JP2006166742A - Two kinds of lipoprotein lipase (lpl) genetic mutations to cause hypertriglyceridemia and lpl mutation detection kit for diagnosing hypertriglyceridemia using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accumulate LPL genetic mutations in Japanese so as to more simply carry out cause elucidation of hypertriglyceridemia and LPL gene analysis useful for tailor-made prevention of hypertriglyceridemia and to obtain a specific diagnostic kit necessary for gene diagnosis targeting the mutations. <P>SOLUTION: The simple primary hypertriglyceridemia diagnosis method comprises finding new mutations S251F and C283F in an LPL gene of subject of Japanese hypertriglyceridemia, providing a probe for detecting the mutations based on the base sequence of LPL gene having mutations in newly found exon 6 and amplifying a base sequence containing a mutation part to detect mutation. The diagnostic kit is obtained. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a lipoprotein lipase gene mutation (S251F and C283S) useful for diagnosis of the cause of hypertriglyceridemia. Furthermore, the present invention relates to a genetic diagnostic reagent for detecting these mutations.

  Lipoprotein lipase (hereinafter referred to as LPL) is a glycoprotein of about 60 kilodaltons, which hydrolyzes triglycerides in chylomicrons produced in the intestine and ultra-low density lipoproteins produced in the liver. It is an enzyme that produces fatty acids.

  Homozygous patients with LPL gene abnormalities may exhibit marked hypertriglyceridemia of 1,000 mg / dl or more with hyperchylomicronemia, resulting in acute pancreatitis and death. Therefore, it is important to clarify the etiology when hyperchylomicronemia or pancreatitis develops in order to determine the subsequent treatment policy.

  In the case of the LPL gene-deficient heterozygote, the LPL activity is about half that of a normal person, but hyperinsulinemia does not result in hyperinsulinemia, and if there is a healthy lifestyle without drinking habits, hypertriglyceridemia (Type IV hyperlipidemia) does not develop. However, it has a genetic background of LPL gene-deficient heterozygotes, and when accompanied by bad lifestyle such as alcohol drinking, lack of exercise, obesity, etc., triglyceride synthesis in the liver is increased and LPL activity is half the level of normal subjects. However, it is insufficient to hydrolyze triglycerides in the blood to maintain a normal lipemia state, and hypertriglyceridemia (type IV hyperlipidemia) develops. If this continues, low density lipoprotein (LDL) will be reduced in size and density, which will be a risk factor for arteriosclerosis and will become a risk factor for heart disease and the like in the future. The etiology of hypertriglyceridemia involves genetic and environmental factors other than LPL gene abnormalities, and not all of the causes have been elucidated. Elucidation of the etiology of hypertriglyceridemia is very important in order to make sure that the subsequent treatment policy is decided and that the patient accepts the treatment and improves the lifestyle. is important.

  In the future, before becoming hypertriglyceridemia, for example, knowing whether you are LPL gene abnormal homozygote or heterozygote before 20 years old who wear bad living habits such as drinking alcohol It will be possible to prevent the development of hypertriglyceridemia, arteriosclerosis and heart disease that may occur in the future. This is the ultimate tailor-made medicine. Rather than improving bad habits once they have been acquired, it is easier to avoid such lifestyles from the beginning. In particular, for women, it is normal for a person with normal LPL genes to become hypertriglyceridemia if they become pregnant, so knowing their LPL gene status in advance prior to strict nutritional management during pregnancy This can prevent the occurrence of pancreatitis and miscarriage caused by hypertriglyceridemia.

  The human LPL gene is located on the short arm of chromosome 8 (p22), consists of 10 exons, and has a length of 35 kb.

About 22 mutations in the LPL gene have been found so far in Japanese. Only a few of them are found outside of Japan, indicating that there are differences in the types of mutations found due to differences in race. In order to easily and accurately diagnose the cause of hypertriglyceridemia in Japanese, it is important to accumulate more lipoprotein lipase gene mutations in Japanese.
Wion et al. Science, 1987, 235, 1638-1641 Lo JY, Smith LC, Chan L, Lipoprotein lipase: role of intramolecular disulfide bonds in enzyme catalysis.Biochemical and Biophysical Research Communications, 206, 266-271, 1995. A. Mori et al. Clinical Biochemistry, Vol. 33, pp. 323-327 (2000)

  Diagnosing whether the cause of hypertriglyceridemia is due to LPL gene abnormality is important in determining the therapeutic strategy for hypertriglyceridemia. Furthermore, in the future, as a tailor-made prevention of hypertriglyceridemia, it is desirable that LPL gene diagnosis is performed before the onset of this disease. However, conventional gene analysis methods such as gene sequencing are not practical because they require time and cost to apply to general diagnosis, and development of simpler diagnostic methods is desired.

