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WO2018187767A1 - Prédiction, diagnostic et traitement de nausées et de vomissements liés à la grossesse - Google Patents

Prédiction, diagnostic et traitement de nausées et de vomissements liés à la grossesse Download PDF

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WO2018187767A1
WO2018187767A1 PCT/US2018/026588 US2018026588W WO2018187767A1 WO 2018187767 A1 WO2018187767 A1 WO 2018187767A1 US 2018026588 W US2018026588 W US 2018026588W WO 2018187767 A1 WO2018187767 A1 WO 2018187767A1
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gdf15
subject
nvp
igfbp7
outlook
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Marlena S. FEJZO
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The Regents Of The University Of California
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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
    • 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
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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

  • NVP Nausea and vomiting of pregnancy
  • NVP Hyperemesis Gravidarum
  • HG Hyperemesis Gravidarum
  • Its clinical presentation includes severe intractable vomiting, often associated with dehydration, weight loss (> 5% pre-pregnancy weight), ketonuria, metabolic alkalosis and electrolyte disturbances.
  • This disease may have killed famous author Charlotte Bronte, was the leading cause of maternal death until intravenous hydration was introduced in the 1950s, and to this day is the second leading cause of hospitalization of pregnancy after preterm labor.
  • the assay reagents and methods described herein meet these needs and others by providing biomarkers for nausea and vomiting in pregnancy (NVP) and hyperemesis gravidarum (HG), An assay as described herein is extremely useful to practicing clinicians to identify patients at risk for NVP and HG.
  • the invention thus provides methods for diagnosis and/or treatment of HG and NVP.
  • Described herein is a method of analyzing genetic expression in a sample obtained from a subject.
  • the method comprises obtaining a sample from the subject; and measuring the level of expression product of at least one of the hyperemesis gravidarum (HG) outlook genes identified in Table 1 in the sample.
  • expression product includes both the genotypic expression (e.g., presence of a variant) and the phenotypic expression (e.g., RNA or protein expression indicative of a genetic variant).
  • measuring an expression product comprises detection of a genetic variant.
  • the method can further comprise assigning a HG outlook score to the measured amount of expression for each of the HG outlook genes, wherein the score is between 0 and 1 ; and calculating a HG status score, wherein the status score is equal to a combination of the outlook scores assigned, in one embodiment, the combination is an average outlook score. In other embodiments, the combination is a total of HG status scores for each outlook gene measured or detected. The combination can be calculated or determined in other ways that result in an HG status score that reflects the number and/or quality of genetic variants detected. In one embodiment, the presence of a genetic variation associated with HG is assigned a score of 1 , and the absence of the genetic variation is assigned a score of 0. Those skilled in the art will appreciate other means of assigning and combining HG outlook scores.
  • patients can be tested for the presence of a risk allele that is predictive of ability or failure to respond to medications, such as ondansetron, promethazine, and metoclopramide.
  • a risk allele that is predictive of ability or failure to respond to medications, such as ondansetron, promethazine, and metoclopramide.
  • failure to respond to ondansetron is linked to the IGFBP7 HG-risk allele;
  • the invention provides a method of treating HG, the method comprising treating a patient having HG with ondansetron or promethazine if the patient is lacking the iGFBP7 HG-risk allele, and treating a patient with metoclopramide if the patient is positive for the GDF15 HG-risk allele.
  • metoclopramide Due to the increased efficacy of ondansetron relative to promethazine, which, in turn, is more effective than metoclopramide, a treating physician may opt to initiate treatment with ondansetron regardless of phenotype. Other factors, such as price differences, insurance coverage, and safety data may also influence which drug is used first. Based on effectiveness, in most instances, a physician would not use metoclopramide before any of the other drugs, unless the patient is homozygous for the GDF15 HG-risk allele, in the latter case, metoclopramide is predicted to be almost as effective as promethazine in patients with at least one of the IGFBP7 risk alleles.
  • the method comprises measuring the expression product of an HG outlook gene, wherein the HG outlook gene is selected from: GDF15, IGFBP7, PGR, and RYR2.
  • the HG outlook gene is selected from: GDF15, IGFBP7, PGR, and RYR2.
  • at least two of the HG outlook genes of Table 1 are measured.
  • three, four, five, six, seven, eight, nine, ten, or more HG outlook genes are measured, including those listed in Table 1 , and other HG outlook genes, such as RYR2.
  • the method comprises measuring the level of GDF15 in a sample obtained from a subject, if the sample shows levels of GDF15 greater than about 10 ng/ml, the subject is likely to have or develop HG.
  • the method further comprises measuring the level of IGFBP7 in a sample obtained from the subject, using either the same or another sample. If the sample shows levels of IGFBP7 greater than about 60 ng/ml, the subject is likely to have or develop HG.
  • Subjects exhibiting greater than 10 ng/ml GDF15 and greater than 60 ng/ml IGFBP7 have a 36-foid greater risk of being hospitalized for HG, and are candidates for treatment with medication, such as ondansetron, promethazine, or metoclopramide.
  • a subject showing greater than about 8 ng/ml GDF15 and/or greater than about 50 ng/ml IGFBP7 are candidates for treatment with medication.
  • Those skilled in the art will know to adjust these cut off levels based on the assays employed and comparison to control or normalization values.
  • the above values are based on data collected from subjects at 12 weeks gestation, and use of a different assay, or collection of samples at a different point in gestation, may involve adjustment to these values.
