WO2014181107A9 - Procédé de dosage - Google Patents

Procédé de dosage Download PDF

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Publication number
WO2014181107A9
WO2014181107A9 PCT/GB2014/051399 GB2014051399W WO2014181107A9 WO 2014181107 A9 WO2014181107 A9 WO 2014181107A9 GB 2014051399 W GB2014051399 W GB 2014051399W WO 2014181107 A9 WO2014181107 A9 WO 2014181107A9
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dna
sequencing
diagnosis
patients
mutation
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WO2014181107A1 (fr
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Timothy J. AITMANN
Jana VANDROVKOVA
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Medical Research Council
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Medical Research Council
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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 invention is in the field of hypercholesterolaemia.
  • the invention relates to the provision of a comprehensive technique for patient management in the field of familial hypercholesterolaemia.
  • Familial hypercholesterolaemia (FH, OMIM #143890) is a common Mendelian disorder that affects 1 in 500 individuals and it is estimated that 12 million of people are affected worldwide (Goldstein et al., 2001). Patients with FH have raised serum cholesterol levels from birth and increased arterial deposition of low density
  • LDL lipoprotein
  • CHD premature coronary heart disease
  • FH FH -causing mutations in LDLR are found throughout the gene and include missense, truncating and splice site mutations, small insertion/deletion mutations, and large insertions/deletions which can encompass multiple exons. Some mutations have been found in many unrelated individuals with FH, while others are found rarely. (Leigh, Foster et al. 2008) Mutations in two other genes, PCSK9 and APOB, can also cause the FH phenotype, but in less than 10% of mutation positive cases. (Taylor, Wang et al. 2010)
  • FH familial hypercholesterolaemia
  • FH diagnostic genes these genes are APOB, LDLR and PCSK9.
  • Standard molecular diagnostic techniques detect mutations in 40% of FH patients and it is believed that a proportion of remaining cases have raised cholesterol due to a combined, polygenic, effect of several LDL-C-raising common variants. This is discussed in more detail below.
  • the current standard test for FH is a genetic test. This involves the analysis of the three diagnostic genes in an individual for mutations. This is currently accomplished by conventional sequencing techniques. This is most often accomplished using capillary sequencing. This is a very laborious process. This is a very time consuming process.
  • the gold standard treatment for hypercholesterolaemia is a prescription of statins, Statins control (reduce) cholesterol levels when administered to patients.
  • Statins are a long term treatment. Many patients will be prescribed statins for the rest of their natural lives when presenting with hypercholesterolaemia.
  • statin toxicity causes a range of harrowing effects, including muscular pain, muscle weakness, death and/or erosion of muscle fibres, and in severe cases a resulting trauma to the kidneys which can result in kidney failure. It is a problem in the art that patients suffering from statin toxicity are typically suffering a long term decline. As the toxicity problem runs on a patient gets weaker and weaker.
  • statin toxicity It is a problem in the prior art that the effects of statin toxicity are prone to end up as a separate diagnostic investigation unless the physician is alert to the risk and recognises the symptoms early in the treatment.
  • hypercholesterolaemia is laborious and expensive.
  • the extra demand for these resource intensive tests is creating a further problem to medical healthcare providers.
  • the present invention seeks to overcome problems associated with the prior art.
  • FH familial hypercholesterolaemia
  • the present inventors have studied FH in detail.
  • the invention is based on a combined test which individually examines multiple factors which are involved in FH.
  • the inventors teach the parallel analysis of three patient attributes - (i) assay of the three main diagnostic genes for FH (APQB, LDLR and PCSK9); (ii) assay of polygenic FH SNPs (a collection of lower penetrance incremental risk indicators); and (iii) assay of the patient's SLCO1B1 genotype (the genetic indicator of statin toxicity).
  • the inventors teach for the first time the combination of these three elements into a single diagnostic test.
  • the advantage of this is that the physician is then appraised with a complete picture of the patient's condition, including their incremental risk indicators, and al so at the same time an indication of whether they are likely to have a negative response to the gold standard statin treatment.
  • This combination has never been attempted in the prior art.
  • This combination has advantages in targeting the treatment to the patient in the correct manner, and also avoiding the extremely negative side effects for that proportion of the population who would typically be placed on statin treatment even though they are at risk of serious side effects from statin toxicity.
  • the present invention advantageously alleviates these problems associated with the prior art.
  • the tests are conducted using a paired approach of micro-fluidics with next generation sequencing.
  • This is also a departure from the prior art techniques which have relied on conventional PCR amplification coupled to ordinary sequencing techniques such as capillary sequencing.