  Therefore, the object of the present invention is to use the LPL gene mutations accumulated in Japanese in order to more easily perform the LPL gene analysis useful for elucidating the origin of hypertriglyceridemia and tailor-made prevention of hypertriglyceridemia. It is to simplify the LPL gene diagnosis by performing gene diagnosis targeting these mutations.

  The present inventors have accumulated 17 types of LPL gene mutations from Japanese hypertriglyceridemic patients. In addition, 22 types of LPL gene mutations in Japan are currently accumulated, including 5 types of LPL gene mutations found only in other facilities in Japan. Incidentally, when these mutations exist, the LPL enzyme activity becomes zero or almost zero.

As a result of further searching for an LPL gene abnormality in Japanese hypertriglyceridemia to achieve the above-mentioned object, the present inventors have found that there are two new mutations previously unknown to exon 6, the S251F mutation. The C283S mutation was discovered, and the present invention was completed based on this fact.
That is, the first of the present invention is a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 1, or a partial sequence comprising the 58th base, preferably a polynucleotide comprising 10 nucleotides or more, more preferably 20 nucleotides or more. Or its complementary sequence. In the base sequence of human LPL gene exon 6, the 58th base, which is c in the wild type, is t, and as a result of this missense mutation (named S251F), the 251st amino acid sequence of human LPL (hereinafter referred to as this) In the specification, the numbering of amino acid residues of LPL is according to Wion et al. (Non-patent Document 1), whereas serine (Ser) in the wild type is phenylalanine (Phe) and loses LPL activity. .

  The second of the present invention is a polynucleotide comprising the DNA sequence represented by SEQ ID NO: 3, or a partial sequence thereof comprising the 154th base, preferably a polynucleotide comprising 10 nucleotides or more, more preferably 20 nucleotides or more Complementary sequence. In the base sequence of human LPL gene exon 6, the 154th base which is g in the wild type is c, and as a result of this missense mutation (named C283S), the 283rd amino acid sequence of human LPL is the wild type. Although it is cysteine (Cys), it becomes serine (Ser) and loses LPL activity.

  Based on the nucleotide sequence of the polynucleotide having the S251F mutation or the C283S mutation of the present invention, it becomes possible to prepare a probe, a primer, or the like useful for diagnosis of primary hypertriglyceridemia due to the mutation at the site.

  A third aspect of the present invention is a hypertriglyceridemia characterized by comprising a primer and a mutation detection reagent for amplifying and detecting the sequence represented by SEQ ID NO: 1, or a partial base sequence including the 58th base thereof. A diagnostic kit for identifying the etiology.

One of the preferred embodiments of the diagnostic kit for identifying the cause of hypertriglyceridemia of the present invention comprises, as primers, an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5 and a base sequence represented by SEQ ID NO: 6. It contains an oligonucleotide and contains the restriction enzyme HinfI as a detection reagent.

  A fourth aspect of the present invention is a hypertriglyceridemia characterized by comprising a primer for amplifying and detecting the sequence represented by SEQ ID NO: 3, or a partial base sequence containing the 154th base thereof, and a mutation detection reagent. A diagnostic kit for identifying the etiology.

One of the preferred embodiments of the diagnostic kit for specifying the etiology of this triglyceridemia is an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 5 and the oligonucleotide consisting of the base sequence represented by SEQ ID NO: 6 as primers. And a restriction enzyme Bst XI as a detection reagent.

  As described above, according to the present invention, a polynucleotide useful for diagnosing primary hypertriglyceridemia due to two types of mutations can be provided. Further, it becomes possible to easily prepare probes, primers and the like useful for diagnosis of primary hypertriglyceridemia based on the base sequences of these polynucleotides, and the cause of hypertriglyceridemia due to mutation of the LPL gene. It is possible to provide a diagnostic kit that allows easy diagnosis.

