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  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention relates to disease-associated mutant genes, and in particular to Lieber congenital amaurosis-associated mutation genes. Background technique
• Leber congenital amaurosis is an autosomal recessive retinal dystrophy that manifests as genetic heterogeneity.
• Leber Congenital Amaurosis is one of the most common inherited cause of blindness in children 1, is a severe retinal dystrophy, it showed at birth or infancy low vision and nystagmus.
• LCA has been found to be associated with at least 17 genes. The molecular cause of the lack of identifiable in 20-30% of cases screened to the LCA, the LCA shows that there are not now causative genes 2--4. Current clinical trials usually look for molecular defects by replacing them, or bypassing metabolic blockade by oral 9-cis retinal in the absence of RPE65 and LRAT.
• the inventors sequenced the exome of individuals with LCA and identified nonsense mutations (c.507G>A, p.Trpl69*) and missense mutations (c.769G>A, p.Glu257Lys) in the NMNAT1 gene, This mutant gene was found to be involved in LCA.
• the invention relates to a biomarker of LCA, i.e., a mutated NMNAT1 gene or a NMNAT1 protein, the biomarker being a NMNAT1 gene or a NMNAT1 protein having a mutation selected from:
• Nonsense mutation (c.507G>A, p.Trpl69*);
• Missense mutation (c.769G>A, p.Glu257Lys).
• sequence of the mutant ⁇ 3 ⁇ 4 ⁇ 7 gene of the invention is SEQ D) NO:l having the following mutations:
• Nonsense mutation c.507G>A and / or
• Missense mutation c.769G>A the sequence of the mutated NMNAT1 protein of the invention is SEQ D) NO: 2 with the following mutation:
• the invention in a second aspect, relates to a method of detecting LCA, the method comprising detecting whether a mutation site is present in a NMNAT1 gene or a NMNAT1 protein of a subject, and if there is a mutation site, the subject is: It is identified as having LCA or susceptible to LCA, or its offspring may have LCA or are susceptible to LCA, and the mutation site is selected from any one of the following or a combination thereof:
• Nonsense mutation (c.507G>A, p.Trpl69*);
• Missense mutation (c.769G>A, p.Glu257Lys).
• the method of detecting an LCA comprises the steps of:
• the DNA sample is subjected to PCR amplification to obtain an amplification product, and the NMNAT1 gene exon and exon/intron boundary are obtained.
• the PCR amplification amplification product is sequenced to obtain a sequencing result.
• the sequencing result is analyzed to determine whether a mutation site exists in the NMNAT1 gene, and if there is a mutation site, the subject is identified as having LCA or susceptible to LCA, or the offspring may have LCA or susceptibility LCA, the mutation site is selected from any one of the following or a combination thereof:
• Nonsense mutation (c.507G>A, p.Trpl69*);
• Missense mutation (c.769G>A, p.Glu257Lys).
• PCR uses KAPA2G Robust HotStart
• the PCR amplified primers are designed with reference to the human genome, such as at least one set of primers as follows:
• the sequencing is a Sanger sequence determination.
• sequence analysis is performed using a Mutation Surveyor program (State College, PA).
• the NMNAT1 protein is a sequence representation of SEQ D) NO:2.
• the V3 ⁇ 4J gene is represented by the sequence of SEQ ID NO: 1.
• the method of detecting LCA of the present invention comprises the step of amplifying at least one set of primers as follows:
• the method for detecting a mutation in the method for detecting LCA of the present invention is carried out by a technique selected from the group consisting of sequencing, electrophoresis, nucleic acid hybridization, in situ hybridization, PCR, reverse transcriptase chain reaction, and denaturing high performance liquid phase. Chromatography.
• the present invention relates to a method for detecting a mutant NMNAT1 gene or NMNAT1 protein, the method comprising detecting whether a mutation site is present in a NMNAT1 gene or a NMNAT1 protein of a subject, wherein the mutation site is selected from the following One or a combination:
• Nonsense mutation (c.507G>A, p.Trpl69*);
• Missense mutation (c.769G>A, p.Glu257Lys).
• the method of detecting a mutant V A3 ⁇ 47 gene of the present invention comprises the steps of: extracting a DNA sample of a subject,
• the DNA sample is subjected to PCR amplification to obtain an amplification product, and the NMNAT1 gene exon and exon/intron boundary are obtained.