  • the forementioned immunoassay is performed, in some embodiments, using detection of one or both of GDF15 and IGFBP7, optionally, in combination with an additional marker, in some embodiments, the assay is part of a multi-marker assay that includes reagents for detecting a panel of up to 20 markers. In some embodiments, the panel consists of no more than 15 markers, or up to 10 markers, and in some embodiments, up to 5 markers are used in combination.
  • the sample is a biological patient sample, representative examples of which include, a urine sample, a blood sample (including serum), or a saliva sample.
  • the sample is tested for genotype and/or associated phenotype (RNA, protein expression levels) for a minimum of 2 alleles in Table 1 (or for an allele with linkage disequilibrium to the allele in Table 1).
  • Treatment for NVP or HG can be administered to the subject based on the assigned score for each test and/or combination of tests, in some embodiments, treatment is
  • cut-off values can be adjusted in accordance with specific adaptations of the methods described
  • the measuring comprises contacting the sample obtained from the subject with reagents for amplifying polynucleotides.
  • the level of expression is measured via hybridization of probes that specifically bind polynucleotides encoding the expression product.
  • the level of expression is measured via binding of antibodies that specifically bind the expression product.
  • Standard immunoassay reagents can be used to detect proteins. Representative immunoassays include enzyme immunoassays (including ELISA), microarray assays, nanosurface assays, western blotting, alphaLISA, radioimmunoassay, competitive binding assays, and automated instrumentation- based assays.
  • the HG outlook gene is GDF15, IGFBP7, PGR, SYN3, MMADHC, TMEM38B, HCRTR2, GFRAL, RYR2 and/or PKHD1.
  • the expression can, in a representative embodiment, be measured via detection of the G allele at rs18982345 (or any alleles in a shared haplotype (in linkage disequilibrium) with the G allele at rs16982345) of GDF15.
  • the HG outlook gene is a combination of two, three, or four of GDF15, IGFBP7, PGR, GFRAL, and RYR2.
  • GDF15 and IGFBP7 examples include, but are not limited to, GDF15 and IGFBP7; GDF15 and PGR; GDF15 and RYR2; GDF15 and GFRAL; IGFBP7 and PGR; IGFBP7 and RYR2; IGFBP7 and GFRAL; PGR and RYR2; PGR and GFRAL;
  • GDF15, IGFBP7, and GFRAL GDF15, PGR, and GFRAL; IGFBP7, PGR, and GFRAL; GDF15, IGFBP7, PGR, and GFRAL; GDF15, PGR, and GFRAL; IGFBP7, PGR, and GFRAL; as well as GDF15, 1GFBP7, and GFRAL.
  • the method further comprises treating the subject for
  • the gene is GDF15 and the treatment comprises administering an anti-GDF15 agent to the subject.
  • the gene is PGR and the treatment comprises administering an anti-PGR and/or anti-progesterone agent
  • the gene is MMADHC and the treatment comprises administering vitamin B12 to the subject.
  • the method can also be used to treat related diseases, including cachexia and low appetite, while the reverse treatment would be applied for detection of the other allele for treating fetal loss and/or overactive appetite.
  • the treatment comprises administering GDF15 if the genotype is A/A at rs16982345 in GDF15, and/or progesterone depending on the genotype at the PGR locus.
  • the method comprises performing the analyzing described herein; and administering a treatment for NVP or HG to the subject if the analyzing results in a status score that is greater than zero.
  • the treatment comprises an inhibitor of GDF15, GFRAL, or PGR.
  • the invention provides a method of treating a subject for HG, wherein the method comprises analyzing a sample obtained from the subject for presence of one or more of the genotype combinations identified in Examples 3 and 5 hereinbelow as associated with HG at a significance of P ⁇ 0.05; and administering an inhibitor of a corresponding gene if the patient is positive for the significant genotype combination. Also provided is a method of treating a subject with an inhibitor of GDF15 or GFRAL, wherein the method comprises assaying a sample obtained from the subject for the presence of a variant of GDF15 or GFRAL as described herein, and administering the inhibitor to the subject whose sample contains the corresponding variant.
  • Figure 1 Genome-wide association scans for nausea and vomiting of pregnancy.
  • the Manhattan plot shows distribution of association test statistics vs. genomic position for (a) SCAN1 (binary phenotype), and (b) SCAN2 (ordinal phenotype).
  • the Manhattan plot shows distribution of association test statistics versus genomic position. Chromosomes are arranged along the X-axis. Log10-scaled p-vaiues are shown on the Y-axis. The loci with positions with p ⁇ 5 x 10 -8 are shown in red and the loci with p ⁇ 10 -6 are labeled with names of nearest genes.
  • Figure 2 is a scatferplot of serum levels of GDF15 (ng/ml), at 12 weeks gestation in patients hospitalized for HG (HG), patients with NVP (NVP), and controls with no NVP before 24 weeks gestation (NO NVP).
  • Figure 3 is a box plot of serum levels of GDF15 (ng/ml), at 12 weeks gestation in patients hospitalized for HG (HG), patients with NVP (NVP), and controls with no NVP before 24 weeks gestation (NO NVP).
  • Figure 4 is a box plot of serum levels of IGFBP7 (ng/ml), at 12 weeks gestation in patients hospitalized for HG (HG), patients with NVP (NVP), and controls with no NVP before 24 weeks gestation (NO NVP).
  • Figure 5 is a box plot of serum levels of beta HCG (mlU/ml), at 12 weeks gestation in patients hospitalized for HG (HG), patients with NVP (NVP), and controls with no NVP before 24 weeks gestation (NO NVP).
