MUTATIONS OF THE PARKIN GENE, COMPOSITIONS, METHODS AND USES
20170183736 ยท 2017-06-29
Inventors
- Alexis Brice (Paris, FR)
- Christophe Lucking (Paris, FR)
- Patrice Denefle (Saint Maur, FR)
- Sylvain Ricard (Paris, FR)
- Nacer Eddine Abbas (Paris, FR)
- Sandrine Bouley (Bletterans, FR)
Cpc classification
C12Y603/02019
CHEMISTRY; METALLURGY
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
Abstract
The invention concerns nucleic acids coding for mutated or truncated forms of the human parkin gene, or forms comprising multiplication of exons, and the corresponding proteins and antibodies. The invention also concerns methods and kits for identifying mutations of the parkin gene, and for studying compounds for therapeutic purposes.
Claims
1-40. (canceled)
41. An oligonucleotide comprising a fragment, or the complementary sequence thereof, of a parkin gene that comprises a genetic alteration selected from the group consisting of: a) a deletion of one or more exons selected from the group consisting of: exon 2, exons 2-3, exons 2-4, exons 3-4, exons 3-6, exons 3-9, exon 5, exons 5-6, exon 6, exons 6-7, exons 7-9, and exon 8; b) a multiplication of exons selected from the group consisting of: a triplication of exon 2, a duplication of exon 3, a duplication of exon 6, a duplication of exon 7, and a duplication of exon 11; c) a point mutation selected from the group consisting of: a mutation from adenine to thymine at a position corresponding to position 584 of SEQ ID NO: 1, a mutation from guanine to adenine at a position corresponding to position 601 of SEQ ID NO: 1, a mutation from adenine to thymine at a position corresponding to position 734 of SEQ ID NO: 1, a mutation from cytosine to thymine at a position corresponding to position 867 of SEQ ID NO:1, a mutation from thymine to adenine at a position corresponding to position 905 of SEQ ID NO: 1, a mutation from cytosine to thymine at a position corresponding to position 924 of SEQ ID NO: 1, a mutation from guanine to adenine at a position corresponding to position 939 of SEQ ID NO: 1, a mutation from thymine to guanine at a position corresponding to position 966 of SEQ ID NO: 1, a mutation from guanine to adenine at a position corresponding to position 1084 of SEQ ID NO: 1, a mutation from cytosine to thymine at a position corresponding to position 1101 of SEQ ID NO:1, a mutation from guanine to cytosine at a position corresponding to position 1239 of SEQ ID NO:1, a mutation from guanine to adenine at a position corresponding to position 1281 of SEQ ID NO: 1, a mutation from cytosine to adenine at a position corresponding to position 1345 of SEQ ID NO: 1, a mutation from guanine to adenine at a position corresponding to position 1390 of SEQ ID NO: 1, and a mutation from guanine to adenine at a position corresponding to position 1459 of SEQ ID NO:1; d) a deletion of 1 or more contiguous base pairs selected from the group consisting of: a deletion of nucleotides adenine and guanine at positions corresponding to positions 202-203 of SEQ ID NO:1, a deletion of adenine at a position corresponding to position 255 of SEQ ID NO: 1, and a deletion of nucleotides guanine and adenine at positions corresponding to positions 1142-1143 of SEQ ID NO:1; and e) an insertion of 1 or more contiguous base pairs selected from the group consisting of: an insertion of guanine and thymine at positions corresponding to 321-322 of SEQ ID NO:1; wherein the oligonucleotide is detectably labeled.
42. The oligonucleotide of claim 41, wherein the detectable label is selected from the group consisting of a radioactive label, a fluorescent label, an enzymatic label, a chemical label.
43. The oligonucleotide of claim 41, wherein the oligonucleotide has less than 500 bp.
44. The oligonucleotide of claim 41, wherein the oligonucleotide has less than 300 bp.
45. The oligonucleotide of claim 41, wherein the oligonucleotide has about 5 to about 100 bp.
46. The oligonucleotide of claim 41, wherein the oligonucleotide has about 5 to about 50 bp.
47. The oligonucleotide of claim 41, wherein the oligonucleotide specifically hybridizes to the parkin gene comprises a genetic alteration under stringent conditions, and does not hybridize to the wild type parkin gene under stringent conditions.
48. The oligonucleotide of claim 41, wherein the genetic alteration comprises a point mutation that results in a nonsense mutation.
49. The oligonucleotide of claim 48, wherein the nonsense mutation affects exon 7, exon 12, or a combination thereof.
50. The oligonucleotide of claim 48, wherein the point mutation comprises the point mutation 905T>A that introduces a stop codon at a position corresponding to the Cys268 residue encoded by the sequence of SEQ ID NO: 1, or the point mutation 1459G>A that introduces a stop codon at a position corresponding to the Trp453 residue encoded by the sequence of SEQ ID NO: 1.
51. The oligonucleotide of claim 41, wherein the genetic alteration is a point mutation that results in a missense mutation.
52. The oligonucleotide of claim 51, wherein the missense mutation causes a nonconservative change in the amino acid sequence encoded by the sequence of SEQ ID NO: 1.
53. The oligonucleotide of claim 51, wherein the missense mutation comprises a point mutation selected from the group consisting of 584A>T at a position corresponding to SEQ ID NO: 1 (Lys161Asn), 601G>A T at a position corresponding to SEQ ID NO:1 (Ser167Asn), 734A>T at a position corresponding to SEQ ID NO:1 (Lys211Asn), 867C>T T at a position corresponding to SEQ ID NO: 1 (Arg256Cys), 924C>T at a position corresponding to SEQ ID NO: 1 (Arg275Trp), 939G>A at a position corresponding to SEQ ID NO: 1 (Asp280Asn), 966T>G (Cys289Gly), 1084G>A at a position corresponding to SEQ ID NO: 1 (Gly328Glu), 1101C>T at a position corresponding to SEQ ID NO: 1 (Arg334Cys), 1281G>A T at a position corresponding to SEQ ID NO:1 (Asp394Asn), and 1390G>A T at a position corresponding to SEQ ID NO:1 (Gly430Asp).
54. The oligonucleotide of claim 51, wherein the missense mutation causes a conservative change in the amino acid sequence encoded by the sequence of SEQ ID NO: 1.