• More PCR amplification products are sequenced to obtain sequencing results.
• the sequencing result is analyzed to determine whether a mutation site exists in the NMNAT1 gene, and the mutation site is selected from any one of the following or a combination thereof:
• Nonsense mutation (c.507G>A, p.Trpl69*);
• Missense mutation (c.769G>A, p.Glu257Lys).
• PCR uses KAPA2G Robust HotStart
• the PCR amplified primers are designed with reference to the human genome, such as at least one set of primers as follows:
• the sequencing is a Sanger sequence determination.
• the sequence analysis is performed using a Mutation Surveyor program (State College, PA).
• the NMNAT1 protein is represented by the sequence of SEQ ID NO: 2.
• the V3 ⁇ 4J gene is represented by the sequence of SEQ ID NO: 1.
• the method of detecting a mutant NMNAT1 gene or NMNAT1 protein of the invention comprises the step of amplifying at least one set of primers as follows:
• the method for detecting a mutation site in the method for detecting a mutant NMNAT1 gene or NMNAT1 protein of the present invention is carried out by the following techniques: sequencing, electrophoresis, nucleic acid hybridization, in situ hybridization, PCR, reverse transcriptase chain reaction And denaturing high performance liquid chromatography.
• the method for detecting a mutant NMNAT1 gene or NMNAT1 protein according to the third aspect of the present invention can be used for the purpose of diagnosing LCA, and can also be used for the purpose of non-diagnostic disease.
• the non-diagnostic diseases described in the present invention include, but are not limited to, studying SNP distribution and polypeptideity for use in family evolution studies. Such applications are understood by those skilled in the art.
• the method of detecting a mutant ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 4 /gene or NMNAT1 protein according to the third aspect of the invention involves detecting a heterozygous mutation.
• the method of detecting a mutant NMNAT1 gene or NMNAT1 protein according to the third aspect of the present invention also includes detecting a homozygous mutation.
• the invention relates to a primer pair for use in detecting a mutant NMNAT1 gene or NMNAT1 protein by PCR, the mutation being selected from any one or combination of the following:
• Nonsense mutation c.507G>A, p.Trpl69*
• Missense mutation c.769G>A, p.Glu257Lys
• the primer pair is designed before and after the genomic sequence or the cDNA sequence based on a position selected from the following positions such that the position is amplified: position 507 and position 769 of the cDNA sequence.
• the primer pair is:
• the invention relates to a nucleic acid probe complementary to a mutant ⁇ 3 ⁇ 4 ⁇ 7 gene, said mutation being selected from any one or combination of the following:
• Nonsense mutation c.507G>A and / or
• Missense mutation c.769G>A position on the cDNA sequence: position 507 and position 769 of the cDNA sequence.
• the invention relates to a kit for detecting a mutant NMNAT1 gene or NMNAT1 protein, comprising one or more sets of primer pairs, wherein the mutation is selected from any one of the following or a combination thereof: nonsense mutation (c.507G) >A,p.Trpl69* ); and
• the primer pair is designed on the genomic sequence or the cDNA sequence based on a position selected from the following, such that the amplification product thereof covers the position: position 507 and position 769 of the cDNA sequence.
• the kit for detecting a mutant NMNAT1 gene or NMNAT1 protein comprises at least one set of primers selected from the group consisting of:
• the invention relates to a kit for detecting a mutant ⁇ 3 ⁇ 4 ⁇ 7 gene comprising one or more nucleic acid probes selected from any one or a combination of the following:
• Nonsense mutation c.507G>A
• the probe is complementary to a region of the mutated NMNAT1 gene comprising a genomic sequence or a cDNA sequence selected from the group consisting of: 507th and 769th sequences of the cDNA sequence.
• the invention relates to a method for detecting a mutation in a gene exon and an exon/intron boundary, comprising the steps of:
• the above sample is subjected to exome capture prior to the second step to collect the CDS region of human genomic DNA, and the exon-enriched DNA library is subjected to a second library construction for sequence determination.