  As already described, the nucleotide sequences represented by SEQ ID NOs: 1 and 3 are the nucleotide sequences of exon 6 of the LPL gene newly discovered by the present inventors from hypertriglyceridemic individuals. In the base sequence represented by SEQ ID NO: 1, the 58th base is c in the wild type, but since it is t, the amino acid sequence 251 of human LPL (19th in SEQ ID NO: 2) is serine in the wild type. What is (Ser) is a missense mutation (named S251F) in which phenylalanine (Phe) is obtained.

In the base sequence represented by SEQ ID NO: 3, the base at position 154 is g in the wild type, but c, so that the amino acid sequence 283 of human LPL in the wild type (51 in SEQ ID NO: 4) Is a missense mutation (named C283S) in which cysteine (Cys) is serine (Ser).
Non-patent document 2 describes that the enzyme activity is lost when Cys at position 283 and Cys at position 278 of human LPL are changed to serine. It does not disclose or suggest the relationship.

  Therefore, examining the presence or absence of the above gene mutations of exon 6, S251F and C283S mutations is useful in the diagnosis and prevention of hypertriglyceridemia.

  Methods for examining the above mutations in the LPL gene include DNA sequencing (normal sequencing, ddNTP extension), PCR (polymerase chain reaction) -RFLP (restriction fragment length polymorphism), SSCP (single strand conformation polymorphisms), ECA (electrochemical) array) -chip method), Allele Specific PCR method, TaqMan Allelic Discrimination method, Hybridization method (DNA microarray, DNA chip), Mass Spectrometry method, Denaturing HPLC method, Invader Assay method, Nano-Invader Assay method, etc. It is not limited to these methods, and there are many other methods.

  Specific gene analysis methods focusing on the above mutations include PCR (polymerase chain reaction) -RFLP (restriction fragment length polymorphism) method, ECA (electrochemical array) -chip method, Allele Specific PCR method, TaqMan Allelic Discrimination method, Hybridization methods (DNA microarray, DNA chip), Mass Spectrometry method, Invader Assay method, Nano-Invader Assay method and the like can be mentioned, but the method is not limited to these methods, and many other methods exist.

  A diagnostic kit for identifying the etiology of hypertriglyceridemia of the present invention (hereinafter simply referred to as a diagnostic kit) is a diagnostic kit for discriminating two types of the above-mentioned single nucleotide substitution mutations in exon 6 of the LPL gene. The primer for detecting the base sequence containing the 58th base of the base sequence represented by this is included. The primer is preferably at least one pair of primers (sense primer and antisense primer) sandwiching a base sequence containing the 58th base. Examples of such a primer include a combination of a primer represented by SEQ ID NO: 5 (sense primer) and a primer represented by SEQ ID NO: 6 (antisense primer).

  The oligonucleotide consisting of 46 bases represented by SEQ ID NO: 5 (Non-patent Document 3) is a sense primer, which is a sense primer of 21M13 sequence 18 bases (base sequence 1 to 18 of SEQ ID NO: 5) and intron 5 of the LPL gene. It includes a partial base sequence on the chain side (base sequence 19 to 46 of SEQ ID NO: 5). The oligonucleotide consisting of 47 bases represented by SEQ ID NO: 6 (Non-patent Document 3) is an antisense primer, which comprises 18 bases of the M13RPI sequence (base sequences 1 to 18 of SEQ ID NO: 6) and intron 6 of the LPL gene. Part of the base sequence on the antisense strand side (base sequence 19 to 47 of SEQ ID NO: 6). By using this primer set, exon 6 of human LPL gene and its surrounding region can be amplified.

  When the primers represented by SEQ ID NOs: 5 and 6 are used to amplify exon 6 of LPL gene and its peripheral portion by PCR from genomic DNA extracted from blood of hypertriglyceridemic subjects by conventional methods, S251F mutation Is obtained, a PCR product having a full length of 359 bp including 243 bp represented by SEQ ID NO: 1 is obtained. When it has a C283S mutation, a full-length 359 bp PCR product containing 243 bp represented by SEQ ID NO: 3 is obtained. A PCR product of the same size can also be obtained when the wild type is used as a template.