55. The oligonucleotide of claim 54, wherein the missense mutation comprises a point mutation selected from the group consisting of 1239G>C T at a position corresponding to SEQ ID NO: 1 (Val380Leu) and 1345C>A T at a position corresponding to SEQ ID NO:1 (Thr415Asn).
56. The oligonucleotide of claim 54, wherein the missense mutation affects a potential phosphorylation site of a polypeptide encoded by said isolated nucleic acid molecule.
57. The oligonucleotide of claim 56, wherein the point mutation is Thr415Asn.
58. The oligonucleotide of claim 41, wherein the genetic alteration comprises a deletion of one or more contiguous base pair(s) that causes a reading frame shift.
59. The oligonucleotide of claim 58, wherein the deletion is selected from the group consisting of: a deletion of the nucleotides adenine and guanine at positions corresponding to positions 202-203 of SEQ ID NO: 1, a deletion of the nucleotide adenine at a position corresponding to position 255 of SEQ ID NO:1, and a deletion of the nucleotides guanine and adenine at positions corresponding to positions 1142-1143 of SEQ ID NO:1.
60. The oligonucleotide of claim 58, wherein the genetic alteration comprises an insertion of one or more contiguous base pair(s) that causes a reading frame shift.
61. The oligonucleotide of claim 60, wherein the insertion is an insertion of nucleotides guanine and thymine at positions corresponding to positions 321-322 of SEQ ID NO:1.
62. A kit for detection of a genetic alteration of a parkin gene, comprising at least one of the oligonucleotides of claim 41.
Description
EXAMPLES
ALegend to the Figures
[0090]
[0091]
[0092]
[0093]
[0094] The peaks represent the exons produced by semi-quantitative multiplex PCR. The encircled figures indicate the height of the peaks. The graduated ruler above the electrophoretograms indicates the size of the PCR products in base pair.
[0095] Table 1: Oligonucleotides used for the ASO technique. The nucleotide changes in the sequence of the oligonucleotides are represented in bold and underlined. WT=wild type; V=variant.
[0096] Table 2: Summary of the mutations identified. The positions of the nucleotides are given according to the cDNA sequence published in the DNA Data Bank of Japan (DDBJ; accession number AB009973) and are illustrated in
[0097] Table 3: Clinical characteristics of patients as a function of the type of genetic alteration. The patients of the IT-020 family who are composite heterozygous for a missense mutation and a truncating mutation do not appear in the table. a: p<0.05 for the comparison between the patients with a homozygous deletion and the patients with truncating mutations.
[0098] Table 4: Frequency and consequences of the deletions/multiplications of exons
del=deletion, het=heterozygote; hom=homozygote
[0099] Table 5: Ratio of the results obtained in
BMaterials and Methods
[0100] 1. Families and Patients
[0101] In a first series of experiments, 38 families were selected according to the following criteria: parkinsonian syndrome reactive to levodopa, ii) starting age at most 45 years for at least one of the affected members, and iii) transmission compatible with an autosomal-recessive heredity.
[0102] In another series of experiments, 77 families were selected according to the following criteria (as indicated in Lcking et al, 1998; Abbas et al, 1999): i) presence of a parkinsonian syndrome with a good response to levodopa (30% improvement) in at least two members of a phratry (or only one if there is a notion of consanguinity); ii) absence of exclusion criteria such as Babinski's syndrome, ophthalmoplegia, dementia or dysautonomia occurring before two years of progression; iii) beginning 45 years in at least one of those affected; iv) heredity compatible with recessive autosomal transmission (several patients in a single generation with or without a notion of consanguinity). The families were from France (n=20), Italy (n=19), Great Britain (n=14), the Netherlands (n=9), Germany (n=9), Lebanon (n=2), Algeria (n=1), Morocco (n=1), Portugal (n=1), Vietnam (n=1).
[0103] Furthermore, 102 isolated cases, with no known consanguinity, were selected with the same clinical criteria. They were from France (n=31), Italy (n=23) and Great Britain (n=26), Germany (n=21)n the Netherlands (n=1).
All the patients were evaluated according to a standard protocol. The informed consent of all the participants was obtained in writing.
[0104] 2. Analysis of the Parkin Gene
[0105] The DNA of the 12 exons encoding the parkin gene was amplified by PCR from peripheral blood leukocytes, for each index case, according to the conditions described in Kitada et al. Briefly, the amplification was carried out on 100 ng of DNA, in the presence of 350 M of each dNTP, 350 M of each primer, and Taq polymerase. The amplification conditions are cycles at 94 C. for 30 sec, at 55-61 C. for 30 sec, and then at 68 C. for 30 sec. For exons 4 and 7, only the pair of intron primers was used. The sequence of the 12 exons was prepared on two strands with the primers used for the PCR amplification, with the sequencing kit Big Dye Terminator Cycle Sequencing Ready Reaction (ABI PRISM) and analyzed after electrophoresis on the ABI 377 sequencer with the sequence analysis 3.0 software (ABI PRISM).
[0106] The detection of the mutations in the samples and the analysis of a population of 45 control individuals was carried out by three techniques, which may be used alone or in combination(s): PCR/restriction with the appropriate restriction enzyme; ASO technique (Allele Specific Oligonucleotide), and polyacrylamide gel electrophoresis (PAGE) as illustrated in Table 2. More particularly, these techniques were carried out as described below.
[0107] The ASO technique: this approach consists in hybridizing two oligonucleotide probes with an amplified sample (for example by PCR), the first specific for and covering a genetic alteration, the second specific for and covering the corresponding wild-type region. Thus, in the presence of a mutated gene, only the first probe allows hybridization with the DNA fragment, whereas in the presence of a nonmutated gene, only the second probe allows hybridization with the DNA fragment. In the case of a heterozygous gene, a hybridization is obtained with each of the probes. This technique may also be carried out concomitantly with the amplification, using two pairs of primers, the first comprising a primer specific for and covering the corresponding wild-type region. In this embodiment, in the presence of a mutated gene, only the first pair allows the amplification of a DNA fragment, whereas in the presence of a nonmutated gene, only the second pair of primers allows the amplification of a DNA fragment. In the case of a heterozygous gene, an amplification product is obtained with each of the pairs of primers.