• the sequencing in the second step is performed as follows:
• the library is subjected to ligation-mediated PCR (LM-PCR) linear amplification and biotin-labeled DNA library for hybridization and enrichment, and then linearly amplified by LM-PCR, then sequenced on the machine, read Take a length of 90 bp and an average sequencing depth of at least 50 per sample.
• LM-PCR ligation-mediated PCR
• the repeated sequencing fragments are removed in the third step.
• Figure 1 Retinal appearance of patient 1 showing retinal vascular reduction, macular pigmentation of the retina, and atrophic macular damage.
• FIG. 1 Schematic diagram of the mutations identified in the NMNAT1 gene. LCA patients without previously identified mutations were screened for ⁇ 3 ⁇ 4 ⁇ 7 gene mutations by Sanger sequencing. Isolation and analysis of genomic DNA is also performed for available family members. Parents from Patient 6 are not available. Patients 9 and 10 are only available for current samples.
• Figure 3 Screening of NMNAT1 gene mutations by Sanger sequence determination in LCA patients without previously identified mutations. Isolation and analysis of genomic DNA is also performed for available family members. Parent samples from patient 6 were not available. Patients 9 and 10 are only available for current samples.
• FIG. 4 Image representation showing residues associated with LCA mutations identified on the human NMNAT1 promer structure. The overall structure is shown as a gray ribbon and the combined NAD is an orange rod. All 8 mutation sites were shown as rods (nonsense blue-green, missense green). Red dotted line shows three detailed description
• c.507G>A indicates that the nucleotide at position 507 of the cDNA changes from G to A; both p.Trpl69* and p.Trpl69stop indicate that the codon at position 169 of the encoded protein is changed from a codon encoding Trp.
• Stop codon; c.769G>A indicates that the nucleotide of position 769 of the cDNA changes from G to A; p.Glu257Lys indicates that the codon 169 of the encoded protein is changed from the codon encoding Glu to the codon encoding Lys, or The 169th position of the protein changed from Glu to Lys.
• the codon of the mutant ⁇ 3 ⁇ 4 ⁇ 7 gene encoding the amino acid 169 of the present invention may be AAA or AAG.
• the cDNA sequence of the wild type UV 3 ⁇ 47 gene is shown in SEQ ID NO: 1.
• the ⁇ column of the wild type human NMNAT1 protein is shown in SEQ ID NO: 2.
• the wild type mRNA3 ⁇ 4 ⁇ 3 ⁇ 4 / gene mRNA sequence is shown in SEQ ID NO: 11.
• sequence includes any one or two of the complementary strands.
• the sequence includes any one or two of the complementary strands.
• the cDNA sequence of the SJV 7 gene is actually included in the sequence and its complementary sequence.
• SEQ ID NO: 1 which actually includes its complementary sequence.
• one chain can be used to detect another chain, and vice versa.
• the gene sequence in the present application includes either the DNA form or the RNA form, and one of them is disclosed, meaning that the other is also disclosed.
• the cDNA sequence of the SJV 7 gene is actually included in the corresponding RNA sequence.
• the exon/intron boundary means 1-200 from the exon-intron boundary, for example 10-100, such as 20-80, such as 30-70, such as 40, 50, 60. Intron nucleotides.
• the inventors mapped the coding region of 17 known LCA-related genes and all exon/intron boundaries (at least 50 inclusive from the exon-intron boundary). Sequencing, the 17 genes are PZJ, CABP4, CEP290, CRB1, CRX, GUCY2D. IQCB1, LCA5. LRAT, RD3, RDH12, RPE65, RPGRIPU SPATA7. TULP IMPDH1 and OTX2, these genes Relevant information is available from the Mendelian Human Genetic Database (OMIM). Among the 220 LCA individuals studied, mutant alleles were found on two chromosomes in 160 cases, and one mutant was found in 10 cases. Gene.
• OMIM Mendelian Human Genetic Database
• NAD nicotinamide adenine dinucleotide
• Nonsense mutations c.507G>A, p.Trpl69*
• missense mutations c.769G>A, p.Glu257Lys
• the assay (PCR primers provided in ⁇ 3) was confirmed.
• the parental test determined that the male parent carries the heterozygote of the nonsense variation, and the female parent carries the heterozygote of the missense variation, and the LCA caused by the mutation of the NMNAT1 gene is an autosomal recessive genetic disease, so the parents do not cause disease.
