WO2018215515A1 - Procédés pour identifier si un sujet est atteint ou est susceptible d'être atteint d'une pancréatite - Google Patents

Procédés pour identifier si un sujet est atteint ou est susceptible d'être atteint d'une pancréatite Download PDF

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WO2018215515A1
WO2018215515A1 PCT/EP2018/063458 EP2018063458W WO2018215515A1 WO 2018215515 A1 WO2018215515 A1 WO 2018215515A1 EP 2018063458 W EP2018063458 W EP 2018063458W WO 2018215515 A1 WO2018215515 A1 WO 2018215515A1
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pancreatitis
subject
risk
allele
nucleic acid
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PCT/EP2018/063458
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Arnaud BOULLING
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Université De Bretagne Occidentale
Etablissement Français Du Sang (Efs)
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Publication of WO2018215515A1 publication Critical patent/WO2018215515A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to methods for identifying whether a subject has or is at risk of having pancreatitis.
  • CP Chronic pancreatitis
  • SPINK1 loss-of-function variants on the SPINK1 gene act as predisposing factors for CP (reviewed by (Chen and Ferec, 2012).
  • Boulling et al. brougth to light that the loss-of-function rs 142703147C>A affects a SPINK1 enhancer and represents an important risk factor for CP (Boulling et al., 2017).
  • This variant turns off the enhancer by disrupting a cis-regulatory module formed by a HNF1A- and a PTF1-L- binding sites ( Figure 1A).
  • the present invention relates to methods for identifying whether a subject has or is at risk of having pancreatitis.
  • the present invention is defined by the claims.
  • the first object of the present invention relates to a method of identifying a subject having or at risk of having pancreatitis comprising determining in a sample obtained from the subject, the presence or absence of the variant allele (C) of the single nucleotide polymorphism (SNP) rs7727286 or one or more polymorphisms in linkage disequilibrium with this allele wherein the presence of the polymorphism indicates that the subject has or is at risk of having pancreatitis.
  • C variant allele
  • SNP single nucleotide polymorphism
  • pancreatitis has its general meaning in the art and refers to a variety of diseases in which the pancreas becomes inflamed. This is believed to occur when powerful digestive enzymes, in particular trypsinogen, are activated before secretion into the duodenum, and attack the pancreatic tissues. Pancreatitis is thus inflammation of the pancreas that progresses from acute (sudden onset; duration ⁇ 6 months) to recurrent acute (>1 episode of acute pancreatitis) to chronic (duration >6 months).
  • Acute pancreatitis is a sudden, usually painful inflammation that occurs over a short period of time, and can be precipitated by a variety of causal factors including gallstones, heavy alcohol use, medications, infections, metabolic - disorders, trauma and surgery, or can occur without known cause.
  • Acute pancreatitis is a rather common complication following endoscopic retrograde cholangiopancreatography (ERCP). Severity of acute pancreatitis can range from mild abdominal discomfort to severe illness that can lead to serious, sometimes life-threatening complications. In most but by no means all cases, treatment with analgesics for management of pain is sufficient.
  • Treatment can also include removal of causal factors (e.g., control of infection with antibiotics, surgery to remove gallstones, abstinence from alcohol, etc.) as appropriate. Over 80% of patients recover completely after receiving the appropriate treatment.
  • the term also includes "acute recurrent pancreatitis" which is defined as more than two attacks of acute pancreatitis without any evidence of underlying pancreatitis.
  • Chronic pancreatitis CP
  • CP causes progressive and irreversible damage to the pancreas and surrounding tissues. Calcification of pancreatic tissues is common and often diagnostic of CP.
  • CP is associated with excessive and prolonged alcohol consumption. While alcoholism is the most common cause of CP, other causes include metabolic disorders and, more rarely, genetic disposition (hereditary pancreatitis). The term also includes "chronic tropical pancreatitis" which is the most common cause, commonly affecting children, in Asia, and more particularly in India.