When the PCR product of the homozygous person of the S251F mutation is digested with Hin fI, the PCR product containing the sequence represented by SEQ ID NO: 1 has lost one Hin fI recognition sequence due to the mutation, so 276 bp, 61 bp , Cleaved into 3 fragments of 22 bp. On the other hand, in the case of the wild type having no S251F mutation, it is cleaved into four fragments of 164 bp, 112 bp, 61 bp, and 22 bp. Therefore, the PCR product of the S251F mutation heterozygote has 5 fragments of 276 bp, 164 bp, 112 bp, 61 bp, and 22 bp.

When a PCR product homozygous for the C283S mutation is digested with Bst XI, the PCR product containing the sequence represented by SEQ ID NO: 3 has a recognition sequence for Bst XI at the mutated location, so two fragments of 219 bp and 140 bp are obtained. Disconnected. On the other hand, in the case of the wild type not having the C283S mutation, it does not have a Bst XI recognition sequence, and thus is not cleaved, and only one fragment of 359 bp. Therefore, the C283S mutation heterozygous PCR product has three fragments of 359 bp, 219 bp, and 140 bp. Since the difference in these fragment sizes can be detected by agarose gel electrophoresis or the like, it is possible to easily determine whether or not there is an S251F mutation and a C283S mutation in exon 6 of the LPL gene.

Diagnostic kits of the present invention comprise in addition to the primers and restriction enzyme Hin fI or Bst XI, thermostable DNA polymerase used in conventional PCR, dNTPs, PCR reagents, such as PCR buffer, the restriction enzyme buffer, etc. You can also

  The diagnostic method and diagnostic kit of the present invention may detect a mutation by a usual hybridization method using a mutation site-specific probe instead of the restriction enzyme as described above as a mutation detection reagent. In this case, the PCR product of the subject DNA prepared in the same manner as above is fixed to a filter, and a labeled oligonucleotide of about 20 bases including the mutation site is used as a probe, and hybridization is detected by a conventional method to detect the presence or absence of the mutation. be able to.

[Example]
EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. In the following examples, PCR reagents (heat-resistant DNA polymerase, dNTP, PCR buffer) from Applied Biosystems were used.

  From the peripheral blood of subject A (TG: 1510 mg / dl, TC: 557 mg / dl, HDL-C: 30 mg / dl) exhibiting hypertriglyceridemia (sample of EDTA blood collection, the same applies hereinafter), QIAamp DNA Blood Kit Genomic DNA was extracted by (QIAGEN).

  Then, by PCR using GeneAmp PCR System 9700 (manufactured by ABI Perkin-Elmer), the primers containing the primers represented by SEQ ID NOs: 5 and 6 were added. Using the primers shown in Mori et al. (Clinical Biochemistry, Vol. 33, p. 323, p. 327, 2000), exon 1 to exon 9 of the LPL gene were amplified.

  Amplified DNA samples are purified by QIAquick PCR Purification (trade name, QIAGEN), then directly sequenced using Big Dye Terminator Cycle Sequencing Kit (trade name, Applied Biosystems), and then Genetic Analyzer 310 (Applied Biosystems) ), And the base sequence was determined.

  As a result, as represented by SEQ ID NO: 1, in the base sequence of human LPL gene exon 6, the 58th base is c in the wild type but becomes t, so that the amino acid sequence 251 of human LPL in the wild type is t. Second, a missense mutation (named S251F) in which serine (Ser) was changed to phenylalanine (Phe) was newly found. The subject was heterozygous for this mutation.

  Genomic DNA was extracted in the same manner as in Example 1 from the peripheral blood of subject A and control subject having a new S251F mutation in exon 6 discovered in Example 1, and PCR was performed under the following conditions. .

PCR reagents
Thermostable DNA polymerase AmpliTaq Gold ^R (1.25 units / μl)
0.2 μl
PCR buffer GeneAmp 10x PCR Buffer 1.0 μl
dNTP Mixture (2.0 mM each) 1 μl
Template (Genomic DNA) 50ng
Primer (SEQ ID NO: 5) (1 pmol / μl) 1 μl
Primer (SEQ ID NO: 6) (1 pmol / μl) 1 μl
Final volume 10 μl with sterile distilled water.