[0108] For carrying out this technique, 10 l of PCR product were denatured at 95 C. for 5 min, deposited on Hybond N+ nylon membranes (Amersham), and then microwave-fixed at 600 W for 2 min. The specific primers (or oligonucleotides) used for the detection (or, where appropriate, for the amplification), are described in the examples (see Table 1). For exon 3, the exon primers Ex3iFor (forward) and Ex3iRev (back) were used. The sequence of these primers is the following:
TABLE-US-00004 Ex3iFor: (SEQIDNo:6) 5-AATTGTGACCTGGATCAGC-3 Ex3iRev: (SEQIDNo:7) 5-CTGGACTTCCAGCTGGTGGTGAG-3
[0109] These oligonucleotides (including the primers, in the case of a simultaneous amplification), labeled with dCTP32 by means of the Terminal Transferase Kit (Boehringer Mannheim) were hybridized with the membranes at 44 C. overnight in a buffer consisting of 5SSC, 5Denhardts and 0.1% SDS. The membranes were then washed twice for 30 min in a 2SSC medium at 59 C. and exposed to an MP film (Amersham) for 3-6 hours.
[0110] PCR/restriction technique: this technique is based on the use of restriction enzymes whose digestion profile becomes modified because of the genetic alteration. Preferably, this technique therefore uses restriction enzymes whose site is modified (destroyed or created) by the genetic alteration. Thus, depending on the nucleic acid digestion profile (generally amplification product), it is possible to distinguish the presence or otherwise of the genetic alteration searched for. Of course, this technique is most particularly appropriate for the search for straightforward genetic alterations, causing a modification in an enzymatic cleavage site. For its use, 15 l of amplification product is digested in the presence of appropriate restriction enzyme(s), according to the manufacturer's recommendations. The particular enzymes used in the examples and the expected size of the restriction fragments are given in Table 2.
[0111] Polyacrylamide gel electrophoresis (PAGE) technique: this technique makes it possible to detect the presence of mutations by measuring the size of the amplification products. It is therefore most particularly appropriate for the detection of genetic alterations of the insertion or deletion type. For its use, a labeled forward primer (5-fluorescent, Hex) was used to amplify exon 2 of the parkin gene. The presence of the 202-203delAG alteration, resulting in a shorter PCR product (306 vs 308 bp) was established by measuring the size of the amplified fragment using an ABI377 automated sequencer equipped with Genescan 2.0.2 and Genotyper 1.1.1 software (ABI PRISM).
[0112] The numbering of the nucleotides used in the present application is given with reference to the sequence of the cDNA which exists in the DNA Data Bank of Japan (DDBJ; accession number: AB009973). The sequence is represented in
[0113] 3. Semi-Quantitative Multiplex PCR for the Detection of Deletions/Multiplications of Exons in the Homozygous and Heterozygous State
[0114] a) Principles
[0115] The detection of heterozygous deletions or multiplications of exons in the Parkin gene cannot be carried out by nonquantitative PCR. Thus, a semi-quantitative PCR which compares the relative amount of template DNA is sufficient to know if 50% of the template DNA is missing for one or more exons or, on the contrary in the case of a heterozygous or homozygous multiplication, if there is for example 50% (heterozygous duplication), 100% (homozygous duplication or heterozygous triplication) or 200% (homozygous triplication) of DNA in excess for one or more exons. To carry out this comparison, several exons from the same individual are simultaneously amplified, in a PCR reaction (multiplex PCR), the coamplified exons serving as internal standard for quantity. The PCR is carried out with fluorescent primers, such that the quantity of PCR product can be measured by the height of peaks on an automated sequencer (ABI Prism 377), as applied for example in the Applied Biosystems LOH (Loss of Heterozygosity) Assay. The quantity of PCR product (height of the peak) is directly linked to the quantity of template DNA as long as the PCR is in its exponential phase which means an absence of limitation by the available substrates. Each multiplex PCR, for a given combination of exons, produces a typical peak height distribution for a control individual as well as defined ratios between the different peaks.
[0116] A homozygous deletion of an exon will be demonstrated by the absence of the corresponding peak. If an exon is deleted in the heterozygous state, the corresponding peak will have half of its normal height, which will change the ratio between the deleted and nondeleted exons by a factor of 2 compared with a control (comparing the high value with the low value;
[0117] b) Establishment of the Appropriate Conditions for Multiplex PCR
[0118] During preliminary experiments, it was noted that the exons which exhibit the best amplification could negatively influence the amplification of other exons, for which the efficiency was not as good. Thus, the exons whose amplification efficiency was comparable were grouped together. Furthermore, as the size of the PCR product can influence the amplification yield (the short sequences being as a rule better amplified than the long sequences), the PCR products of comparable size were grouped together in the multiplex reaction. Thus, three combinations of exons were chosen:
Ex 4o (261 bp)+7o (239 bp)+8 (206 bp)+11 (303 bp),Comb 1:
Ex 5 (227 bp)+6 (268 bp)+8 (206 bp)+(165 bp) andComb 2:
Ex 2 (308 bp)+3i (243 bp)+9 (278 bp)+12 (255 bp)+C328Comb 3:
[0119] (external control of 328 base pairs).
[0120] The primers used are those described by Kitada et al (1998). For exon 3, a pair of exonic primers was used:
TABLE-US-00005 (SEQIDNo:8) For:5-(Hex)AATTGTGACCTGGATCAGC-3 and (SEQIDNo:9) Rev:5-CTGGACTTCCAGCTGGTGGTGAG-3.
[0121] The primers for C328 being:
TABLE-US-00006 TheprimersforC328being: TTRForHex: (SEQIDNo:10) 5-(Hex)ACGTTCCTGATAATGGGATC-3 and TTR328Rev: (SEQIDNo:11) 5-CCTCTCTCTACCAAGTGAGG-3.
[0122] In order to obtain peaks of comparable heights in each multiplex PCR and to be situated in the exponential phase for each exon, the PCR conditions were adjusted permanently (by partly following the recommendations of Henegariu et al (Henegariu et al, 1997). In particular, the hybridization and extension temperatures were reduced and the concentration of MgCl.sub.2 and the duration of the extension were increased. Furthermore, the concentrations of primers were adjusted from a standard concentration of 0.8 M, according to the amplification efficiency (the concentrations of primers being reduced for the exons which amplify well, and increased for the others).