  • the subject can be male or female.
  • a subject can be one who has been previously diagnosed as having some symptoms of pancreatitis.
  • the subject can also be one who has not been previously diagnosed as having symptoms of pancreatitis.
  • a subject can be one who exhibits one or more risk factors for pancreatitis (e.g. alcohol consumption or cigarette smoking), or a subject who does not exhibit risk factors, or a subject who is asymptomatic for pancreatitis (e.g. in case of a screening test).
  • the subject is an American or African subject.
  • risk relates to the probability that an event will occur over a specific time period, as in the conversion to pancreatitis, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the . probability of event and (1- p) is the probability of no event) to no- conversion.
  • Alternative continuous measures which may be assessed in the context of the present invention include time to pancreatitis conversion and therapeutic pancreatitis conversion risk reduction ratios.
  • Risk evaluation in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to pancreatitis or to one at risk of developing pancreatitis.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of pancreatitis, such as alcohol consumption or cigarette smoking, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to pancreatitis, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for pancreatitis.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk for pancreatitis.
  • the present invention may be used so as to discriminate those at risk for developing pancreatitis from those having pancreatitis, or those having pancreatitis from normal.
  • sample refers to any biological sample isolated from the subject liable to contain nucleic acid for the purpose of the present invention. Samples can include by way of example and not limitation, bodily fluids (e;g. saliva) and/or tissue extracts such as homogenates or solubilized tissue obtained from the subject. In some embodiments, the sample is a blood sample.
  • blood sample means any blood sample derived from the patient that contains nucleic acids. Peripheral blood is preferred, and mononuclear cells (PBMCs) are the preferred cells.
  • PBMC peripheral blood mononuclear cells
  • unfractionated PBMC refers to whole PBMC, i.e.
  • PBMC can be extracted from whole blood using a hypotonic lysis which will preferentially lyse red blood cells.
  • the template nucleic acid need not be purified. Nucleic acids may be extracted from a sample by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington D.C.). .
  • single nucleotide polymorphism or "SNP” has its general meaning in the art and refers to a single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population. There are millions of SNPs in the human genome. Most commonly, these variations are found in the DNA between genes. When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting the gene's function.
  • allelic variant has its general meaning in the art and refers to an alternative form of a gene (one member of a pair) that is located at a specific position on a specific chromosome which, when translated result in functional or dysfunctional (including non-existent) gene products.
  • polymorphism or "allelic variant” means an alteration in the normal sequence of a gene or in the sequences that control expression of this gene.
  • Complete gene sequencing often identifies numerous allelic variants (sometimes hundreds) for a given gene. The significance of allelic variants is often unclear until further study of the genotype and corresponding phenotype occurs in a sufficiently large population.
  • rs7727286 refers to the NCBI reference variant present in the SPINK1 gene Typically rs7727286 is characterized by the nucleic acid sequence as set forth in SEQ ID NO: l.
  • linkage disequilibrium refers to a population association among alleles at two or more loci.
  • Populations considered for LD calculation include African, American, European, east Asian and south Asian meta-populations and any corresponding sub- populations, as described in the 1000 Genome project Phase 3 data. Alleles will be considered in LD if a significant association is observed in at least one of these meta-populations or subpopulations, or in any newly described population.
  • LD is a measure of co- segregation of alleles in a population.
  • Linkage disequilibrium or allelic association is the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population.
  • Determining the presence of the allele may be determined according to any genotyping method known in the art.
  • common genotyping methods include, but are not limited to, TaqMan assays, molecular beacon assays, nucleic acid arrays, allele- specific primer extension, allele- specific PCR, arrayed primer extension, homogeneous primer extension assays, primer extension with detection by mass spectrometry, pyrosequencing, multiplex primer extension sorted on genetic arrays, ligation with rolling circle amplification, . homogeneous ligation, OLA, multiplex ligation reaction sorted on genetic arrays, restriction- fragment length polymorphism, single base extension-tag assays, and the Invader assay.