  • Figures 6A-6C Three of five HG families show segregation of alleles at GDF15 locus associated with increased expression of GDF15.
  • Fig. 8A) Family 1 is of
  • Case (1A) reported iv fluid, total parenteral nutrition, antiemetic medication, home health care, and a 5% weight loss due to HG.
  • Her cousin (1 B) reported normal NVP with no weight loss and no medication to treat NVP.
  • Her uncle (1 C) reported antiemetic medication and weight loss due to HG, and her great uncle (1 D) reported antiemetic medication and unrelenting nausea that kept her bedridden for 8 months due to HG.
  • Fig. 6B Family 2 is of English/German/Scottish/Irish decent.
  • Case (2A) reported iv fluid, hospitalization, >5% weight loss, and antiemetic medication to treat her HG.
  • Her uncle (2B) reported iv fluid, hospitalization, weight loss, and antiemetic medication to treat her HG.
  • Her cousin [affected aunt's daughter, (2C)] reported antiemetic medication and a 24-pound weight loss in the first trimester due to HG. Participant 2D, the unaffected sister of 2C, reported 2 easy pregnancies with no weight loss nor treatment for NVP.
  • Fig. 6C) Family 3 is of English/Irish descent.
  • Case (3A) reported iv fluid, hospitalization, >10% weight loss, and antiemetic medication to treat HG.
  • One affected sister (3B) reported iv fluid, hospitalization, and antiemetic medication to treat HG.
  • the other affected sister (3C) reported iv fluid, hospitalization, weight loss, and antiemetic medication to treat HG,
  • Their unaffected aunt (3E) reported mild NVP with no medication nor weight loss.
  • Figure 7 is Table 1.2, which shows the results of the genome-wide association study of no NVP vs HG (SCAN1) with p ⁇ 10-6. The directions of odds ratios (OR) correspond to the minor allele, listed second.
  • Figure 8 is Table 1.4, which shows the results of GWAS of NVP as an ordinal response (SCAN2) with p ⁇ 5 x 10 -8 , The directions of effect correspond to the minor allele, listed second.
  • NVP nausea and vomiting in pregnancy
  • HG hyperemesis gravidarum
  • markers and methods of using these markers of NVP and/or HG address a long-felt need for diagnostic tests that can predict individual risk of NVP and HG, as well as treatment options for severe nausea and vomiting (HG) that is associated with poor outcomes including preterm birth, neurodevelopmental delay, and vitamin K deficient embryopathy.
  • the invention provides genetic analyses as well as serum tests, e.g. for GDF15 and 1GFBP7, that can be used to identify patients at risk for HG. Definitions
  • a "control" or “reference” sample means a sample that is representative of normal measures of the respective marker, such as would be obtained from normal, healthy control subjects, or a baseline amount of marker to be used for comparison. Typically, a baseline will be a measurement taken from the same subject or patient. The sample can be an actual sample used for testing, or a reference level or range, based on known normal measurements of the corresponding marker.
  • a "significant difference” means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a difference that is of sufficient magnitude that, under the circumstances, can be detected with a reasonable level of reliability.
  • an increase or decrease of 10% relative to a reference sample is a significant difference
  • an increase or decrease of 20%, 30%, 40%, or 50% relative to the reference sample is considered a significant difference
  • an increase of two-fold relative to a reference sample is considered significant.
  • Nucleotide sequence refers to a heteropolymer of deoxyribonucieotides
  • ribonucleotides or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of expressing the encoded protein.
  • pharmaceutically acceptable carrier includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.
  • examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
  • compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).
  • the term "subject” includes any human or non-human animal.
  • the term "non-human animal” includes ail vertebrates, e.g., mammals and non-mammals, such as non- human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In a typical embodiment, the subject is a human.
  • "a” or “an” means at least one, unless dearly indicated otherwise.
  • the invention provides methods for analyzing genetic expression, or assaying gene expression status in a sample obtained from a subject, for diagnosing NVP and/or HG in a subject, as well as methods for predicting, treating, and/or monitoring NVP and/or HG.
  • the method comprises obtaining a sample from the subject; and measuring the level of expression product of at least one of the hyperemesis gravidarum (HG) outlook genes identified in Table 1 in the sample.
  • the method can further comprise assigning a HG outlook score to the measured amount of expression for each of the HG outlook genes, wherein the score is between 0 and 1 ; and calculating a HG status score, wherein the status score is equal to the average outlook score assigned, in other embodiments, the combination is a total of HG status scores for each outlook gene measured or detected.
  • the presence of a genetic variation associated with HG is assigned a score of 1
  • the absence of the genetic variation is assigned a score of 0.
  • the combination can be calculated or determined in other ways that result in an HG status score that reflects the number and/or quality of genetic variants detected.
  • the method comprises measuring the expression product of an HG outlook gene, wherein the HG outlook gene is selected from: GDF15, IGFBP7, PGR, and RYR2.
  • the HG outlook gene is selected from: GDF15, IGFBP7, PGR, and RYR2.
  • at least two of the HG outlook genes of Table 1 are measured.
  • three, four, five, six, seven, eight, nine, ten, or more HG outlook genes are measured, including those listed in Table 1 , and other HG outlook genes, such as RYR2.
  • the method comprises measuring the expression product of GDF15 and IGFBP7.
  • the method comprises measuring the expression product of at least 3 of the HG outlook genes selected from GDF15, IGFBP7, PGR, and RYR2.