  • NGS next generation sequencing
  • the invention provides a method for collecting information useful in aiding the diagnosis and treatment of familial hypercholesterolaemia (FH), the method comprising providing a sample of nucleic acid such as DNA from the subject, assaying said nucleic acid such as DNA for FH.
  • FH familial hypercholesterolaemia
  • w erein assaying said nucleic acid such as DNA comprises the step of contacting said nucleic acid such as DNA sample with one or more primers for amplification and/or sequencing of the relevant segment(s) of said nucleic acid such as DNA
  • the invention relates to a method as described above wherein assaying said nucleic acid such as DNA further comprises determining the nucleotide sequence of the relevant segment(s) of said nucleic acid such as DNA, and inferring from said nucleic acid such as DNA sequence whether said mutations of steps (i) and (iii), and said one or more SNPs of step (ii), are present.
  • the invention may be applied to any nucleic acid; suitably the nucleic acid is DNA.
  • the invention is described with reference to DNA as an exemplary nucleic acid.
  • 'single assay cycle' is meant that the assay steps are carried out on the same sample. Suitably this means in a multiplexed assay.
  • the single assay cycle which may consist of multiple tubes or reactions as necessary, is carried out as a single coherent test event.
  • the invention relates to a method as described above wherein said one or more genes indicative of FH diagnosis is selected from the group consisting of APOB, LDLR and PCSK9. Each gene in this group of genes shares the common property of being indicative of monogenic FH.
  • the invention relates to a method as described above wherein said mutation in one or more genes which is indicative of FH diagnosis is selected from those listed in Table 2.
  • the invention relates to a method as described above wherein said SNP is selected from Table A or Table B.
  • SNP is selected from Table A or Table B.
  • Each SNP in this group of SNPs shares the common property of being indicative of incremental risk of high cholesterol.
  • each SNP in this group of SNPs shares the common property of being indicative of polygenic FH.
  • the invention relates to a method as described above wherein said gene indicative of risk of statin toxicity is SLCO1B1.
  • step (iii) comprises determining the presence of the rs.4149056 or 1 * 54149056 variant of SLCO1B1.
  • step (iii) comprises determining the presence of the ⁇ 4149056 variant of SLCO1B1.
  • the invention relates to a method as described above wherein said DNA sample is subjected to micro-fluidic PGR amplification of the DNA segment(s) of interest.
  • PCR is carried out using at least one primer selected from Table 3.
  • PCR is carried out using each of the primers in Table 3.
  • Enrichment is suitably accomplished by PCR.
  • the invention relates to a method as described above wherein sequence information is determined by next generation sequencing (NGS).
  • NGS next generation sequencing
  • the invention in another aspect, relates to a method of treating a subject comprising performing the method according to any preceding claim wherein if an increased likelihood of FH is identified, then cholesterol lowering medicament is administered to said subject, wherein said medicament is selected according to the subject's SLCO1B1 genotype.
  • selection of the medicament comprises selection of one or more of the upper limit of the dose of said medicament for said subject, the dose of said medicament to be administered to said subject, or the particular medicament to be administered to said subject
  • said medicament is a statin.
  • the invention relates to a primer selected from Table 3.
  • the invention relates to a set of primers comprising at least one pair of the primers in Table 3. In another aspect, the invention relates to a set of primers according to claim 17 comprising each of the primers in Table 3.
  • the invention may relate to a method for determining whether a subject has a predisposition for monogenic familial hypercholesterolemia by virtue of a mutation in one of the genes selected from LDLR, PCSK9, APOB or polygenic familial hypercholesterolaemia by virtue of combination of genotypes in (rs6s 11720) LDLR, (rs2479409) PCSK9, (1 S1367117) APOB, (JS629301) CELSR2, (rs4299376) ABCG8, (TS1564348) SLC22A1, (XS1800562) HFE, (rs3757354) MYLIP, (1-511220462) ST3GAL4, (rs8oi7377) NYNRIN, ( «429358, rs74i2) APOE and for determining individual's response to statin treatment by virtue of a variant (rs4149056) in SLCO1B1 gene comprising:
  • nucleic acid amplification step uses all primer pairs, the primer pairs comprising a forward primer with a forward primer sequence
  • the amplification product being a sequence of at least a part of one of the genes LDLR, PCSK9, APOB, CELSR2, ABCG8, SLC22A1, HFE, MYLIP, ST3GAL4, NYNRIN, APOE and SLCO1B1
  • APOE amplification products have SNPs that are indicative of a risk for a polygenic familial hypercholesterolaemia determining whether the SLCO1B1 amplification product represents a sequence that has an SNP that is indicative of a risk for adverse statin effect,
  • the exemplary PGR primers described may be used to assist in sequencing the full gene LDLR and selected genomic regions CELSR2, ABCG8, SLC22A1, HFE, MYLIP, ST3GAL4, NYNRIN, APOE and SLCOiBiof the subject.