  • Such methods may be used in combination with detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection.
  • detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection.
  • Various methods for detecting polymorphisms include, but are not limited to, methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA, comparison of the electrophoretic mobility of variant and wild type nucleic acid molecules, and assaying the movement of polymorphic or wild- type fragments in polyacrylamide gels containing a gradient of denaturant using denaturing gradient gel electrophoresis. Sequence variations at specific locations can also be assessed by nuclea
  • genotyping is performed using the TaqMan assay, which is also known as the 5' nuclease assay.
  • the TaqMan assay detects the accumulation of a specific amplified product during PCR.
  • the TaqMan assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye.
  • the reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the excited reporter dye does not emit a signal.
  • the proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter.
  • the reporter dye and quencher dye may be at the 5' most and the 3' most ends, respectively, or vice versa.
  • the reporter dye may be at the 5' or 3' most end while the quencher dye is attached to an internal nucleotide, or vice versa.
  • both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced.
  • the 5' nuclease activity of DNA polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye.
  • the DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target SNP-containing template which is amplified during PCR, and the probe is designed to hybridize to the target SNP site only if a particular SNP allele is present.
  • Preferred TaqMan primer and probe sequences can readily be determined using the SNP and associated nucleic . acid sequence information provided herein. A number of computer programs, such as Primer Express (Applied Biosystems, Foster City, Calif.), can be used to rapidly obtain optimal primer/probe sets. It will be apparent to one of skill in the art that such primers and probes for detecting the nucleic acids of the present invention are useful in diagnostic assays for stenosis and related pathologies, and can be readily incorporated into a kit format.
  • Another method for genotyping the nucleic acids of the present invention is the use of two oligonucleotide probes in an Oligonucleotide Ligation Assay (OLA).
  • OVA Oligonucleotide Ligation Assay
  • one probe hybridizes to a segment of a target nucleic acid with its 3' most end aligned with the nucleic acid site.
  • a second probe hybridizes to an adjacent segment of the target nucleic acid molecule directly 3' to the first probe.
  • the two juxtaposed probes hybridize to the target nucleic acid molecule, and are ligated in the presence of a linking agent such as a ligase if there is perfect complementarity between the 3' most nucleotide of the first probe with the nucleic acid site.
  • OLA may also be used for performing nucleic acid detection using universal arrays, wherein a zipcode sequence can be introduced into one of the hybridization probes, and the resulting product, or amplified product, hybridized to a universal zip code array.
  • OLA may be used where zipcodes are incorporated into OLA probes, and amplified PCR products are determined by electrophoretic or universal zipcode array readout.
  • OLA is carried out prior to PCR (or another method of nucleic acid amplification). In some other embodiments, PCR (or another method of nucleic acid amplification) is carried out prior to OLA.
  • Mass spectrometry takes advantage of the unique mass of each of the four nucleotides of DNA. Nucleic acids can be unambiguously genotyped by mass spectrometry by measuring the differences in the mass of nucleic acids having alternative nucleic acid alleles. MALDI-TOF (Matrix Assisted Laser Desorption Ionization— Time of Flight) mass spectrometry technology is preferred for extremely precise determinations of molecular mass, such as for SNPs. Numerous approaches to genotype analysis have been developed based on mass spectrometry. Preferred mass spectrometry-based methods of nucleic acid genotyping include primer extension assays, which .
  • the primer extension assay involves designing and annealing a primer to a template PCR amplicon upstream (5' ) from a target nucleic acid position.
  • a mix of dideoxynucleotide triphosphates (ddNTPs) and/or deoxynucleotide triphosphates (dNTPs) are added to a reaction mixture containing template.
  • this is a SNP-containing nucleic acid molecule which has typically been amplified, such as by PCR.
  • Primer and DNA polymerase may further be added.