[0123] Each combination of exons was tested in order to verify that the exponential phase was established, this being in two multiplex PCRs in parallel for the 3 combinations of primers, on a control individual with 22, 23 and 24 cycles. The peak heights were corrected for the variations in loadings according to the internal molecular weight marker (Applied Biosystems TAMRA 500 XL). The corrected peak heights were compared to the number of cycles, and represent, on a logarithmic scale, an ascending straight line which demonstrates that the exponential phase was established for the following conditions:
[0124] 5 minutes at 95 C. for one cycle,
[0125] 30 seconds at 95 C., 45 seconds at 53 C. and 2.5 minutes at 68 C. for 23 cycles,
[0126] 5 minutes at 68 C. for one cycle.
[0127] The reaction was carried out with 40 ng of DNA in a volume of 25 l of PCR solution, with 3 mM MgCl.sub.2, 0.2 mM dNTP and 1 U Taq/25 l. The concentration of each primer was:
Ex 2 (0.8 M), Ex 3 (0.4 M), Ex 4 (1.0 M), Ex 5 (0.6 M), Ex 6 (1.4 M), Ex 7 (0.44 M), Ex 8 (in comb 1:1.0 M and in comb 2:0.8 M), Ex 9 (0.4 M), Ex 10 (1.04 M), Ex 11 (0.8 M), Ex 12 (1.2 M) and C328 (1.92 M).
[0128] c) Applications of Multiplex PCR, Internal Controls and Electrophoresis
[0129] As a general rule, the multiplex PCRs were carried out at least in two parallel reactions for each individual. For each series of patient, at least one positive control (with a heterozygous deletion of known exons) and one negative control (control individual) were treated in parallel in order to obtain the normal and pathological values for each reaction premix, so as to avoid erroneous results due to possible differences in the premix (variation of pipetting). Two additional controls were added, which did not contain template DNA. 1.5 to 2.5 l of the PCR product were mixed with 4 l of loading buffer (comprising 0.3 l of the Applied Biosystems TAMRA 500 XL size marker). 1.5 l of this mixture was loaded onto a 4% denatured polyacrylamide gel containing 96 wells on an ABI 377 automated sequencer. The gels are analyzed by the GeneScan 3.1 and Genotyper 1.1.1 software packages (Applied Biosystems). The peak heights are measured as indicated in Genotyper. For the double peaks with one base pair difference (caused by the fact that Taq polymerase inconstantly adds an A to each end), the two peak heights are added. The ratios of each combination of peaks are calculated for each reaction, using the Excel 5.0 software (Table 5) and the mean values are calculated for two reactions.
[0130] d) Interpretation
[0131] For the deletions, the results are interpreted as pathological if the difference in ratio was a factor of at least 1.6 or 0.625 (=1/1.6) in all the respective ratios between the control and the case (ratio of the subject/ratio of the controlTable relating to
[0132] For the duplications, a change in the ratios by a factor of 1.30-1.65 or >1.75 is interpreted as a heterozygous or homozygous duplication respectively (by comparing the high value with the low value).
[0133] For a triplication, a change in the ratios by a factor of 1.6-2.4 or >2.6 is interpreted as a heterozygous or homozygous triplication respectively (by comparing the high value with the low value).
[0134] However, as the conditions were continuously adjusted during the development of the method, some of the results were obtained under slightly different conditions. These results are taken into account when they are clearly normal or pathological and reproducible. In ambiguous situations, the experiment was repeated under appropriate conditions.
[0135] 4. Analysis of Cosegregation and of a Control Population
[0136] a) Point Mutations
[0137] The variants of the Parkin sequence were tested for their cosegregation in the families (according to the availability of other samples) and for their presence in a population of controls without Parkinsonian syndrome (61 to 73 individuals). Because of the certainly pathogenic character of the 1142-1143delGA mutation, controls were not tested for this mutation. The techniques used are PCR and digestion with the appropriate restriction enzyme or polyacrylamide gel electrophoresis (PAGE) (see Table 2). When the variant did not cause any change in restriction site by itself, a site was artificially created with the aid of a primer with a mismatch. The primers were designed so as to introduce the change of base near the position of the sequence variant, so as to create a restriction site which includes this variant. The primers are indicated in the table below.
[0138] Modified primers (not complementary to the wild-type sequence for one base) for PCR:
TABLE-US-00007 Restriction Mutation enzyme PrimerF PrimerR Asp280Asn AlwI Ex70For 5-GGCAGGGAGTAGCCAAGTTGAGGAT-3 (SEQIDNO.12) wild-typesequenceG Arg334Cys BstUI Ex9For 5-AGCCCCGCTCCACAGCCAGCGC-3 (SEQIDNO.13) wild-typesequence
The change in base pair introduced is underlined by comparison with the wild-type sequence.
[0139] b) Deletions or Multiplications of Homozygous or Heterozygous Exons
[0140] The cosegregation of a deletion or of a multiplication of exons in the families was analyzed with the aid of the methods described above. A control population was not tested because of the highly probable pathogenic character of the mutations, which causes an internal deletion of the protein, with or without a reading frame shift.
[0141] 5. Linkage Analysis
[0142] To test the linkage to the PARK2 locus, four microsatellite-type markers, situated near the locus, were tested (D6S1579, D6S411, D6S1550 and D6S305) as described by Tassin et al (1998).
CResults
[0143] a) In a First Series of Experiments, the Analysis of the Parkin Gene was Carried Out in the Index Case of 38 Families with AR-JP which Contain 87 Patients.
[0144] 1. Detection of Deletions of Exons
[0145] The amplification of the exons revealed the presence of a deletion in the homozygous state in three families: deletion of exon 3 in a French family (SAL-024) and a Portuguese family (SAL-711), and of exons 8 and 9 in an Algerian family (DEL-001). These deletions are transmitted with the disease because they are detected in each family in the homozygous state in all patients but not in the healthy related ones sampled (
[0146] 2. Detection of Point Mutations
[0147] The sequence analysis in the families without homozygous deletion revealed the presence of 16 variants of the nucleic sequence: 12 in the exons and 4 in the introns (Tables 2 and 3,
[0148] The variants identified in introns 2, 3, 6 and 7 (IVS2+25T>C (272+25T>C), IVS3-20C>T (514-20C>T), IVS6+19T>C (835+19T>C) and IVS7-35A>G (973-35A>G)) constitute polymorphisms (Table 2). They are not located near splicing sites and are detected in the control chromosomes.