  • the subject's gene sequence(s) is then compared with known variants of these genes associated with monogenic or polygenic FH. From the comparison, it is determined whether any particular DNA variant or variants exist in the subject. It is then possible to correlate the variants to risk of FH and statin related myopathy.
  • the comparison of the sequence information of the subject with known allele sequences can be carried out by making comparisons using established and freely available bio- info rmatic techniques (e.g. GATK or platform-specific software) with comparison to the reference human genome, 1000 genomes data and in high-quality controls, which we have established.
  • bio- info rmatic techniques e.g. GATK or platform-specific software
  • pathogenicity are well established. In short, if a mutation is known to cause the disease from previous publications that have shown segregation of the mutation in a family or enrichment of a mutation in cases versus controls then it is assigned as disease causing. New mutations, not present in the reference human genomes, that segregate in a family may also be assigned pathogenicity in combination with pathogenicity scoring tools (conservation, polyphen and sift). Variants of unknown significance are assigned as such and common benign variants are also identified.
  • Polygenic predisposition to FH is established by calculating a score, an LDL-c gene score, based on presence or absence of selected variants in LDLR, PCSK9, APOB,
  • the invention provides a method of determining whether a subject has a predisposition for familial hypercholesterolaemia and adverse reaction to statin treatment.
  • Amplification of polynucleotides may utilise methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. Also useful are strand displacement amplification (SDA), thermophilic SDA, and nucleic acid sequence based amplification (3SR or NASBA).
  • PCR polymerase chain reaction
  • LCR ligation amplification
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • thermophilic SDA thermophilic SDA
  • NASBA nucleic acid sequence based amplification
  • PCR polymerase chain reaction
  • polymerase-driven amplification assays can achieve over a million-fold increase in copy number through the use of polymerase-driven amplification cycles.
  • the resulting nucleic acid can be sequenced or used as a substrate for DNA probes.
  • These methods are well known and widely practiced in the art. See, e.g., U.S. Patents 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); Wu and Wallace, 1989 (for LCR); U.S. Patents 5,270,184 and 5,455,166 and Walker et al., 1992 (for SDA); Spargo et al, 1996 (for thermophilic SDA) and U.S. Patent 5,409,818, Fahy et al., 1991 and Compton, 1991 for 3SR and NASBA. Reagents and hardware for conducting PCR are commercially available.
  • Primers useful to amplify sequences from the LDLR, PCSK9, APOB, CELSR2, ABCG8, SLC22A1, HFE, MYLIP, ST3GAL4, NYNRIN, APOE and SLCO1B1 genes are suitably complementary to, and hybridise specifically to, sequences in the LDLR, PCSK9, APOB, CELSR2, ABCG8, SLC22A1, HFE, MYLIP, ST3GAL4, NYNRIN, APOE and SLCO1B1 genes or in regions that flank a region therein.
  • Sequences in the LDLR, PCSK9, APOB, CELSR.2, ABCG8, SLC22A1, HFE, MYLIP, ST3GAL4, NYNRXN, APOE and SLCO1B1 genes generated by amplification may be sequenced directly.
  • the amplified sequence(s) may be cloned prior to sequence analysis.
  • a method for the direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf et ah, 1986. The most popular method used today is target amplification.
  • the target nucleic acid sequence is amplified with polymerases.
  • fragment of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridisation to a target of interest, e.g., a sequence that is at least 10 nucleotides in length.
  • the fragments may comprise 10, preferably 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide.
  • a fragment of a polynucleotide sequence can be used as a primer, a probe, included in a mieroarray, or used in polynucleotide-based identification methods.
  • hybridise under stringent conditions refers to the ability of a polynucleotide molecule to hybridise to a target polynucleotide molecule (such as a target polynucleotide molecule immobilised on a DNA or RN A blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration.
  • a target polynucleotide molecule such as a target polynucleotide molecule immobilised on a DNA or RN A blot, such as a Southern blot or Northern blot
  • the ability to hybridise under stringent hybridisation conditions can be determined by initially hybridising under less stringent conditions then increasing the stringency to the desired stringency.
  • typical stringent hybridisation conditions are no more than 25 to 30°C (for example, io°C) below the melting temperature (Tm) of the native duplex (see generally,
  • Typical stringent conditions for a polynucleotide of greater than 100 bases in length would be hybridisation conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridising at 6s°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in iX SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.