• the offspring also passed on these two mutations, which caused the NMNAT1 gene to function abnormally and cause disease. Analysis shows that unaffected cell members do not carry mutations. Together, these results support that the 7 ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 41 gene is an LCA candidate gene. Table 1. Screening of LCA genes by exome sequencing
• RNA expression patterns were analyzed by GeneCards.
• X indicates the termination of the translation.
• NMNAT1 (nicotinamide mononucleotide adenylyltransferase 1) contains four coding exons, encoding a 279 residue protein, which plays an important role in NAD+ biosynthesis, in which it catalyzes a single nucleotide from nicotinamide (NMN) ) and ⁇ form NAD+.
• the VMA3 ⁇ 47 gene was not previously associated with LCA but was associated with axonal degeneration 6 .
• TVw homozygous knockout mice to not live birth, homozygous mutations nmnat Drosophila (DrosophUa melanogaster) is lethal; These results support the Vw «a gene essential function in some species views 7 .
• the genetic map of the ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 4/gene is at 1 ⁇ 36.22, near the LCA9 locus where the genetic map was previously identified.
• the affected individuals in the LCA9 family have congenital non-progressive severe visual impairments 5 .
• the 11 LCD cases reported herein have severe early visual loss of vision (Table 5).
• allele clamping as used herein relates to a process by which a phenotype of an individual having the same variation (eg, p.Glu257Lys) is examined in the presence of a second variant allele to infer the The effect of the second allele. All four individuals carrying the nonsense mutation p.Trpl69* were blind at birth and still had varying degrees of light perception. For 5 individuals who only carried the missense variation, the visual reduction was reduced within a few years after birth. The retina of all affected individuals has a marginal macula appearance and atrophic macular degeneration-like lesions
• Fig. 1 description of macular scar formation in patient 8
• the enlargement of macular lesions was recorded during follow-up examinations at patients 2 years 6-9 months and patients 4 years 5-7 years old.
• Patient 4 has two variant alleles on the second chromosome.
• At 8 years old there are 20/200 OD (right eye) and 20/400 OS (left eye) visual acuity, which is the only retinal current map (ERG).
• EEG retinal current map
• a case where the primary cone is dysfunctional rather than a deep loss of all responses; the individual's diagnosis is corrected for cone dystrophy.
• Patient 5 developed left eye exudative retinitis (Coats' disease) at age 3 - patient 6 was the only one carrying c.451G>T
• the allele frequency of c.769G>A (p.Glu257Lys) is estimated to be 0.001 (dbSNP: rsl50726175).
• dbSNP rsl50726175
• the inventors have not found that the individual is a homozygous c.769G>A (p.Glu257Lys) allele. None of the other variants identified by the inventors have been reported in any public database.
• the p.Trpl69* variant occurs in four of the nine families; the cause of such a high incidence is unknown.
• the NMNAT1 gene is Puda and shows the highest specific activity in the final step of catalyzing NAD+ biosynthesis.
• Human NMNAT1 is a homohexamer, each of which contains a central six-parallel beta-sheet with a number of helices 12 13 (Fig. 4). In this article, all 11 individuals with LCA carry the p.Glu257Lys variant. According to the crystal structure of human NMNAT1, Glu257 is located in short The outer surface in the C-terminal helix. It is worth noting that adjacent Ser256 is predicted to be a plate acidification site and may be involved in physical interaction with other related proteins such as poly (ADP-ribose) polymerase or protein kinase 12 ' 13 .
• Displacement of a negatively charged glutamic acid with a positively charged lysine may alter the electrostatic properties of the surface, thereby affecting the proposed physical interaction.
• Asn273 is located in the last short helix (residues 267-274) and is involved in coordinating the active site water molecule 12 i3 . Displacement of this residue with an acidic residue (aspartic acid) will likely affect enzyme activity.
• Met35 and Vall51 are located in the center of the protein, while Val98 and Leul53 are very close to the ligand binding site (distance of about 6-7 A).
• LCA The diagnosis of LCA is based on clinical evaluation (lack of limited imaging, nystagmus and infantile pupillary weak response, fundus examination and diagnosis), and suspected cases are re-examined by electroretinogram (ERG).