[0149] 3. Functional Domains of Parkin
[0150] A study of the functional domains of parkin was undertaken by analysis and comparison of sequences. This study shows that the conservative change in amino acid Thr415Asn is located in the consensus sequence of a cAMP- and cGMP-dependent protein kinase (KKTT) and in the phosphorylation site of a protein kinase C (TTK). This study shows, in addition, that the nonsense mutation Trp453Stop is located in an N-terminal myristoylation site (GCEWNR).
[0151] 4. Phenotype Genotype Correlations
[0152] The homozygous deletions and the point mutations were detected in 12 families which contain 26 patients. The average age at onset is 36.7 years with extremes of 7 to 56 years (Table 3). The comparison between the families according to the functional consequences of the mutations (homozygous deletion, truncating mutation and missense mutation) does not reveal any significant difference in the age at onset, in the severity or the frequency of the associated signs, except for tremor which is significantly less frequent in families with a homozygous deletion, compared with families with truncating mutations (Table 3).
[0153] b) Detection of New Point Mutations
[0154] Eight new point mutations in exons were identified, of which six are missense mutations one truncating and one nonsense: 734A>T (Lys211Asn) in exon 6, 905T>A (Cys268Stop), 939G>A (Asp280Asn) and 966T>G (Cys289Gly) in exon 7, 1084G>A (Gly328Glu), 1142-1143delGA and 1101C>T (Arg334Cys) in exon 9 and 1390G>A (Gly430Asp) in exon 12. Five of the missense mutations lead to nonconservative amino acid changes and one to a conservative change (Cys289Gly). Furthermore, a deletion of five base pairs in intron 8, located at positions 21 to 17 relative to exon 9 was detected. All these sequence variants were not detected in 61 to 73 control individuals (the 1142-1143delGA mutation was not tested) and do not therefore represent polymorphisms. The results are detailed in Table 2.
[0155] c) Detection of New Homozygous Deletions of Exons
[0156] Homozygous deletions of exons were detected in 3 families in addition to the deletions previously reported by Hattori et al (1998a) and Lcking et al (1998) for exon 3 and by Hattori et al (1998a) for exons 3+4. These deletions relate to exons 3 (FDP-ANG-GEO-141), 3+4 (IT-064) and 5+6 (SPD-LIB-HAG-076). The consequences of the deletions of exons on the reading frame and their relative frequency in the sample are indicated in Table 4.
[0157] d) Detection of Homozygous and Heterozygous Duplications/Triplications
[0158] Five new types of duplications of exons were detected: a duplication of exon 3 in the homozygous state (SPD-NIC-AIT-091) and a duplication of exon 3 in the heterozygous state (SAL 399 213). In addition, heterozygous duplications of exon 6 (FPD-LIL-CHA-171), of exon 7 (DE 4001) and of exon 11 (SAL 399 213) were detected. Two types of triplication were detected: a triplication of exon 2 in the homozygous state (RM 347) and a the heterozygous state (RM 330).
[0159] e) Detection of New Heterozygous Deletions
[0160] Thirteen different combinations of heterozygous deletions of exons were detected in 21 families. The following deletions were observed: exons 2, 2+3, 2+3+4, 3, 3+4, 36, 39, 4, 5, 6, 6+7, 7+8+9 and 8. The deletions of exons 2, 2+3, 2+3+4, 3-6, 3-9, 6, 6+7, 7+8+9 and 8 are new.
[0161] For two families (Sal-Hab-436 and UK 12416), it was not possible to establish with certainty if the heterozygous mutations of exons 2+3 or 6-7, respectively, were situated on the same chromosome or if they were composite heterozygous cases because of the absence of DNA for other members of these families. The consequences of the deletions and of the multiplications of exons described on the reading frame and their relative frequency in our sample are indicated in Table 4.
[0162] f) Recurring Point Mutations
[0163] Five point mutations were detected in more than one family. These mutations are 202-203delAG (in the heterozygous or homozygous state in 5 families), 255delA (in the homozygous or heterozygous state in 6 families), Lys211Asn (in the heterozygous state in 2 families), and Arg275Trp (in the heterozygous state in families).
[0164] g) Frequencies of the Different Types of Mutations and of the Composite Heterozygotes
[0165] Among the families with Parkin mutations, homozygous deletions of exons were detected in 8 families, point mutations in the homozygous state in 10 families, a duplication of homozygous exon in one family and a triplication of exon in one family. The patients from 21 families were composite heterozygotes for two different mutations (3 times for the different point mutations, 6 times for a point mutation and an exon deletion, twice for a point mutation and a duplication, once for a triplication and an exon deletion, once for two different duplications of exon and 6 times for two different deletions of exons; see example
DDiscussion
[0166] The present invention relates to variants of the Parkin gene, their diagnostic and/or therapeutic use, as well as techniques for the detection of alterations (in particular of deletions of heterozygous exons and of multiplications of exons) of the Parkin gene.
[0167] The detection of different causal genetic alterations (in particular of homozygous deletions, point mutations, insertions and multiplications of exons) demonstrate that the abnormalities in the parkin gene constitute a frequent cause of AR-JP.
1. FIRST STUDY ON 38 EUROPEAN FAMILIES
[0168] A first study made it possible to demonstrate the existence of deletions, mutations and insertions in the parkin gene.
[0169] The pathogenic role of the homozygous deletions appears to be easy to establish. In the 2 mutations described, deletions of exon 3 and of exons 8-9, the loss of the exon is accompanied by a reading frame shift leading to the appearance of a premature stop codon. In the absence of alternative splicing, a truncated protein results therefrom.
[0170] Eight of the exon variants constitute causal mutations. First, these mutations segregate with the disease in the families. Secondly, these variants are not detected by ASO, PAGE or PCR/restriction in 90 control chromosomes. Thirdly, the functional consequences of the mutations appear to be deleterious. It is easy to understand that the 4 truncating point mutations (Gln34Arg(Stop37), Asn52Met(Stop81), Trp74Cys(Stop81), Trp453Stop) detected in the homozygous state in the patients of 3 of the 5 families will cause a loss in the parkin function in accordance with the autosomal-recessive transmission of the disease. Three of the four missense mutations cause nonconservative changes in amino acids. One of them (Lys161Asp) is associated with a truncating mutation on the other allele which reinforces the assumption of a pathogenic role. A missense mutation is conservative (Thr415Asn), but affects a potential phosphorylation site. Three of the missense mutations are present in the heterozygous state in patients whose other mutation has not been characterized. It is probable that deletions of one or more exons in the heterozygous state are involved which cannot be visualized with the techniques used for this study.