PCR reaction conditions 95 ° C. After 10.0 minutes, the following steps were repeated 40 times.
94 ° C, 0.5 min 55 ° C, 1.0 min 72 ° C, 2.0 min, then 72 ° C, 7.0 min once.

To the resulting each PCR product (10 [mu] l), 1 [mu] l restriction enzyme buffer _{(500mM NaCl, 100mM Tris-HCl} , 100mM MgCl 2, 10mM DDT, pH7.9) to 1 [mu] l and restriction enzyme Hin fI (manufactured by New England Biolabs) And digested by reacting overnight at 37 ° C. After heat inactivation of the enzyme at 80 ° C. for 20 minutes, this digest was analyzed by electrophoresis on 4% NuSieve GTG agarose (CAMBREX) gel (containing ethidium bromide). FIG. 1 shows the migration pattern.

  As shown in FIG. 1, in the control person (lane 3), a 164 bp fragment and a 112 bp fragment common to the wild type and the S251F mutation were detected (the 22 bp fragment was too short to be detected). On the other hand, in the subject (lane 2, heterozygote), a 276 bp fragment derived from the S251F mutant allele was further detected, and this diagnostic kit makes it possible to easily identify the etiology of hypertriglyceridemia in the subject. became.

  Genomic DNA was extracted in the same manner as described above from the peripheral blood of subject B (TG: 657 mg / dl, TC: 277 mg / dl) exhibiting hypertriglyceridemia.

  Then, exon 1 to exon 9 of the LPL gene were amplified in the same manner as described above.

  The base sequence of the amplified DNA sample was determined in the same manner as described above.

  As a result, in the base sequence of the human lipoprotein lipase gene exon 6 represented by SEQ ID NO: 3, the 154th base was g in the wild type but became c, so that the amino acid sequence of the human LPL in the wild type was 283. Was newly found a missense mutation (named C283S) in which cysteine (Cys) became serine (Ser). This subject was a complex heterozygote having this mutation on the LPL gene and the same mutation as previously reported.

  Example 4: Genomic DNA was extracted in the same manner as in Example 2 from the peripheral blood of subject B and a control subject having a new C283S mutation in exon 6 discovered in Example 3, and PCR was performed.

1 μl of restriction enzyme buffer (1000 mM NaCl, 500 mM Tris-HCl, 100 mM MgCl ₂ , 10 mM DDT, pH 7.9) and 1 μl of restriction enzyme Bst XI (manufactured by New England Biolabs) were added to each obtained PCR product (10 μl). And digested by reacting at 55 ° C. overnight. After heat inactivation of the enzyme at 65 ° C. for 20 minutes, this digest was analyzed by electrophoresis on 4% NuSieve GTG agarose (CAMBREX) gel (containing ethidium bromide). FIG. 2 shows the migration pattern.

As shown in FIG. 2, a 359 bp fragment was detected in the control (lane 2). On the other hand, in subject B (lane 3, heterozygote), in addition to the 359 bp fragment derived from the wild-type allele, the 219 bp and 140 bp fragments derived from the C283S mutant allele were detected. As a result, it was possible to easily identify the etiology of hypertriglyceridemia in subjects.
In addition, since it was clarified by another method that subject B has the same mutation as that already reported in other exons on the LPL gene, subject B is a complex heterozygote. I understood.

  The two types of novel LPL gene mutations of the present invention are important in the development of genetic diagnosis chips and the like that easily detect LPL mutations. In the development of a kit for early diagnosis of Japanese LPL gene mutations, a genetic diagnosis chip capable of detecting all LPL mutations found by Japanese is required. We have already developed an invader-based genetic diagnosis system for 22 types of mutations found in Japanese. This time, by adding the newly found mutation into the diagnostic kit for Japanese LPL gene mutation, the mutation detection power can be enhanced. The mutation of the present invention is considered to be useful for companies considering the development of diagnostic kits for LPL gene mutations. The rapid diagnosis of LPL gene mutations will contribute to tailor-made medical care for the prevention of lifestyle-related diseases as well as the elucidation of the cause of hypertriglyceridemia and the determination of the treatment policy.