  • stringent conditions use 50% formamide, 5 x SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ / ⁇ 1), o.i% SDS, and 10% dextran sulphate at 42°C, with washes at 42°C in 0.2 x SSC and 50% formamide at 55°C, followed by a wash comprising of 0.1 x SSC containing EDTA at 55°C.
  • exemplar stringent hybridisation conditions are 5 to io°C below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) °C.
  • nucleic acid as used herein, means a single or double-stranded
  • deoxyribonucleotide or ribonucleotide polymer of any length include as non- limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, and fragments thereof.
  • Reference to a polynucleotide(s) is to be similarly understood. Most suitably nucleic acid is DNA.
  • NGS next generation sequencing
  • CTR cyclic reversible termination
  • SNA single-nucleotide addition
  • real-time sequencing The imaging coupled with these techniques often range from measuring bioluminescent signals to four-colour imaging of single molecular events.
  • NGS technologies include, but are not limited to, Massively Parallel Signature Sequencing (MPSS), polony sequencing, 454 pyrosequencing, Alumina (Solexa) sequencing (sequencing based on reversible dye- terminators), Sequencing by Oligonucleotide Ligation and Detection (SOLID) sequencing, ion semiconductor sequencing, DNA nanoball sequencing, HelioscopeTM single scope sequencing, single molecule real time (SMRT) sequencing, single molecule real time (RNAP) sequencing and nanopore DNA sequencing.
  • MPSS Massively Parallel Signature Sequencing
  • polony sequencing 454 pyrosequencing
  • Alumina (Solexa) sequencing sequencing (sequencing based on reversible dye- terminators)
  • SOLID Oligonucleotide Ligation and Detection
  • ion semiconductor sequencing DNA nanoball sequencing
  • HelioscopeTM single scope sequencing single molecule real time (SMRT) sequencing
  • RNAP single molecule real time sequencing and nanopore DNA sequencing
  • oligonucleotide(s) are nucleic acids that are usually between 5 and 200 contiguous bases, and often between 5-10, 5-20, 10-20, 10-50, 5-50, 15-100, 20-50, 20-100, 20-200, 50-200, or 100-200 contiguous bases.
  • An oligonucleotide that is longer than about 20 contiguo s bases may be referred to as a polynucleotide.
  • a polymorphic site can occur at any position within an oligonucleotide.
  • a "polymorphic site” refers the position in a nucleic acid sequence at which a polymorphism occurs.
  • a polymorphic site may be as small as one base pair.
  • the term "polymorphism” refers to a genetic variation, or the occurrence of two or more genetically determined alternative sequences at a single genetic locus in a population. Each version of the sequence with respect to the polymorphic site is referred to as an "allele" of the polymorphic site.
  • Preferred polymorphisms ha ve two alleles, with the minor allele occurring at a frequency of greater than 1%, and more preferably greater than 5% or 10% of a selected population.
  • the allelic form occurring most frequently in a selected population is sometimes referenced as the "wildtype” form.
  • the allelic form occurring less frequently in a selected population is sometimes referenced as the "mutant” form. Diploid organisms may be homozygous or
  • a biallelic polymorphism has two forms.
  • a triaileiic polymorphism has three forms.
  • Examples of polymorphisms include restriction fragment length polymorphisms (RFLPs), variable number of tandem repeats (VNTRs), single nucleotide polymorphisms (SNPs), dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
  • RFLPs restriction fragment length polymorphisms
  • VNTRs variable number of tandem repeats
  • SNPs single nucleotide polymorphisms
  • dinucleotide repeats trinucleotide repeats
  • tetranucleotide repeats simple sequence repeats
  • insertion elements such as Alu.
  • an individual's susceptibility to FH refers to a statistically higher, or lower, frequency of FH in an individual carrying a particular polymorphic allele, or genotype (i.e. allelic or polymorphism pattern) in comparison to the frequency in a member of the population that does not carry the particular polymorphic allele, or genotype.
  • An individual that carries one or both high-risk alleles at a polymorphic site is said to have a heterozygous or homozygous "high-risk" genotype for that particular
  • primer refers to a polynucleotide, usually having a free 3' OH group, that may be hybridised to a template and used for priming polymerisation of a
  • Probes In general, it is expected that analysis of the amplification products will take place by sequencing. However, it may also be desirable to analyse the amplification products by "wet" binding probing to detect the presence or absence of specific sequences. Analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte. The region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of the gene of interest such as LDLR, PCSK9, APOB, CELSR2, ABCG8, SLC22A1, HFE, IVfiT.JP,
  • ST3GAL4, NYNRFN, APOE or SLCO1B1 genes Therefore, high stringency conditions are desirable in order to prevent false positives.
  • conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome.
  • the stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide. These factors are outlined in, for example, Maniatis et al., 1982 and Sambrook et al., 1989.
  • SNP single nucleotide polymorphism
  • SNPs single nucleotide polymorphism
  • SNPs are the plural of SNP. SNPs are most frequently diallelic. A most common allele of a SNP is called a "major” or “wild-type” allele and an alternative allele of said SNP is called a "minor” or “mutant” allele.
  • a SNP usually arises due to substitution of one nu cleotide for another at the polymorphic site.
  • a transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine or vice versa.
  • SNPs can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • SNPs tend to be evolutionarily stable from generation to generation and, as such, can be used to study specific genetic abnormalities throughout a population. If SNPs occur in the protein coding region it can lead to the expression of a variant, sometimes defective, form of the protein that may lead to development of a genetic disease. Such SNPs can therefore serve as effective indicators of the genetic disease. Some SNPs may occur in non-coding regions, but nevertheless, may result in differential or defective splicing, or altered protein expression levels. SNPs can therefore be used as diagnostic tools for identifying individuals with a predisposition for certain diseases, genotyping the individual suffering from the disease in terms of the genetic causes underlying the condition, and facilitating drug development based on the insight revealed regarding the role of target proteins in the pathogenesis process.
  • SNP location or "SNP locus” is a polymorphic site at which a SNP occurs.
  • biological sample means a biological sample derived from a patient to be screened.
  • the biological sample may be any suitable sample known in the art in which the expression of the selected markers can be detected. Included are individual ceils or cell populations such as obtained from bodily tissues or fluids.
  • the biological sample may comprise for example a blood, tissue, saliva or buccal smear sample,
  • the biological sample comprises blood.
  • the biological sample comprises saliva.
  • a biological sample comprising nucleic acid is obtained from a subject.
  • Nucleic acid is suitably extracted from the biological sample for assay/analysis according to any suitable method known in the art. if further guidance is required, the reader is referred to the examples section.
  • the sample for analysis comprises nucleic acid.
  • the sample for analysis consists essentially of nucleic acid.
  • the sample for analysis consists of nucleic acid.
  • the sample is an in vitro sample.
  • the sample is an extracorporeal sample.
  • the method is an in vitro method.
  • the method is an extracorporeal method.
  • the actual sampling of the subject is not part of the method of the invention.
  • the method does not involve collection of the biological sample.
  • the sample is a sample previously collected.
  • the method does not require the presence of the subject whose nucleic acid is being assayed.
  • the sample is an in vitro sample.
  • the method does not involve the actual medical decision, stricto sensu; such a decision stricto sensu would typically be taken by the physician.
  • the inventors teach the application of emerging technologies such as micro-fluidic amplification and next generation sequencing (NGS) in the field of familial
  • hypercholesterolaemia It is an advantage of these techniques that the tests can be performed on a small amount of DNA, meaning a smaller sample needing to be collected form the patient. In addition the combined method is more efficient on both time and labour.
  • the inventors teach for the first time the comprehensive joining together of mutation detection for FH with the assay for SLCO1B1 and the polygenic FH genes. This is a novel approach and has not been taught previously and it is this tripartite assay that provides numerous advantages as set out herein.
  • the combined method of the invention advantageously provides a comprehensive picture of the patient's need for treatment, coupled to their tolerance of the
  • the invention advantageously provides a comprehensive management package for patients with high cholesterol.
  • the assay of the invention is advantageously carried out as a multiplex.
  • the separate and individual test which make up the assay of the invention are advantageously carried out on a single DNA sample.
  • the assays of the invention are carried out in a single run of a sequence analyser.
  • the assays of the invention are enriched in a single PCR run, such a micro-fluidic PGR run.
  • the assays are conducted in a multiplex format. This provides the advantage of presenting a complete set of information to the physician so that they may diagnose and design a suitable treatment regime on the basis of the output of the multiplexed assay.
  • a multiplex assay has its normal meaning in the art of molecular biology.
  • the individual tests being carried out as taught by the invention are performed by a single sample.
  • the individual tests of the invention are performed simultaneously.
  • the individual tests of the invention are performed in a single PGR assay.
  • Suitable the assays of the invention are performed in a single sequencing step.
  • the combination of the various tests taught into a single simultaneous (multiplexed) analysis is a departure from the prior art because the phenomenon of familial hypercholesterolaemia (whether monogenic or polygenic) is a separate medical condition from the status of sensitivity to statin toxicity. It is an advantage of the invention that analysis of these two phenomena is brought together in a single step procedure. This is more efficient. This is cheaper. This saves labour. This provides a greater armoury of information to the physician at a single time point, bringing together diagnosis and design of an appropriate treatment regime.