  • the method comprises measuring the expression product of at least 3 of the HG outlook genes selected from GDF15, IGFBP7, PGR, SYN3, MMADHC, TMEM38B, and RYR2. In one embodiment, the method comprises measuring the expression product of each of GDF15, IGFBP7, PGR, and RYR2. In one embodiment, the method comprises measuring GDF15 and/or IGFBP7. [0036] In one embodiment, the method comprises detecting the presence of a genetic variant as described in Table 1. The detection of a genetic variant as described herein can be used to predict, detect and/or diagnose NVP, HG, cachexia, fetal loss, or an appetite disorder.
  • the method comprises detection of at least one SNP per haplotype.
  • the sample is a biological patient sample, representative examples of which include, a urine sample, a blood or serum sample, or a saliva sample.
  • the sample is tested for genotype and/or associated phenotype (RNA, protein expression levels) for a minimum of 2 alleles in Table 1 (or for an allele with linkage disequilibrium to the allele in Table 1).
  • Treatment for NVP or HG can be administered to the subject based on the assigned score for each test and/or combination of tests.
  • the measuring comprises contacting the sample obtained from the subject with reagents for amplifying polynucleotides, in some embodiments, the level of expression is measured via hybridization of probes that specifically bind polynucleotides encoding the expression product. In yet other embodiments, the level of expression is measured via binding of antibodies that specifically bind the expression product. In one embodiment, the levels of GDF15 and/or IGFBP7 are assayed, in one embodiment, the method comprises an immunoassay for GDF15 and IGFBP7, wherein levels of these proteins are detected in a patient sample, such as, for example, a serum sample.
  • the HG outlook gene is GDF15, IGFBP7, PGR, SYN3, MMADHC, TMEM38B, HCRTR2, GFRAL, RYR2 and/or PKHD1.
  • the expression can, in a representative embodiment, be measured via detection of the G allele at rs18982345 (or any alleles in a shared haplotype (in linkage disequilibrium) with the G allele) at rs16982345 in GDF15.
  • the method comprises:
  • step (d) referring the subject for treatment of NVP and/or HG if the status score is significantly greater than 1.
  • step (a) is performed with one HG outlook gene from Table 1 and one HG outlook gene from Table 2.
  • step (a) is performed with at least two HG outlook genes from Table 1 and at least one HG outlook gene from Table 2.
  • step (a) is performed with one of the combinations of HG outlook genes listed in Table 3 or Example 5.
  • the method further comprises treating the subject for
  • the gene is GDF15 and the treatment comprises administering an anti-GDF15 agent to the subject
  • the gene is PGR and the treatment comprises administering an anti-PGR and/or anti-progesterone agent.
  • the gene is MMADHC and the treatment comprises administering vitamin B12 to the subject.
  • the method can be adapted for related diseases, including cachexia and low appetite, while the reverse treatment would be applied for detection of the other allele for treating fetal loss and/or overactive appetite.
  • the method comprises administering GDF15 if the genotype is A/A at rs18982345 in GDF15, and/or progesterone depending on the genotype at the PGR locus.
  • treatment include, but are not limited to, administering to the subject one or more antiemetic agents (e.g., ondansetron, diciegis, propranolol, or a related drug that inhibits the pathway that includes RYR2 and other calcium channel signaling proteins).
  • antiemetic agents e.g., ondansetron, diciegis, propranolol, or a related drug that inhibits the pathway that includes RYR2 and other calcium channel signaling proteins.
  • a method of monitoring NVP and/or HG, and/or cachexia, an appetite disorder, and/or fetal loss comprises:
  • the method comprises performing the analyzing described herein; and administering a treatment for NVP or HG to the subject if the analyzing results in a status score that is greater than zero.
  • the treatment comprises an inhibitor of GDF15, GFRAL, or PGR.
  • the invention provides a method of treating a subject for HG, wherein the method comprises analyzing a sample obtained from the subject for presence of one or more of the genotype combinations identified in Table 3 or Example 5 hereinbelow as associated with HG at a significance of P ⁇ 0.05; and administering an inhibitor of a corresponding gene if the patient is positive for the significant genotype combination.
  • a variant indicative of HG/NVP that can be detected via genotypic and/or phenotypic measures.
  • a variant associated with HG/NVP can be detected, for example, by sequencing DNA obtained from a sample (or running an allelic discrimination assay), or, in some cases, by detecting an expression product that reveals the presence of the variant.
  • the HG outlook gene is at least one HG outlook gene selected from Table 1 or at least one combination of genes listed in Table 3 or Example 5. in one
  • At least two or more HG outlook genes of Table 1 is used in combination.
  • Examples of combinations of HG outlook genes include: one or more of the combinations listed in Table 3 or Example 5; GDF15 and an additional HG outlook gene selected from Table 1 ; PGR and an additional HG outlook gene selected from Table 1 ; IGFBP7 and an additional HG outlook gene selected from Table 1 ; RYR2 and an additional HG outlook gene selected from Table 1 ; SYN3 and an additional HG outlook gene selected from Table 1 ; MMADHC and an additional HG outlook gene selected from Table 1 ; TMEM38B and an additional HG outlook gene selected from Table 1 ; MAP3K7 and an additional HG outlook gene selected from Table 1 ; SPECC1 L and an additional HG outlook gene selected from Table 1 ; SPECC1 L and an additional HG outlook gene selected from Table 1 ; GDF15 and IGFBP7 and an additional HG outlook gene selected from Table 1 ; any combination of 3 or more HG outlook genes selected from Table 1 ; any combination of 4, 5, 8, 7, 8, 9, 10, or 1 1 HG outlook genes of Table 1.