  • Extension of the primer terminates at the first position in the template where a nucleotide complementary to one of the ddNTPs in the mix occurs.
  • the primer can be either immediately adjacent (i.e., the nucleotide at the 3' end of the primer hybridizes to the nucleotide next to the target SNP site) or two or more nucleotides removed from the nucleic acid position. If the primer is several nucleotides removed from the target nucleic acid position, the only limitation is that the template sequence between the 3' end of the primer and the nucleic acid position cannot contain a nucleotide of the same type as the one to be detected, or this will cause premature termination of the extension primer.
  • primers are designed to bind one nucleotide upstream from the SNP position (i.e., the nucleotide at the 3' end of the primer hybridizes to the nucleotide that is immediately adjacent to the target SNP site on the 5 ' side of the target SNP site). Extension by only one nucleotide is preferable, as it minimizes the overall mass of the extended primer, thereby increasing the resolution of mass differences between alternative SNP nucleotides.
  • mass-tagged ddNTPs can be employed in the primer extension reactions in place of unmodified ddNTPs. This increases the mass difference between primers extended with these ddNTPs, thereby providing increased sensitivity and accuracy, and is particularly useful for typing heterozygous base positions. Mass-tagging also alleviates the need for intensive sample- preparation procedures and decreases the necessary resolving power of the mass spectrometer.
  • the extended primers can then be purified and analyzed by MALDI-TOF mass spectrometry to determine the identity of the nucleotide present at the target SNP position. In one method of analysis, the products from the primer extension reaction are combined with light absorbing crystals that form a matrix.
  • the matrix is then hit with an energy source such as a laser to ionize and desorb the nucleic acid molecules into the gas-phase.
  • the ionized molecules are then ejected into a flight tube and accelerated down the tube towards a detector.
  • the time between the ionization event, such as a laser pulse, and collision of the molecule with the detector is the . time of flight of that molecule.
  • the time of flight is precisely correlated with the mass-to-charge ratio (m/z) of the ionized molecule. Ions with smaller m/z travel down the tube faster than ions with larger m/z and therefore the lighter ions reach the detector before the heavier ions.
  • the time-of-flight is then converted into a corresponding, and highly precise, m/z.
  • SNPs can be identified based on the slight differences in mass, and the corresponding time of flight differences, inherent in nucleic acid molecules having different nucleotides at a single base position.
  • Nucleic acids can also be scored by direct DNA sequencing.
  • a variety of automated sequencing procedures can be used, including sequencing by mass spectrometry.
  • the nucleic acid sequences of the present invention enable one of ordinary skill in the art to readily design sequencing primers for such automated sequencing procedures.
  • Commercial instrumentation such as the Applied Biosystems 377, 3100, 3700, 3730, and 3730x1 DNA Analyzers (Foster City, Calif.), is commonly used in the art for automated sequencing.
  • Nucleic acid sequences can also be determined by employing a high throughput mutation screening system, such as the SpectruMedix system.
  • SSCP single-strand conformational polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • Sequence-specific ribozymes can also be used to score nucleic acids, in particular SNPs, based on the development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. Thus, for example, if the SNP affects a restriction enzyme cleavage site, the SNP can be identified by alterations in restriction enzyme digestion patterns, and the corresponding changes in nucleic acid fragment lengths determined by gel electrophoresis .
  • Genotyping can include the steps of, for example, collecting the sample, isolating nucleic acids (e.g., genomic DNA, mRNA or both) from the cells of the sample, contacting the nucleic acids with one or more primers which specifically hybridize to a region of the isolated nucleic acid containing a target nucleic acid region of interest under conditions such that hybridization and amplification of the target nucleic acid region occurs, and determining the nucleotide present at the nucleic acid position of interest, or, in some assays, detecting the presence or absence of an amplification product (assays can be designed so that hybridization and/or amplification will only occur if a particular nucleic acid sequence allele is present or absent).