  • monogenic and polygenic FH in a single assay.
  • the patient will require treatment if they have either monogenic FH or polygenic FH. It is exceptionally rare for a patient to have both monogenic FH and polygenic FH. Even if they do possess both genetic predispositions to FH, in any case the treatment would typically be the same (although clearly the investigation of related family members would be different if they were diagnosed as having monogenic as opposed to polygenic FH). It is a result of the insight of the present inventors that it is taught to carry out the tests for both monogenic FH and polygenic FH in the same multiplexed sample. Clearly, this leads to an inbuilt redundancy since individual patients will typically only have either have only monogenic FH or polygenic FH as explained above.
  • Micro-fluidics offers a method for conducting highly parallel PGR. Instead of carrying out conventional PGR which allows amplification of (for example) 48 or 96 samples per ran, parallel PGR permits up to approximately 2500 amplifications to be carried out in the same machine. For example, up to approximately 500 primer pairs can be used in a single run of the machine. This technique provides a massively parallel PGR
  • micro-fluidics/ highly parallel PGR is an especially suitable approach to the enrichment of the sample for sequencing.
  • Other techniques for enrichment may equally be used if desired.
  • hybridisation techniques which may be used to enrich the nucleic acids of interest.
  • This is a lengthy and expensive approach to enrichment. If is an advantage to use micro- fluidic PGR based enrichment as described in more detail herein.
  • mutations in the exons of genes have been found to have the greatest effects on disease, and can also act as indicators of disease risk. Mutations in the introns of a gene (the non-coding regions) can also affect disease and indicate disease risk but are less readily interpreted.
  • GS next generation sequencing
  • primers In order for the genomic DNA to be amplified, it is necessary for appropriate primers to be provided. A skilled person working the invention may design their own primers, for example using commercially available software to aid the process. The invention should not be regarded as limited solely to the particular exemplary primer sequences provided herein. However, determining the sequences to be used for such primers is not always simple: long amplicons can incorporate erroneous bases; mutations in the gene can interfere with primer annealing, and tertiary structure of the DNA can make certain sequences inaccessible. Automated platforms for primer design can have variable performance and custom design is often important. In this regard, the inventors have also provided a set of especially useful exemplary primers according to the present invention. These are presented in Table 3.
  • the present inventors have designed a new series of primers which together provides complete coverage of the LDLR gene and clinically relevant parts of PCSK9, APOB, CELSR2, ABCG8, SLC22A1, HFE, MYLIP, ST3GAL4, NYNRIN, APOE and SLCO1B1 genes.
  • This level of completeness to be achieved by a single genetic assay provided by the complete new series of primer pairs set out in Table 3 has not previously been described.
  • the assay is applicable to screening large numbers of patients for genetic causes of hypercholesterolaemia where previous assays were unsuitable.
  • the primers of SEQ ID NO i to SEQ ID NO 46 and SEQ ID NO 61 to SEQ ID N O 106 make up a set of primers for the sequencing of the coding regions of the currently screened genes LDLR, PCSK9 and APOB. That set of primers however does not cover certain genomic regions that are important in determining polygenic FH and/or in outcome of statin treatment. Primers of sequences SEQ ID NO 47 to SEQ ID NO 60 and SEQ ID NO 107 to SEQ ID NO 120 provide coverage of these genomic regions, and are specifically taught to achieve improved and/or complete coverage of the clinically important regions in FH,
  • Primers SEQ ID NO 1 to SEQ ID NO 120 have been specifically designed to allow simultaneous PGR amplification and subsequent sequencing to achieve a greater than 4-fold reduction in time required for FH genetic testing,
  • the LDLR gene encodes low density lipoprotein receptor protein, which binds low density lipoprotein (LDL) particles at the cell membrane and internalises them for hepatic processing and excretion.
  • LDL low density lipoprotein
  • LDLR spans nearly 45W3 and is made up of 18 exons, with pathogenic mutations found throughout the gene, some of which have been found in many unrelated individuals with FH, while many others are rare.
  • the intronic regions of LDLR contain multiple Alu repeat sequences, which can recombine incorrectly leading to large FH-causing indels which encompass one or many exons.
  • Patients with homozygous or compound heterozygous mutations in LDLR have a severe phenotype with onset of cardiovascular disease in childhood requiring aggressive early treatment of lipid levels.
  • the gene is located on chromosome 19 between base pairs 11,200,225 and 11,241,992 on forward strand.