  • the combination of HG outlook genes is 2, 3, 4, or 5 HG outlook genes selected from Table 1.
  • the combination of HG outlook genes is at least one HG outlook gene from Table 1 , and at least one HG outlook gene from Table 2.
  • the combination of HG outlook genes is at least one HG outlook gene from Table 1 , and at least one combination of HG outlook genes from Table 3.
  • the combination of HG outlook genes is at least one HG outlook gene from Table 2, and at least one combination of HG outlook genes from Table 3.
  • the combination of HG outlook genes is at least one HG outlook gene from each of Tables 1 , 2, and 3.
  • kits comprising a set of reagents as described herein, such as antibodies that specifically bind one or more HG outlook genes of the invention (or their expression products), and optionally, one or more suitable containers containing reagents of the invention.
  • Reagents include molecules that specifically bind and/or amplify and/or detect one or more HG outlook genes or related expression products of the invention. Such molecules can be provided in the form of a microarray or other article of manufacture for use in an assay described herein.
  • a reagent is an antibody or nucleic acid probe that is specific for the HG outlook gene (or its expression product, e.g. GDF15 and/or IGFBP7).
  • Kits of the invention optionally comprise an assay standard or a set of assay standards, either separately or together with other reagents.
  • An assay standard can serve as a normal control by providing a reference level of normal expression for a given HG outlook gene that is representative of a healthy individual.
  • Kits can include probes for detection of alternative gene expression products in addition to antibodies for protein detection.
  • the kit can optionally include a buffer.
  • Reagents and standards can be provided in combinations reflecting the combinations of HG outlook genes described herein as useful for the detection of HG and/or NVP.
  • This Example a describes a genome-wide association study (GWAS) for binary (HG) and ordinal (severity of nausea and vomiting) phenotypes of pregnancy complications.
  • Two loci, chr19p13.1 1 and chr4q12, are genome-wide significant (p ⁇ 5 x 10-8) in both association scans and are replicated in an independent cohort.
  • the genes implicated at these two loci are GDF15 and 1GFBP7 respectively, both known to be involved in placenfation, appetite, and cachexia. This GWAS provides insights into the genetic risk factors contributing to the disease.
  • Supplementary materials referenced in this Example can be found online in connection with the published version of this work at Fejzo et al., Nature Communications 9, Article Number 1 178 (2016) doi: 10, 1038/s41467-018-03258-0, and can also be found in related provisional patent application number 62/596,704, filed December 8, 2017.
  • SNPs single-nucleotide polymorphisms
  • indeis insertion or deletion polymorphisms
  • Table 1.2 shows the results of the genome-wide association study of no NVP vs HG (SCAN1) with p ⁇ 10-6.
  • the directions of odds ratios (OR) correspond to the minor allele, listed second.
  • the first replication cohort included 789 women with HG requiring IV fluid treatment and 606 controls reporting normal NVP.
  • the second replication cohort included only women at the extreme ends of the clinical spectrum, 1 10 women requiring total parenteral nutrition (TPN) and 143 women reporting no NVP.
  • Basic demographics of the replication cohorts are shown in Table 1.5. Over 90% of participants self-reported that they were of European descent.
  • Table 1.5 Demographic characteristics of the replication cohorts HG-IV consisting of HG patients treated with intravenous (IV) fluids and controls with untreated NVP, and HG-TPN consisting of HG patients treated with total parenteral nutrition (TPN) and controls with no NVP.
  • rs16982345 was successfully genotyped in 789 individuals with clinically defined HG and 581 controls reporting normal NVP using the TaqMan genotyping platform (Table 1 .6). The call rate was >95%. The replication results for rs16982345 were supportive of the GWAS result
  • the number (N) in Table 1.6 for each group is the total number of samples that were successfully genotyped.
  • the call rate was >95%.
  • OR estimates, 95% confidence intervals, and p-values were computed using 2 x 2 contingency tables in R. The effect allele is assumed to be the allele in the left-most (i.e., first) homozygous cell.
  • Conditional analysis detected secondary associations in the chr19p13.1 1 locus in SCAN1 and the chr19p13.1 1 and PGR/TRPC6 loci in SCAN2 (Supplementary Table 3 and Supplementary Figures 4 and 5).
  • LD between the primary and secondary signals is low (r 2 ⁇ 0.01), suggesting that they are independent signals corresponding to multiple risk variants within the locus.
  • the secondary signal in the chr19p13.11 locus identified from SCAN1 maps within the 3' UTR of GDF15.
  • GDF15 Supplementary Data 1 17, 18,19,20,39,40.41 .42.44 with the ful list of variants in LD with the lead SNP identified using
  • Expression levels of GDF15 were compared in an in vitro model between the ceil line DU145 transfected with wild-type GDF15 (rs1058587, C allele) and GDF15 (rs1058587, G allele) 20 .
  • the C allele of rs1058587 was associated with increased GDF15 levels in the in vitro model and was also the risk allele associated with HG, providing evidence that the direction of effect is toward higher levels of GDF15 for HG compared to controls.
  • GDF15 encodes a TGF- ⁇ superfamily member that is expressed at its highest levels in the trophoblasf cells of the placenta 23 .
  • the protein is found in maternal serum and increases significantly in the first two trimesters 23 .