  • nucleic acids e.g., genomic DNA, mRNA or both
  • the size of the amplification product is detected and compared to the length of a control sample; for example, deletions and insertions can be detected by a change in size of the amplified product compared to a normal genotype.
  • the nucleic acid, or in particular the SNP, found may then be compared to the nucleic acids of other individuals whom have also received the antigen to induce an immune response.
  • Methods of comparing the identity of two or more sequences may be performed by any reasonable means, including programs available in the Wisconsin Sequence Analysis Package version 9.1 (Genetics Computer Group, Madison, Wis., USA). Other programs such as BESTFIT may be used to find the "local homology" algorithm of Smith and Waterman and finds the best single region of similarity between two sequences.
  • programs such as GAP may be used, which aligns two sequences finding a "maximum similarity.”
  • % identities and similarities are determined when the two sequences being compared are optimally aligned.
  • Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs, available from the National Center for Biotechnology Information (NCB), Bethesda, Md., USA) and FASTA, available as part of the Wisconsin Sequence Analysis Package.
  • Exemplary diagnostic tests for pancreatitis typically include biochemical measurements, such as measurements for abnormal levels of isoamylase, lipase, trypsin, elastase, or secretin, quantitative measurement of fecal fat, measurement of plasma cholecystokinin (CCK), tests for pancreatic exocrine function, radiological testing such as plain abdominal film, transabdominal ultrasound, or CT scanning, magnetic resonance cholangiopancreatography (MRCP), or endoscopic diagnosis, e.g., endoscopic retrograde cholangiopancreatography (ERCP) or endoscopic ultrasonography. .
  • the physician can prescribe a treatment (curative or prophylactic treatment).
  • the treatment includes, for example, pain management, abstinence from alcohol and cigarette smoking, cholecystectomy, biliary sphincterotomy, endoscopic retrograde cholangiopancreatography (ERCP), administration of intravenous fluids, nutritional support, antibiotics, carbapenems, enzyme therapy, surgery, such as longitudinal pancreaticojejunostomy or pancreatoduodenectomy, distal pancreatectomy, celiac nerve block, endoscopic therapy, percutaneous drainage and/or antioxidant therapy (e.g. N-acetylcysteine, selenium, ⁇ -carotene, vitamin C, vitamin E, or methionine).
  • antioxidant therapy e.g. N-acetylcysteine, selenium, ⁇ -carotene, vitamin C, vitamin E, or methionine.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 The C allele at rs7727286 decreases the activity of the SPINKl enhancer.
  • B) PWM -based analysis reveals that the presence of the minor rs7727286C allele decreases the weight of the HNFIA binding site.
  • the P Value of the predicted HNFIA binding site is above the threshold (1.0e-04) to be considered as biologically relevant.
  • Luciferase reporter constructs carrying the SPINKl promoter alone (hSPINKlpp) or in cis with the SPINKl enhancer in the context of the T (+E[rs7727286T]) or C (+E[rs7727286C]) alleles at rs7727286 were co-transfected with expression plasmids encoding the HNFIA transcription factor and the components of the PTF1- L transcriptional complex in HEK293T cells. Expression level of the SPINKl promoter alone is set to 100. Results represent means + SD of three independent experiments. Bars, SD. *, P ⁇ 0.05.
  • Genomic coordinates correspond to the human GRCh37/hgl9 assembly (https://genome.ucsc.edu/).
  • the build 147 of dbSNP was explored via the UCSC Genome _
  • the PWM-based analysis was performed with the RSAT - matrix-scan (full options) tool and the HNF1A (MA0046.2) position frequency matrix, as described in (Boulling et al., 2017).
  • the luciferase reporter plasmid harbouring the SPINK1 promoter alone (hSPINKlpp) or in cis with the SPINK 1 enhancer (+E[rs7727286T]), in the context of SPINK 1 promoter and enhancer wild-type sequences, have been described previously (Boulling et al., 2017).