  • APOB apolipoprotein B
  • FDB familial defective apolipoprotein-B1oo
  • the defective APOB ligand has reduced affinity for LDLR, reducing LDL clearance and causing hypercholesterolaemia with a similar phenotype to LDLR mutations.
  • APOB is a highly polymorphic gene, complicating identification of pathogenic variants. The gene is located on chromosome 2 between base pairs 21,224,301-21,266,945 on reverse strand.
  • the mutations discussed herein are exemplary. For example there are more than 1000 different mutations reported in LDLR. Many of them are only found in one or two families. It is very likely that more mutations may be employed. See
  • SLCOIBI substitute carrier organic anion transporter family member 1B1
  • statin-induced myopathy particularly in patients on high dose treatment (The Search Collaborative Group, 2008).
  • Statin drugs are the mainstay of treatment for FH, and muscular side-effects ranging from muscle pa through to life-threatening
  • SLC01B1 encodes the organic anion-transporting polypeptide OATPiBi, which mediates hepatic uptake of many drugs including most statins. It has been suggested that patients being treated with statins should be routinely genotyped for this variant and that an upper limit on statin doses should be applied according to genotype (NiemL 200£s).
  • the ⁇ 4149056 variant is located on chromosome 12 at position 21,368,722 on forward strand.
  • LDLR LDL receptor
  • novel variants that cause premature stop cod on, frameshift or overlap with splice donor or acceptor sites are expected to be mutations causing FH.
  • Non-synonymous substitutions, in-frame deletions or insertions and variants that overlap splice regions may or may not be causal.
  • These variants should be accessed for their pathogenicity using algorithms that predict possible impact of such variants on protein function.
  • Most commonly used pathogenicity predictive web-sites include SIFT and PoiyPhen (Kumar et aL, 2009; Adzhubei et aL, 2010).
  • variants with unclear pathogenicity should also be followed up by segregation analyses and in vitro functional studies. Functional studies include LDL binding and/or uptake assays (Soutar et. al., 1982; Goldstein et. al., 1972).
  • SNPs single nucleotide polymorphisms located in 11 genes should be genotyped in order to establish the risk of polygenic FH. Identifying such individuals will improve the efficiency of cascade testing, since this type of test will only benefit patients with monogenic FH. Much less than the expected half of children of individuals with polygenic FH are expected to inherit the disease.
  • Table A Single nucleotide polymorphisms identified as risk indicators for polygenic FH
  • Table B The SNPs used to determine predisposition to polygenic FH and their genomic locations are listed in Table B.
  • Table B The position of single nucleotide polymorphisms identified as risk indicators for polygenic FH
  • SNPs in Table A and Table B are the same 12 S Ps, the tables present different information in connection with them.
  • one or more of these SNPs are assayed; suitably two or more; suitably three or more; suitably four or more; suitably five or more; suitably six or more; suitably eight or more; suitably ten or more; suitably all twelve SNPs of Table A or Table B are assayed.
  • statins are prescribed statins. They are typically then sent away with their prescription. At most the patient may be advised to return if they experience side effects such as muscle pain etc. Patients are not automatically given a statin toxicity test.
  • statin toxicity is a serious problem, A certain proportion of the population carries a genetic predisposition to statin toxicity. Symptoms of statin toxicity include muscle weakness, pain such as muscle pain, death of muscle fibres, and kidney trauma which can lead to kidney failure from decomposing muscle fibre. Up to 5% of patients exhibit some form of statin toxicity. As many as 1 in iooo patients can experience the most severe statin toxicity including loss of muscle fibre and kidney trauma or kidney failure.
  • the in ventors teach the parallel assay of the statin toxicity predisposition locus SLCO1B1 at the same time as collecting the information useful in aiding the diagnosis of FH together with the incremental risk factors. As many as 15-20% of the population carry an SLCO1B1 allele which might predispose them to statin toxicity. A proportion of these people will actually go on to experience statin toxicity.
  • SLCOiBi is avoided. More importantly, there is additional value to the physician who is better informed when designing the treatment for the patient under examination, Patients presenting and undergoing the combined tests of the invention showing a predisposition to stain toxicity can be counselled in great detail about the side effects to be alert to.
  • the patient can be prescribed a re-examination at early time points. The patients can be placed under a more frequent surveillance regime. The patient can be prescribed smaller doses of the statin. It is even possible to design a lower dose/better tolerated statin treatment regime based on their SLCO1B1 status.
  • subjects who are heterozygous for an SLCO1B1 allele predisposing to statin toxicity show approximately a three times greater risk of stain toxicity then a patient with no risk allele.