  • GDF15 is believed to suppress production of proinflammatory cytokines in order to facilitate placentation and maintain pregnancy 24 .
  • GDF15 has been shown to be a regulator of physiological body weight and appetite via activation of neurons in the hypothalamus and area postrema (vomiting center) of the brainstem 25 26 . It is also notable that abnormal overproduction of GDF15 in cancer was recently found to be the key driver of cancer anorexia and cachexia which, like HG, exhibits symptoms of chronic nausea and weight loss 27,28 . Of particular clinical interest, inhibition of GDF15 restored appetite and weight gain in a mouse model of cancer cachexia 28 , suggesting a therapeutic strategy that may be applicable to patients with HG, if GDF15 proves to be the implicated gene.
  • IGFBP7 insulin-like growth factor binding protein 7
  • GDF15 insulin-like growth factor binding protein 7
  • IGFBP7 is a gene involved in implantation and decidualization of the pregnant uterus 29 .
  • IGFBP7 is significantly upregulated after implantation and highly expressed in the developing placenta 30 , inhibition of IGFBP7 caused pregnancy loss in a mouse model by shifting uterine cytokines to Th1 type dominance and repressing uterine decidualization 30 .
  • IGFBP7 may play roles in both miscarriage and in the severity of NVP, providing a genetic mechanism for the protective effect of NVP.
  • GDF15 evidence points to IGFBP7 as a promising biomarker for cachexia associated with end-stage disease 29 .
  • An interesting finding is that the Drosophila homolog of IGFBP7 has been suggested to play a role in neuronal coordination between metabolic status and feeding behavior, potentially, like with HG, causing food aversion to normally attractive food, even when starving, and vice versa 31 .
  • the strengths of this study include the well-defined phenofype and large sample sizes in two scans and the HG-IV replication sample.
  • Another caveat is that while the conditional analysis yielded additional association signals at chr19p13.1 1 that may act independently of the lead association signal, they were not replicated. Replication is necessary to determine if additional independent variants are associated at this locus.
  • the lack of eQTL data and considerable distance between the chr4q12 association signal and the closest gene IGFBP7 is a noteworthy limitation.
  • GWAS participants were customers of 23andMe, Inc. who consented to participate in research, and provided answers to the morning sickness-related questions. Two genome-wide association scans were performed, for (1) a binary HG phenotype and (2) an ordinal phenotype related to HG. Phenotype definitions are described below. Due to the nature of the phenotype definitions, some research participants were included in both association scans. All research participants included in the analysis provided informed consent and answered on-line surveys according to a human subjects protocol approved by Ethical & Independent Review Services, a private institutional review board.
  • a total of 17,062 females in SCAN1 and 53,731 females in SCAN2 were genotyped on one of four custom lllumina genotyping arrays and additional genotypes were imputed using the September 2013 release of the 1000 Genomes Project Phase 1 33 reference hapiotypes as described previously 34 35 . 16, 165 individuals in SCAN1 were also included in SCAN2.
  • SCAN2 are shown in Supplementary Table 1a. Fields that contain 5 or fewer individuals have been masked to protect the privacy of 23andMe customers. Ail females were filtered to select for European ancestry, and close relatives were removed. We performed logistic (SCAN1) and linear (SCAN2) regression assuming an additive model for allelic effects on NVP, using age and five principal components of genetic ancestry as covariates. Our previously published analysis of categorical phenotypes using ordinal and linear regression showed high concordance between resulting p-values 36 . Due to comparative ease of implementation and lower computational burden, vve used linear regression in SCAN2. The SNP-levei quality control information is shown in Supplementary Tables 1 b and 1c.
  • the index SNPs were identified by choosing the SNPs with the strongest association in each associated region. Each region contained SNPs with p ⁇ 10 -5 that were grouped into intervals separated by a gap of a minimum of 250 kbp. The SNP with the smallest p-vaiue within each interval was chosen as the lead SNP.
  • the first replication cohort included 789 HG cases treated with IVs and 606 controls with NVP that did not require treatment.
  • the second replication cohort included 110 cases requiring TPN due to HG and 143 controls reporting no NVP in any of at least two pregnancies. Ail participants gave informed consent. This study was approved by the UCLA institutional Review Board.
  • Affected individuals were included if they had an HG diagnosis and were treated with IV fluids and/or TPN. Participants affected by HG recruited acquaintances that were non-blood related and had at least two pregnancies lasting more than 27 weeks gestation. Controls were eligible to participate in the study if they reported normal or no NVP, did not lose weight due to nausea/vomiting, and did not receive medical attention due to NVP.
  • Saliva samples were collected for DNA analysis from all cases and controls.
  • DNA Genotek saliva kits (Oragene, Ottawa, Canada) were mailed to ail cases and controls.
  • the saliva collection kit is self-administered and comes with directions for submitting 2 mi of saliva into a collection vial and returning the sample to the study site via an addressed and postage- paid return envelope provided with the collection kit.
  • DNA extraction [0106] DNA was extracted from the saliva samples according to manufacturer's instructions (Oragene, Ottawa Canada). Using the kit, we have successfully isolated, on average, 197 pg of DNA of high quality (280/280 1.84) from 2 mi of saliva. The low end of expected DNA quantity reported by the manufacturer is 30 pg/ml of saliva or 60 Mg/sample. After the extraction, the DNA was stored at -20 °C. [0107] Quality control for replication data
  • the TaqlVlan genotyping platform was performed on 384-weil plates with a minimum of two blank samples per plate and a minimum of two duplicate samples per plate. Once genotypes were determined from the first 384-weil plate, a minimum of three positive controls or one positive control for each genotype was added to the remaining plates. The minimum call rate for each SNP was >95%.