  • the luciferase reporter construct carrying the SPINK 1 promoter in cis with the SPINK 1 enhancer in the context of the minor rs7727286C allele (+E[rs7727286C]) was obtained by site directed mutagenesis using the Quick Change Site Directed Mutagenesis Kit (Stratagene, Massy, France) and primer forward 5 ' -CAAGGACTAATTATCCACCTGAGTAAAGAGTGATG-3 ' (SEQ ID NO:2) and primer reverse 5'-homo-3' (SEQ ID NO:3).
  • transcription factor expression plasmids Construction of transcription factor expression plasmids, cell culture, co-transfection transactivation experiments and luciferase reporter gene assay
  • luciferase reporter constructs carrying the SPINK 1 promoter alone or in cis with the enhancer was co-transfected with plasmids expressing HNF1A and PTF1-L transcription _ factors in HEK293T cells ( Figure 1C).
  • the activity of the construct carrying the rs7727286C allele was compared to that of the construct carrying the rs7727286T allele.
  • Both constructs showed an enhancer activity in terms of luciferase expression.
  • the C allele was associated to a lower relative activity (+29 % in comparison to the SPINK1 promoter alone) than the T allele (+50 % in comparison to the SPINK1 promoter alone). It means that, in our system, the minor C allele results in a loss of 42 % of the enhancer activity. Based on these observations, rs7727286T>C can be considered as a SPINK1 loss-of-function variant.
  • the rs7727286T>C variant is not equally distributed in the five human meta-populations (Table 1). Its highest allele frequency is observed in african population, in which more than 7 % of the individuals are carrying at least one rs7727286C allele. This allele is also observed at the heterozygous state in 1.7 % of americans, but is rare in Europeans and absent in both east and south asians. We suggest that the presence of the rs7727286T>C variant should be tested at least in africans and americans with CP, or at risk to develop CP.

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Abstract

L'invention concerne la pancréatite, qui est une inflammation du pancréas. L'établissement du diagnostic et de la cause de la pancréatite repose sur les antécédents médicaux, les antécédents familiaux et/ou un test génétique moléculaire, qui peut être soit un test de gène unique (monogène), soit l'utilisation d'un panel multigénique. Un variant pathogène commun de "perte de fonction" dans un amplificateur de SPINK 1 a été décrit en tant qu'augmentation du risque de pancréatite. Par la navigation dans la base de données de Variations Génétiques Courtes (dbSNP), les inventeurs ont recherché d'autres variants communs situés dans cet amplificateur et n'ont découvert qu'un variant commun de "perte de fonction" (c'est-à-dire rs7727286T>C) à une fréquence supérieure à 0,01 in. Sa fréquence d'allèle la plus élevée est observée dans une population africaine, dans laquelle plus de 7 % des individus sont porteurs d'au moins un allèle rs7727286C. La détermination de la présence dudit variant (par exemple par un test de TaqMan) est ainsi appropriée pour identifier un sujet présentant ou risquant d'être atteint d'une pancréatite.
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Cited By (2)

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JP2020178539A (ja) * 2019-04-23 2020-11-05 ジェネシスヘルスケア株式会社 膵炎のリスクを判定する方法
WO2022140380A1 (fr) * 2020-12-21 2022-06-30 Pancreomics Llc Signatures métabolomiques pour prédire, diagnostiquer et pronostiquer la pancréatite chronique

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JP2020178539A (ja) * 2019-04-23 2020-11-05 ジェネシスヘルスケア株式会社 膵炎のリスクを判定する方法
JP7137520B2 (ja) 2019-04-23 2022-09-14 ジェネシスヘルスケア株式会社 膵炎のリスクを判定する方法
WO2022140380A1 (fr) * 2020-12-21 2022-06-30 Pancreomics Llc Signatures métabolomiques pour prédire, diagnostiquer et pronostiquer la pancréatite chronique

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