It is a figure which shows the result of having carried out the agarose gel electrophoresis of the restriction enzyme Hin fI digest of the PCR product obtained using the primer set represented by sequence number 5 and sequence number 6 using the genome of test subject A and a control person as a template. . It is a figure which shows the result of having carried out the agarose gel electrophoresis of the restriction enzyme Bst XI digest of the PCR product obtained by using the primer set represented by sequence number 5 and sequence number 6 using the genome of test subject B and a control person as a template. .

Claims (26)

  A nucleic acid comprising the base sequence of human lipoprotein lipase gene exon 6 represented by SEQ ID NO: 1, a partial base sequence containing the 58th base, or a complementary sequence thereof.   The nucleic acid according to claim 1, wherein the nucleic acid is 10 nucleotides or more.   The nucleic acid according to claim 1, wherein the nucleic acid is 20 nucleotides or more.   The nucleic acid according to claim 1, wherein the nucleic acid is 20 nucleotides or more and 200 nucleotides or less.   A method for diagnosing human lipoprotein lipase, comprising: collecting DNA from a test object to prepare a DNA preparation; detecting the presence or absence of the base sequence according to claim 1 in the DNA preparation; A method for diagnosing a human lipoprotein lipase gene, comprising:   6. The diagnostic method according to claim 5, wherein the detection step uses the nucleic acid according to claim 1 as a probe.   The detection step amplifies a portion containing a region corresponding to the nucleic acid according to claim 1 from the DNA preparation, digests with a restriction enzyme that changes the number of cleavage sites depending on the presence or absence of mutation, 6. The diagnosis method according to claim 5, wherein presence or absence is determined. The diagnostic method according to claim 7, wherein the restriction enzyme is Hin fI.   A diagnostic kit for identifying the etiology of hypertriglyceridemia, comprising a primer set for amplifying the base sequence of SEQ ID NO: 1 or a partial base sequence including the 58th base and a mutation detection reagent.   The diagnostic kit according to claim 9, wherein the mutation detection reagent is a probe comprising the nucleic acid according to claim 4.   The diagnostic kit according to claim 9, wherein the mutation detection reagent is a restriction enzyme. The diagnostic kit according to claim 11, wherein the restriction enzyme is Hin fI.   The diagnostic kit according to any one of claims 9 to 12, wherein the primer set has the sequences of SEQ ID NOs: 5 and 6.   A nucleic acid comprising the base sequence of human lipoprotein lipase gene exon 6 represented by SEQ ID NO: 3, or a partial base sequence containing the 154th base, or a complementary sequence thereof.   The nucleic acid according to claim 14, wherein the nucleic acid is 10 nucleotides or more.   The nucleic acid according to claim 14, wherein the nucleic acid is 20 nucleotides or more.   The nucleic acid according to claim 14, wherein the nucleic acid is 20 nucleotides or more and 200 nucleotides or less. A human lipoprotein lipase gene diagnosis method comprising:
Collecting DNA from the test object and preparing a DNA preparation;
A method of detecting the presence or absence of the base sequence according to claim 14 in a DNA preparation, and a human lipoprotein lipase gene diagnostic method.   The diagnostic method according to claim 18, wherein the detection step uses the nucleic acid according to claim 17 as a probe.   The detection step amplifies a portion containing a region corresponding to the nucleic acid according to claim 14 from the DNA preparation, digests with a restriction enzyme that changes the number of cleavage sites depending on the presence or absence of mutation, The diagnosis method according to claim 18, wherein presence or absence is determined. The diagnostic method according to claim 20, wherein the restriction enzyme is Bst XI.   A diagnostic kit for identifying the etiology of hypertriglyceridemia, comprising a primer set for amplifying the base sequence of SEQ ID NO: 3 or a partial base sequence thereof containing the 154th base and a mutation detection reagent.   The diagnostic kit according to claim 22, wherein the mutation detection reagent is a probe comprising the nucleic acid according to claim 17.   The diagnostic kit according to claim 22, wherein the mutation detection reagent is a restriction enzyme. The diagnostic kit according to claim 24, wherein the restriction enzyme is Bst XI. The diagnostic kit according to any one of claims 22 to 25, wherein the primer set has the sequences of SEQ ID NOs: 5 and 6.

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