  • rs16982345 the SNP with the strongest association to NVP in the chr19p13.1 1 locus was selected for genotyping in the replication sample HG-IV consisting of 789 cases and 606 controls.
  • HG-TPN extreme cohort
  • TaqMan genotyping primers for rs16982345 were available from Thermo Fisher Scientific and Applied Biosystems PRISM 7900HT Sequence Detection System (TaqMan) was used for large-scale screening. The call rate was >95%.
  • Genotypes were tested for significant association with HG using Fisher's exact test using 2 x 2 contingency tables counting the number effect vs non-effect alleles for cases and controls using R.
  • the SNP rs4885234 was genotyped as a proxy for the most significant association signal at chr4q12 because the most significant association signal, rs143409503, is an indei, which cannot be assayed using the TaqMan genotyping system.
  • the 3 additional loci that reached significance p ⁇ 10 -5 in the binary trait scan (SCAN1 ) were also genotyped in the two replication cohorts (HG-IV and HG-TPN) using the TaqMan genotyping platform.
  • the lead SNP rs56108151 was commercially available and used for genotyping the two replication cohorts.
  • Conditional analysis detects statistically independent signals in a GWAS locus. At each step, we assessed association between variants within 20 kbp around a GWAS locus and the HG or related ordinal phenotype, using top SNPs from preceding steps as additional covariates. This was repeated until no significant association was defected at p ⁇ 1 orx. Finally, we fit a joint model with all of the primary and secondary signals and evaluated its goodness of fit through the likelihood ratio test (LRT) against the model with just the primary signal. The reported effect sizes of the secondary signals are from the joint model, and the p- vaiues are from the likelihood ratio test comparing the full model with the ieave-one-out models.
  • LRT likelihood ratio test
  • HapioReg v4.1 uses LD information from the 1000 Genomes Project 33 .
  • HapioReg v4.1 includes GWAS and eQTL (including GTEx) and GRASP eQTL updates which were queried for the SNPs and included in Supplementary Data 1.
  • GTEx Genotype-Tissue Expression
  • Example 2 1GFBP7 and PGR, in addition to GDF15, associated with nausea and vomiting of pregnancy and hyperemesis gravidarum [0129]
  • This Example provides a replication study confirming the results described in Example 1 , particularly with respect to 1GFBP7 and PGR.
  • Combinations of genotypes of selected risk genes are compared for HG cases requiring feeding tube (TPN) vs Controls with no nausea/vomiting in at least 2 pregnancies.
  • Example 4 Analysis of GDF15 and IGFBP7 in Hyperemesis Gravidarum lends further support for a role in the etiology of severe nausea and vomiting of pregnancy
  • This Example shows that GDF15 and IGFBP7 can be used to identify patients at risk for HG.
  • the GWAS shows placentation, appetite, and cachexia genes GDF15 and IGFBP7 are linked to Hyperemesis Gravidarum (HG).
  • HG Hyperemesis Gravidarum
  • This Example shows that GDF15 and IGFBP7 are upreguiated in HG patients.
  • the Example also shows that alleles are associated with symptoms, medication effectiveness, and segregation with disease in families.
  • GDF15 serum levels are significantly increased in women with HG compared to controls.
  • GDF15 alleles may be linked to metoclopramide response and segregate with HG in 3 of 5 families.
  • the IGFBP7 risk allele may be associated with ondansetron and promethazine response, and prolonged symptoms.
  • This study supports GDF15 and possibly IGFBP7 in the pathobioiogy of HG and provides a tool for prediction and diagnosis.
  • the GDF15-GFRAL brainstem-activated pathway was recently identified, and therapies to treat conditions of abnormal appetite are under intense investigation. HG should be added to the list.
  • GDF15, 1GFBP7, and HCG were analyzed at 12 weeks gestation in 1 1 women hospitalized for HG, compared to 9 women with normal NVP, and 20 women with no NVP.
  • the scatterplot shown in Figure 2 displays the levels (ng/ml) of GDF15 (protein 1) and IGFBP7 (protein 2) from women with HG, NVP, or no NVP. Serum levels at 12 weeks gestation were compared for 1 1 women hospitalized for HG compared to 29 women who did not have HG (see Figures 3-5). Evaluation of odds ratios shows that patients are 36-fold more likely to be hospitalized for HG with values of GDF15 above 10 ng/ml and IGFBP7 above 80 ng/ml.
  • the fisher test tests the table for significance.
  • the p-vaiue is 0.00074, thus there is a significant difference for HG vs. no HG in the categories high and low serum values.
  • GDF15 10 and IGFBP7 > 60
  • the SNPs linked to HG in the GWAS replication study were rs16982345 (closest gene GDF15) and rs4865234 (closest gene IGFBP7). The study also explored whether the SNPs were associated with medication effectiveness, HG symptoms, and whether they segregated with disease in HG families. [0153] Table 4.2. Demographic characteristics of genofyped cohort.
  • HG patients were divided into two groups depending on whether they had a recurrence or not.
  • the SNPs tested were not predictive of recurrence.
  • the first GWAS of HG identified two candidate genes, GDF15 and IGFBP7, but causality may only be hypothesized as functional studies are prohibitory in pregnant women.