• ERG electroretinogram
• the patient was diagnosed with a macular defect, which is atrophic damage, not a true congenital defect.
• Patients 1 - 6 and patient 11 were single cases.
• Patients 7 and 8 are cell relatives.
• Patients 9 and 10 are cell relatives. Patients 1, 7 and 8 were from USA; patients 2, 3 and 5 were from Brazil; patients 4, 6, 9 and 10 were from.
• Patient 11 is from Australia.
• Written informed consent was signed by their parents or guardians and the study was approved by the BGI and OHSU (P-WC) Institutional Ethnics Committee.
• the male is a Brazilian Caucasian ancestor whose maternal ancestors are from Tru, Portugal, Lebanon and Italy. The paternal ancestors are from Turkey, Germany, Italy and Portugal, born for normal pregnancy and cesarean section.
• Parents noticed the child's eye tremor in the first 3 months. At 6 months, due to nystagmus and no imaging consultation with Sao Paulo's eye genetics expert, the expert found that the child's eyes were light, the macula atrophy (so-called macular defect) and retinal macular pigmentation, hence the LCA. By the age of 9 months, the photo shows an increase in the area of macular atrophy.
• Patient 2 has no cell relatives and is the only patient with LCA in the family. During the first year of life, there is no light.
• Patient 3 was a Brazilian baby girl whose mother noticed that she was pressing at 4 months. Examination revealed that the Teller card test was visually invisible, had Franceschetti eye-catching signs, nystagmus, eye socket depression, pigmentation of the retina, and the appearance of a yellowed hammered copper. When she was a child, she was already blind. She is the only patient in the family.
• Patient 4 provided by EH
• Patient 4 is a male whose ancestor is west, has a cell relative, and is the only family member who is ill.
• Horizontal tremor and weak vision were found at 2 months.
• a 6-month-old examination showed pigmentation to reduce macular damage and the patient was diagnosed with LCA.
• night vision weakens. Examination showed an increase in hypertrophic macular damage and atrophy of the retinal vasculature.
• the field of view is about 145 degrees.
• the ERG shows the main functional obstacles of the cone system, with a slight rod-focal b-wave latency delay, all the amplitudes of the reaction and the b-wave separation of the cone are reduced and the 30Hz flicker response is delayed.
• the 20/200 OD and 20/400 OS are 20/100 on both sides and the field of view is reduced to 95 degrees.
• the diagnosis was changed to cone-rod malnutrition, not LCA. You can still see the color.
• Parents ERG is normal. One parent has an indeterminate significant mid-peripheral pigment spot.
• Patient 5 is a Brazilian male whose maternal ancestors are Vietnameserds, Caucasians, and the paternal background is African and Portuguese. He ⁇ L month pregnancy and normal birth. At 3 months of age, his mother observed that he did not see her and any objects. At the age of 1 he was sent to Sao Paulo Hospital for diagnosis of LCA. Initially, the omentum showed macular atrophy and fine retinal pigmentation. At the age of 4, he had exudative retinitis in his left eye. Atrophic macular degeneration-like lesions in Patient 5 were similar to Patient 2. Patient 5 is the only family member with LCA.
• Patient 7 was a female, aged 10 years old. After her brother's skin was diagnosed with LCA, she was sent to the Retina Foundation for visual function testing and evaluation. At the time of testing, binocular sensitivity was evaluated as LP. The full-field ERG showed no response to the single-flash blue light, and the cone's response to 30 Hz scintillation light decreased by 99% with a delay. These symptoms are consistent with LCA.
• Patient 7's brother (patient 8) was diagnosed at the Retina Foundation when he was 1 year old. He was discovered by the pediatrician at 4.5 months due to the H3 ⁇ 4 movement and lack of a dermatologist.
• the visual acuity is LP.
• the left eye full field of view ERG did not respond to the rod response test, and the cone response reduced the 30 Hz flash amplitude by 99% and the b wave latency time was significantly delayed.
• Patient 9 is a Caucasian male who has a weak visual appearance from infancy and is diagnosed with Lieber congenital amaurosis (LCA) during childhood. He was only able to identify bright colors when he was learning to walk, but soon lost that ability.