[0171] The abnormalities detected in the parkin gene are varied and there are no hot spot mutations. It should be noted that the truncating point mutations preferably correspond to the N- and C-terminal regions of parkin (comprising in particular the ubiquitin-like and ring RING-finger units, respectively) whereas the missense-type mutations affect the central region. Only two of the 11 mutations described in this first study are found in several families. The homozygous deletion of exon 3 is detected in the French SAL-024 and Portuguese SAL-711 families. The mutation with a reading frame shift 202-203delAG (Gln34Arg(Stop37)) is visualized in the heterozygous state in the Italian IT-020 and English UK-086 families. The different 2.0 origin of the families is in favor of the hypothesis for the independent occurrence of these mutations.
[0172] The mutations described affect families from 6 countries: Algeria, Germany, England, France, Italy and Portugal. The study of the phenotype in the families with a mutation shows that the clinical spectrum associated with the abnormalities of parkin is broader than in the Japanese families [Kitada et al., 1998]. These results confirm the observations made in the European and North African families studied by genetic linkage [Tassin et al., 1998]. The age of onset is above 50 in several patients, ranging up to 56. Certain clinical signs such as dystonia or pyramidal signs in the lower limbs are not always present in the carriers of mutation even after periods of evolution of several decades. Overall, the phenotype remains very similar between the groups of patients classified according to the functional consequences of the mutations. However, the presence of painful dystonia episodes appears to be encountered exclusively in patients carrying homozygous deletions. The absence of a significant difference for the age of onset, the severity and the frequency of the associated signs between the truncating point mutations and the missense mutations suggests that the modified amino acids in the latter play an important role in the physiology of parkin.
[0173] In conclusion, this first study underlines the frequency of the mutations in the parkin gene in early-onset familial parkinsonian syndromes in Europe. Abnormalities in this gene are also responsible for more tardive or atypical parkinsonian syndromes. The role of mutations of parkin or of its polymorphisms in the isolated cases remains to be determined. The mutations detected are very diverse both by their nature and by their location. The study of their location in parkin suggests that many regions of the protein contribute to its as yet unknown function.
2. METHOD FOR THE DETECTION OF DELETIONS OF EXONS AND MULTIPLICATIONS OF EXONS
[0174] For the first time, the detection of deletions of exons in the heterozygous state and of multiplications of exons (for example duplication, triplication) in the homozygous and heterozygous state in the Parkin gene is described. This aspect is advantageous because exon deletions are relatively frequent (see later). As a method of detection, a semiquantitative multiplex PCR protocol was chosen and developed. This method had previously been validated for gene assay, for example for the detection of deletions of the PMP22 gene (Poropat and Nicholson, 1998), provided that the PCR amplification is in the exponential phase. In all these experiments, the choice of coamplified controls which serve as a standard for the quantification is critical (Prior, 1998). In the experiment, the nondeleted exons serve as internal controls in the multiplex PCR amplification in the same individual. Combinations of 4 or 5 exons were chosen so as not to contain more than 2 adjacent exons, because such exons cannot serve as controls in the case of a deletion of the two. The exon on a different gene (Transthyretin) was coamplified in one of the three combinations in order to identify heterozygous deletions of the entire Parkin gene. This external control was indirectly represented in the other two combinations, which include exons on either side of exon 9; the latter being tested with Transthyretin.
[0175] The results obtained by this method were very reproducible and the abnormal results show differences in the ratios of a factor, which corresponds to that expected in theory. These results show that this is a simple and validated method for rapid screening. Furthermore, small deletions or insertions in the PCR product, which are relatively frequent (see below) may be simultaneously detected by this method.
3. DELETIONS OF EXONS AND MULTIPLICATIONS OF EXONS
[0176] It was possible to identify four duplications of exons and one triplication of exons which had never been described in the Parkin gene before, but whose relative frequency is low. Furthermore, 10 combinations of new deletions of exons were identified with, for the first time, the demonstration of the deletions which carry exon 2. The relative frequency of the point mutations and of the deletions of exons was estimated at about 50%. Thus, the deletions of exons (heterozygous or homozygous) may represent up to 50% of the Parkin mutations, emphasizing the importance of the technique described here. In fact, this technique has made it possible to detect mutations in 26 of the 53 families. Thus, in the sample studied, the point mutations and the deletions in the Parkin gene have the same frequency, whereas the deletions of exons are predominant in the Japanese population (Hattori et al, 1998). The functional consequences of the deletions or of the multiplications of exons described (reading frame shift or deletion/multiplication in phase) were deduced from the published cDNA sequences for Parkin (Kitada et al, 1998) and are speculative, because the absence of a PCR product does not indicate that there is necessarily a deletion of the exon in its entirety (see above). However, the pathological role of the modifications detected is highly probable, because they are transmitted with the disease in the families which have been able to be tested, they are associated with point mutations in composite heterozygotes and the deletions are identified with a frequency similar to that of point mutations. Likewise; in the isolated cases, the frequency of the heterozygous deletions and of the point mutations is similar. In the case of exon 3, exonic primers were used, which demonstrate the alteration of this exon when there is no PCR product. Furthermore, in some of the cases, several juxtaposed exons were simultaneously deleted, which is an argument for a large genomic deletion.
[0177] Heterozygous deletions or multiplications of the entire Parkin gene were not observed. This is probably a rare event given the very large size (about 500 kb) of this gene (Kitada et al, 1998).
[0178] The exon deletions observed frequently effect exons 3 to 5. This observation has been confirmed in European families. Furthermore, it has been demonstrated that exon 2 alone or associated with others is also frequently involved in European families (Table 2).