  • This Example provides further evidence of a role for these loci by showing that GDF15, and also IGFBP7, are dysregulated in HG pregnancies, while beta HCG, which was the leading hypothesis for the cause of HG, is not. Additional supportive evidence comes from previous studies showing both GDF15 and IGFBP7 are upreguiated in early pregnancy when HG occurs, play critical roles in placentation, decrease prior to miscarriage, and are drivers of stress- induced feeding behavior and cachexia, a disease characterized by symptoms similar to HG: nausea, weight loss, and muscle wasting.
  • This Example analyzes the frequencies of various genotype combinations.
  • the genes analyzed were (1) GDF15, corresponding to SNP rs18982345; (2) 1GFBP7, corresponding to SNP rs4885234; (3) PGR, corresponding to SNP rs7948518; (4) RYR2; and (5) GFRAL, corresponding to SNP rs7761177.
  • the data in the table below shows the significantly increased risk of HG for certain allele combinations.
  • Inhibitors of GDF15, GRAL, PGR can be selected as medications to block the corresponding gene product if a patient is positive for those variants.
  • Example 8 Genetic analysis of hyperemesis gravidarum reveals association with intracellular calcium release channel CRYR2)
  • This Example identifies a link between HG and RYR2.
  • Whoie-exome sequencing of 5 families was performed and followed by analysis of variants in 584 cases/431 controls.
  • Variants in RYR2 segregated with disease in 2 families.
  • the novel variant L3277R was not found in any case/control.
  • Replication of G1886S using Norwegian/Australian data was supportive.
  • Common variants rs790899 and rs1891246 were significantly associated with HG and weight loss. Copy-number analysis revealed a deletion in a patient.
  • RYR2 encodes an intracellular calcium release channel involved in vomiting, cyclic-vomiting syndrome, and is a thyroid hormone target gene. Additionally, RYR2 is a downstream drug target of Inderal, used to treat HG and CVS. These results provide genetic evidence for a pathway and therapy for HG, More details relating to this example can be found in Fejzo et al., Molecular and Cellular

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Abstract

La présente invention concerne des dosages permettant d'identifier des patientes présentant un risque de nausée et de vomissement au cours de la grossesse (NVP) et de l'hyperemesis gravidarum (HG), ainsi que des procédés de diagnostic et/ou de traitement de HG et NVP. L'invention concerne un procédé d'analyse d'expression génétique dans un échantillon obtenu à partir d'un sujet. Le procédé comprend l'obtention d'un échantillon à partir du sujet ; et la mesure du taux de produit d'expression d'au moins l'un des gènes de prédiction de HG. Les produits d'expression à détecter comprennent des protéines et des séquences d'acide nucléique. Des anticorps et/ou des sondes peuvent être utilisés pour détecter des produits d'expression génique de prédiction de HG, tels que GDF15 et IGFBP7, dans des échantillons, comprenant du sérum, de l'urine et du sang, obtenus à partir de patients. Le traitement de NVP et/ou HG peut être sélectionné sur la base du dosage.
PCT/US2018/026588 2017-04-06 2018-04-06 Prédiction, diagnostic et traitement de nausées et de vomissements liés à la grossesse WO2018187767A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197329B1 (en) * 1999-05-03 2001-03-06 Drugtech Corporation Anti-nausea compositions and methods
WO2001081928A1 (fr) * 2000-04-20 2001-11-01 St Vincent's Hospital Sydney Limited Methode diagnostique et procede de traitement impliquant la cytokine-1 inhibitrice macrophage (mic-1)
US20140065648A1 (en) * 2010-08-26 2014-03-06 Roche Diagnostics Operations, Inc. Use of biomarkers in the assessment of the early transition from arterial hypertension to heart failure
US20160048632A1 (en) * 2013-05-30 2016-02-18 Genomic Health, Inc. Gene expression profile algorithm for calculating a recurrence score for a patient with kidney cancer
US20160258018A1 (en) * 2004-06-04 2016-09-08 The Chinese University Of Hong Kong Marker for prenatal diagnosis and monitoring

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197329B1 (en) * 1999-05-03 2001-03-06 Drugtech Corporation Anti-nausea compositions and methods
WO2001081928A1 (fr) * 2000-04-20 2001-11-01 St Vincent's Hospital Sydney Limited Methode diagnostique et procede de traitement impliquant la cytokine-1 inhibitrice macrophage (mic-1)
US20160258018A1 (en) * 2004-06-04 2016-09-08 The Chinese University Of Hong Kong Marker for prenatal diagnosis and monitoring
US20140065648A1 (en) * 2010-08-26 2014-03-06 Roche Diagnostics Operations, Inc. Use of biomarkers in the assessment of the early transition from arterial hypertension to heart failure
US20160048632A1 (en) * 2013-05-30 2016-02-18 Genomic Health, Inc. Gene expression profile algorithm for calculating a recurrence score for a patient with kidney cancer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BLITZ ET AL.: "Gonadotropin-Releasing Hormone And Luteinizing Hormone Receptor Polymorphisms And Risk Of Hyperemesis Gravidarum", AMERICAN JOURNAL OF OBSTETRICS & GYNECOLOGY, vol. 201, no. 6, 2009, pages S296, XP085455094 *
FEJZO, M ET AL.: "Placenta and Appetite Genes GDF15 and IGFBP7 are Associated with Hyperemesis Gravidarum", NATURE COMMUNICATIONS, vol. 9, no. 1178, 21 March 2018 (2018-03-21), pages 1 - 9, XP055544233 *

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