• LCA Lieber congenital amaurosis
• retinal findings revealed scarring of the macula in both eyes, pale optic nerve head, and retinal vascular atrophy.
• the visual acuity report has a light perception.
• the pupillary response was sluggish, and the reaction was sometimes abnormal, with the pupil becoming larger as the light came in.
• Retinal enlargement examination revealed macular degeneration-like damage, reduced retinal blood vessels, small and pale papilla, and surrounding pigmentation.
• the intraocular pressure is normal. Double darkness and lightness ERG has no test results for all stimuli above noise. He could not complete the Goldmann field of view test. Other conveniences such as health and intelligence are normal.
• a brother, 30 years old, has a suspected LCA; a sister is not sick.
• Parents and ancestors were British and Dutch. Mother has 1 sister and 2 brothers. The father is adopted, with one brother and one sister.
• the female infant whose mother is a descendant of British origin, whose father is of Australian descent, produces full-term pregnancy without complications. At 8 weeks, she could not watch and follow, and observed nystagmus and patrolling eye movements. The pediatric ophthalmologist then diagnosed as suspected LCA. Other aspects of the baby are healthy. ERG testing showed severe lateral retinal dysfunction with light perception but no graphic perception. Brain MRI is normal. At 2 years and 7 months, there is a certain sense of light. The patient had two unaffected brothers who did not have a family history of LCA. Similar to the clinical appearance of patients 1, 2 and 3, patient 11 presented with severe LCA. Importantly, macular atrophy (macular scar formation or macular degeneration) was also observed in their parents. In fact, suspected ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 4 / gene mutations, based on analysis of fundus imaging, suggest testing the V 3 ⁇ 47 gene.
• Sample preparation Collect peripheral blood of patients and related relatives, and use the kit to extract the genome of peripheral blood leukocytes
• the OD260/OD280 values of each specimen genomic DNA are between 1.7-2.0.
• the concentration is not less than 100 ng / ⁇ ⁇
• the total amount is not less than 30 ⁇ 1.
• the above samples were subjected to exome capture using an Agilent 38M (Agilent) chip to collect the CDS region of human genomic DNA.
• the exon-encoding DNA library was then subjected to a second library construction for Illumina GA sequencing.
• the sequencing platform is the Illumina Genome Analyzer, which is sequenced according to the Illumina/Solexa standard library instructions (see http://www.illumina.com/), which is briefly described as follows:
• the library is subjected to ligation-mediated PCR (LM-PCR) linear amplification and biotin-labeled DNA library for hybridization and enrichment, and then linearly amplified by LM-PCR, then sequenced on the machine, read Take a length of 90 bp and an average sequencing depth of at least 50 per sample.
• LM-PCR ligation-mediated PCR
• the human reference genome and its gene annotations were downloaded from the UCSC database (http://genome.ucsc.edu/), version hgl9 (build 37).
• the sequence from the LCA patient sample was aligned with the reference sequence using BWA (Burrows-Wheeler Alignment) and then the duplicated reads were read.
• the SNP was transferred to the default SOAPsnp component (Li R, Li Y, Fang X, Yang H, et al, SNP detection for massively parallel whole-genome resequencing. Genome Res 2009, 19(6): 1124-1132).
• the threshold value for filtering SNPs ⁇ )
• SEQ ID NO: 1 cDNA sequence of wild type AV 3 ⁇ 47 gene (where underlined indicates that the position may be mutated) (http: ⁇ www.ncbi.nlm.nih.gov/nuccore/95113669) ATGGAA AATTCCGAGA AGACTGAAGT GGTTCTCCTT GCTTGTGGTT CATTCAATCC CATCACCAAC ATGCACCTCA GGTTGTTTGA
• V3W03V3V1 V31W13VVV V310013V33
• V03V3103V1 1VVWVV0V1
• VV3VV10V10 101V0000VVV V31331V1VV 00331003V0
• VVW033310 03V0010V03 ⁇ 0V00313 ⁇ 33VV003V31 3V001133V0 V033V33300 L9S L
• ITC100/Z10ZN3/X3d 80 ⁇ 6 ⁇ 0/ 0 ⁇ OAV 3301 CTTTTCACTT TTCTAGAAGT CCAGAAGTTG TTATATGATG AAATAGCCTC CTTTAACGTT

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