4. NEW POINT MUTATIONS
[0179] The identification of 8 new point mutations (6 of the missense type, 1 truncating and 1 of the nonsense type) increases the diversity of the point mutations in the Parkin gene. The mutations described are pathogenic, as the segregation with the disease has shown, and are not detected in 122 to 147 control chromosomes (mutation 1142-1143delGA not included). Even if the Cys289Gly change is conservative, this change in amino acid may have substantial deleterious consequences, if the cysteine at position 289 is involved in a disulphide bridge, which is important for the function of the protein.
[0180] Interestingly, 2 patients of the UK-040 family exhibit 3 different mutations (see Table 5): one Arg334Cys missense mutation in exon 9 in the homozygous state, one homozygous deletion of 5 base pairs at position 17 to 21 of intron 8, and one nonconservative Asp280Asn missense mutation in the heterozygous state. It may be suspected that the Arg334Cys mutation in the homozygous state is causal, but the deletion of five base pairs in the homozygous state, near the acceptor splicing site of exon 9, could also have functional consequences.
[0181] Five point mutations are present in several families analyzed. The three most frequent are 255delA (detected in 6 families) and 202-203delAG (found in 5 families) and Arg275Trp (detected in 5 families). A foundation effect could be suspected for the 255delA mutation which affects 5 French families. However, this hypothesis can only be verified by analysis of the haplotypes.
5. EPIDEMIOLOGICAL GENETICS
[0182] The results obtained show that 34 of the 77 families with an early-onset parkinsonian syndrome exhibit mutations of the Parkin gene, emphasizing the importance of this gene in European families. The detection of mutations in 18 of the 102 cases isolated and analyzed is more difficult to interpret because the number is smaller and the analysis of some cases is not complete. However, it is striking to note that the age at onset of the 7 cases for which it is known is particularly early (13 to 22 years) and that there are very few cases with very early onset without mutation in the Parkin gene (for example IT-NA-JMP-3). This result suggests that the frequency of the mutations of the Parkin gene in isolated cases increases when their age decreases, especially before the age of 25. The observation of mutations in the Parkin gene in isolated cases is not surprising if it is considered that in small families, an autosomal recessive disease has every chance of appearing as an isolated case. Analyzing a larger sample will be useful for determining precisely the frequency of the Parkin mutations in the isolated cases, according to the age at onset.
[0183] Mutations were identified in families from a wide variety of origins: France, Italy, Great Britain, Germany, The Netherlands, Algeria, Portugal. These results show that the mutations in the Parkin gene are detected in all the populations tested so far.
6. PATIENTS WITH AN ABNORMALITY IN THE PARKIN GENE IN THE HETEROZYGOUS STATE
[0184] Although the technique for the detection of heterozygous deletions of exons or of multiplications of exons allowed us to identify composite heterozygous cases, in about of the families (13 out of 53), a single mutation was detected. This includes 6 cases with a point mutation in the heterozygous state and 7 with an exon deletion in the heterozygous state. The pathogenic role of these mutations is highly probable because they cause a nonconservative change in amino acid or a truncated protein. Furthermore, one of these mutations of the missense type (Arg275Trp) is associated with another heterozygous point mutation (Gly430Asp) and with heterozygous exon deletions (exon 3-6 or exon 5+6), carried by the other allele in three different families. The absence of detection of a mutation on the other allele in 13 families suggests that a second undetected mutation affects another region of the gene. This hypothesis is strengthened by the fact that in 6 families probably linked to the PARK2 locus, because the patients are haploidentical for 4 markers for the region, no mutation was detected. Thus, other regions of the gene could be affected, such as the promoter regions, the untranslated 5 and 3 regions, or intron sequences.
7. GENETIC HETEROGENEITY OF THE EARLY ONSET AUTOSOMAL RECESSIVE PARKINSONIAN SYNDROMES
[0185] In 5 of the 77 families, it has been possible to exclude a genetic linkage at the Parkin locus. Furthermore, no mutation was identified in 21 families for which this locus could not be conclusively excluded. These results suggest that there may be at least one other locus for families with an early onset autosomal recessive parkinsonian syndrome in Europe. This hypothesis had been proposed by Leroy et al (1998), which reports a family with two branches, of which one exhibits deletions of the Parkin gene, whereas the other does not exhibit either these deletions or the same haplotype, which excludes a linkage to this locus.
8. CONCLUSIONS
[0186] A novel method for the detection of heterozygous deletions of exons or multiplications of exons is reported. In particular, the duplications/triplications of exons and deletions of exon 2 and of other combinations of exons are novel. In combination with the sequencing of exons, it has been possible to identify eight novel point mutations and an intron deletion which could affect a splicing site. Thus, 34 of the 77 families analyzed (about 50%) exhibit mutations in the Parkin gene. Furthermore, the mutations in this gene were detected in 19 isolated cases. In the European population, the proportion of point mutations and deletions of exons appear to be identical. Two mutation hot points which correspond to deletions at exons 3 to 5 and to three point mutations (202-203delAG, 255delA and Arg275Trp) were in addition detected.
