fluorescein (FITC) conjugated AffiniPure goat anti-rabbit IgG (green fluorescence) (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) diluted 1:75 for 1 h and then treated with the nucleic acid marker Propidium Iodide (red fluorescence) (Sigma, Ronkonkoma, N.Y.) for 1 h. A confocal laser-scanning microscope (LSM 410 invert, Carl Zeiss, Oberkochen, Germany) equipped with a 25-mW krypton-argon laser (488 and 568 maximum lines) was used to analyze the slides.
Genomic DNA was isolated from the whole blood of patient I and from cultured fibroblasts of patient II. In addition, mRNA was isolated from fibroblast and reverse transcribed to cDNA
The cDNA was amplified using primer pairs as described in Table 2. Primer pairs used for amplifying genomic HEXA (exons and their flanking introns) were as described before (Triggs-Raine et al 1991).
Genomic DNA from the patients was first screened for some common mutations: 1278insTATC, IVS9+1G>A, IVS12+1G>C and 805G>A (Myerowitz et al 1988; Akerman et al 1992; Ohno and Suzuki 1988; Navon and Proia 1989; Paw et al 1989). Both patients were found to carry the common 805G>A (G269S) mutation. Sequencing the amplified cDNA and genomic HEXA of the patients revealed that they are both compound heterozygotes. They both carried the common 805G>A (G269S) mutation, but each carried an additional point mutation: a novel 638A>C transversion (Y213S) in exon 6 in patient I, and a novel 181C>T transition (L61S) in exon 1 in patient II. The patients' ethnicity and genotype are summarized in Table 3.
The mutations, 638A>C, and 181C>T were confirmed by restriction analyses with NdeI, and MboII. The 638A>C mutations abolishes a restriction site for NdeI, and the 181C>T mutation creates a restriction site for MboII respectively.
To examine the role of the different identified mutations in the pathology of the disease, we performed expression studies. Transfections with the wild-type construct pcDNA3α resulted in ˜8.5-fold increase in Hex S activity as compared to the background activity of the mock-transfected cells. Transfections with the mutant constructs pcDNA3α638A>C and pcDNA3α181C>T all resulted in reduced Hex S activities, which were virtually indistinguishable from one another and from the background activity of the mock-transfected cells. Results are summarized in Table 4.
The reduction in Hex S activity obtained following transfections with the mutant constructs could result from either an inactive enzyme in the lysosomes or a defect in the synthesizing route of the protein. The α subunit protein level in COS-7 transfected with pcDNA3α638A>C cells was evaluated by Western blotting yielding an undetectable level of the mature α subunit (data not shown). This ruled out the possibility of an inactive enzyme.
Direct immunofluorescence was performed to determine the fate of the mutant proteins in COS-7 cells (
GOR-IV software (Garnier et al 1996) was used to predict the possible consequences of the identified mutations on the secondary structure of the precursor α subunit.
*One unit is defined as the activity that releases 1 nmol of 4-methylumbeliferone per hour.
**Values were calculated by subtracting the mock transfection background activity and are given as the percentage of the wild-type transfection.
***Was performed in a separate experiment. Calculation of Hex S activity was performed according to the Hex S of the wild type and mock in that experiment.
Taken together, the results confirm the involvement of the newly identified mutations in Tay-Sachs disease pathology (summarized in Table 5)
NA - not performed
Patient II (HEXA 181C>T) is unique with respect to her phenotype, age of onset and expression of very mild symptoms, which were not observed in other Ashkenazi LOTS patients. This can perhaps be explained by her different genotype, seen for the first time, since all Ashkenazi LOTS patients reported to date harbor the 805G>A (G269S) in compound heterozygosity with either 1278+TATC or IVS12+1G>A. Interestingly, the very mild unique manifestation displayed by this patient implies that 181C>T may enable residual Hex A activity.
This application claims the benefit of prior U.S. provisional patent application No. 60/722,427 filed Oct. 3, 2005, the contents of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60722427 | Oct 2005 | US |