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TABLE-US-00008 TABLE1 Posi- Nucleotide tion change Oligonucleotidesequence Ex3 321-322insGT WT:5TGCAGAGACC-GTGGAGAAAA-3 (SEQIDNo:14) V:5GCAGAGACCGTGTGGAGAAA-3 (SEQIDNo:15) Ex4 584A> T WT:5-GCCGGGAAAACTCAGGGTA-3 (SEQIDNo:16) V:5-GCCGGGAAATCTCAGGGTA-3 (SEQIDNo:17) Ex7 867C> T WT:5-TGCAACTCCCGCCACGTGA-3 (SEQIDNo:18) V:5-TGCAACTCCTGCCACGTGA-3 (SEQIDNo:19) Ex10 1239G> C WT:5-TGCAGTGCCGTATTTGAAG-3 (SEQIDNo:20) V:5-TGCAGTGCCCTATTTGAAG-3 (SEQIDNo:21) Ex11 1345C> A WT:5-AGAAAACCACCAAGCCCTG-3 (SEQIDNo:22) V:5-AGAAAACCAACAAGCCCTG-3 (SEQIDNo:23)
TABLE-US-00009 TABLE 2 Amino acid changed Expected length Nucleotide (position of the Detection of the fragment changed stop codon) Type of mutation technique (bp) Exon Ex2 202-203delAG Gln34Arg(Stop37) reading frame PAGE WT: 308 V: 306 Ex2 255delA Asn52Met(Stop81) reading frame Fok 1 WT: 278 + 30 creation of the V: 222 + 57 + 30 site Ex3 321-322insGT Trp74Cys(Stop81) reading frame ASO Ex4 584A > T Lys161Asn missense ASO (nonconservative) Ex4 601G > A Ser167Asn missense Alw NI WT: 164 + 97 (nonconservative) loss of the site V: 261 Ex6 734A > T Lys211Asn missense Dra I WT: 171 + 98 (nonconservative) loss of the site V: 269 Ex7 867C > T Arg256Cys missense ASO (nonconservative) Ex7 905T > A Cys268Stop nonsense Dde I WT: 141 + 100 gain of the site V: 117 + 100 + 24 Ex7 924C > T Arg275Trp missense Sau3A I WT: 142 + 97 (nonconservative) loss of the site V: 239 Ex7 939G > A Asp280Asn missense Alw I with WT: 153 + 30 (nonconservative) mismatched primer V: 183 loss of the site Ex7 966T > G Cys289Gly missense BstN I WT: 177 + 64 (nonconservative) gain of the site V: 118 + 64 + 59 Ex9 1142-1143delGA Arg348Glu(Stop368) reading frame PAGE WT: 278 V: 276 Ex9 1084G > A Gly328Glu missense Mnl I WT: 124 + 80 + 74 (nonconservative) gain of the site v: 124 + 74 + 60 + 20 Ex9 1101C > T Arg334Cys missense BstU I with WT: 123 + 21 (nonconservative) mismatched primer V: 144 loss of the site Ex10 1239G > C Val380Leu missense ASO (conservative) Ex11 1281G > A Asp394Asn missense Taq I WT: 221 + 84 (nonconservative) loss of the site V: 303 Ex11 1345C > A Thr415Asn missense ASO (conservative) Ex12 1390G > A Gly430Asp missense Mnl I WT: 191 + 65 (nonconservative) loss of the site V: 256 Ex12 1459G > A Trp453Stop nonsense Nla IV WT: 142 + 17 + 35 + 61 loss of the site V: 159 + 35 + 61 intron Intron 2 IVS2 + 25T > C BstN I WT: 308 bp (272 + 25T > C) creation of the V: 264 + 44 bp site Intron 3 IVS3 20C > T Mnl I WT: 201 + 60 (514 20C > T) loss of the site V: 261 Intron 7 IVS7 35A > G Mae III WT: 206 (973 35A > G) creation of the V: 159 + 47 site Intron 8 IVS8 21 17del splice site PAGE (1035 21 17del) TCTGC
TABLE-US-00010 TABLE 3 CLINICAL CHARACTERISTICS OF FAMILIES WITH MUTATIONS IN THE PARKIN GENE Homozygous Missense Truncating deletions mutations mutations Total Families (patients) 3 (8) 3 (8) 4 (9) 10 (25) Average age at onset (extremes) 30 16 (7-55) 44 9 (30-56) 37 6 (29-45) 37 12 (7-56) Average duration of evolution 13 6 (3-20) 13 7 (0.5-27) 16 10 (3-29) 14 8 (0.5-29) (extremes) Hoehn and Yahr scale 3.1 1.2 2.6 0.8 2.0 0.6 2.5 0.98 Akinesia 8/8 8/8 8/9 96% Rigidity 8/8 8/8 9/9 100% Tremor 3/8 4/8 8/9 60% Dystonia 4/8 0/5 1/7 25% Good reaction to levodopa 7/7 (1) 6/6 (2) 7/7 100% (case de novo) Dyskinesia 4/7 5/6 6/9 68% Fluctuations.sup.a 7/7 ND 2/6 62% Sharp reflexes in the 2/8 3/4 0/6 28% lower limbs
TABLE-US-00011 TABLE 4 exon(s) deleted/multiplied Number of families Consequences 2 del 3 het Reading frame shift 2 triplication 1 hom + 1 het No reading frame shift 2 + 3 del 1 het No reading frame shift 2 + 3 + 4 del 1 het Reading frame shift 3 del 3 hom + 7 het Reading frame shift 3 duplication 1 hom + 1 het Reading frame shift 3 + 4 del 1 hom + 3 het No reading frame shift 3 6 del 1 het Reading frame shift 3 9 del 1 het No reading frame shift 4 del 1 hom + 3 het Reading frame shift 5 del 3 het No reading frame shift 5 + 6 del 2 hom Reading frame shift 6 del 1 het Reading frame shift 6 duplication 1 het Reading frame shift 6 + 7 del 1 het Reading frame shift 7 duplication 1 het Reading frame shift 7 + 8 + 9 del 1 het Reading frame shift 8 del 1 het Reading frame shift 8 + 9 del 1 hom Reading frame shift 11 duplication 1 het Reading frame shift
TABLE-US-00012 TABLE 5 C328/ C328/ Ex3i/ Case 3i 12 9 2 C328 3i 12 C328/9 C328/2 12 Ex3i/9 Ex3i/2 Ex12/9 Ex12/2 Ex9/2 T2 743 838 1040 935 455 0.61 0.54 0.44 0.49 0.89 0.71 0.79 0.81 0.90 1.11 FR 155 5 608 1245 1588 1466 635 1.04 0.51 0.40 0.43 0.49 0.38 0.41 0.78 0.85 1.08 T2/FR 0.59 1.06 1.09 1.12 1.82 1.87 1.92 1.03 1.06 1.03 155 5 FR 155 6 759 861 1120 540 498 0.66 0.58 0.44 0.92 0.88 0.68 1.41 0.77 1.59 2.07 T2/FR 0.93 0.94 0.98 0.53 1.01 1.05 0.57 1.05 0.56 0.54 155 6 FR 155 8 597 1185 1467 766 623 1.04 0.53 0.42 0.81 0.50 0.41 0.78 0.81 1.55 1.92 T2/FR 0.59 1.03 1.03 0.60 1.76 1.76 1.02 1.00 0.58 0.58 155 8 FR 155 9 495 1200 1438 754 688 1.39 0.57 0.48 0.91 0.41 0.34 0.66 0.83 1.59 1.91 T2/FR 0.44 0.95 0.91 0.53 2.15 2.08 1.21 0.97 0.56 0.58 155 9