  • a subject who is homozygous for an SLCO1B1 allele predisposing to statin toxicity has a ten times greater risk of statin toxicity.
  • statin dose or treatment regime it is possible to design a statin dose or treatment regime according to the number of copies of an SLCO1B1 allele predisposing to statin toxicity which the patient is carrying.
  • an upper limit on statin doses may be set for subjects in whom a risk of statin toxicity is detected according to the present invention.
  • a particular statin dose may be set for subjects in whom a risk of statin toxicity is detected according to the present invention.
  • a particular statin may be selected for subjects in whom a particular SLCO1B1 mutation is detected.
  • statin choice a measure of statin choice of statin choice.
  • dose choice or dose limits are found in (Niemi, 2009 - Clinical Pharmacology & Therapeutics (2010) 87 1, 130-133), which is incorporated herein by reference expressly for the teachings of alternate statin regimes according to SLCO1B1 genotype, as well as Wilke et al 2012, Clinical Pharmacology and Therapuetics 92:112-7.
  • the invention relates to a method for determining whether a subject has a predisposition for monogenic familial hypercholesterolaemia by virtue of a mutation in one of the genes selected from LDLR, PCSK9, APOB or polygenic familial hypercholesterolaemia by virtue of combination of genotypes in (rs6s 11720) LDLR, ( s2479409) PCSK9, ( ⁇ 1367117) APOB, (rs62930i) CELSR2, 084299376) ABCG8, (rsi504348) SLC22A1, (1-51800562) HFE, ( ⁇ 3757354) MYLIP, (rsii220402) ST3GAL4, (rs8oi7377) NYNRIN, ( ⁇ 429358, rs74i2) APOE and for determining individual's response to statin treatment by virtue of a variant ( ⁇ 4149056) in SLCO1B1 gene comprising:
  • nucleic acid amplification step uses all primer pairs, the primer pairs comprising a forward primer with a forward primer sequence selected from SEQ ID NO: 1 to SEQ ID NO:6o; or a reverse primer with a reverse primer sequence selected from SEQ ID NO: 61 to SEQ ID NO: 120; sequencing at least part of the amplification product, the amplification product being a sequence of at least a part of one of the genes LDLR, PCSK9, APOB,
  • the amplification step uses at least one primer pair, the pair comprising a forward primer with a forward primer sequence selected from SEQ ID NO: 1 to SEQ ID NO: 60; and its accompanying reverse primer, with appropriate sequence selected from SEQ ID NO: 61 to SEQ ID NO: 120.
  • the invention may also relate to assessing a subject for the diagnosis and/or treatment of familial hypercholesterolaemia (FH).
  • the invention may also relate to designing a treatment for familial
  • hypercholesterolaemia FH.
  • the invention may also relate to managing a subject suspected of having familial hypercholesterolaemia (FH).
  • FH familial hypercholesterolaemia
  • the invention may also relate to determining an appropriate programme for the diagnosis and/or treatment of familial hypercholesterolaemia (FH).
  • FH familial hypercholesterolaemia
  • a method for collecting information useful in aiding the diagnosis and treatment of familial hypercholesterolaemia comprising providing a sample of DNA from the subject, assaying said DNA for
  • assaying said DNA comprises the step of contacting said DNA sample with one or more primers for amplification and/ or sequencing of the relevant segment(s) of said DNA
  • FH familial hypercholesterolaemia
  • assaying said DNA comprises the step of contacting said DNA sample with one or more primers for amplification and/or sequencing of the relevant segment(s) of said DNA characterised in that each of said assays of steps (i) and (2) is carried out in a single assay cycle.
  • Figure 1 shows the depth of coverage of primer pairs (SEQ ID NO: 1 - 120), as shown in Table 3, in an in vitro experiment using a sample of human DNA.
  • Figure 3 shows the time improvements of the current test compared to standard methods.
  • Figure 4 shows Figure Si. Family pedigree showing affected individuals as filled black boxes/circles and unaffected individuals as unfilled boxes/circles. The arrow indicates the proband. Total cholesterol (TC) levels (mmol/1) are shown for each individual as well as mutation status. WT, wild type; NA, not available;
  • Micropipettes capable of dispensing volumes in the range of 1-1000 ⁇ 1, with compatible sterile filtered tips.
  • Primer solutions are valid for use for one year after resuspension, stored at -20°C
  • Genomic DNA templates were amplified using the Access Array IFC, according to the manufacturers instructions (http://www.fluidigram.com).
  • the technique employed a microfluidic chip that systematically combined 48 sample DNAs, along with 120 primer pairs (SEQ ID NOs 1 - 120). PGR reagents were then drawn into the chip reaction chambers prior to PCR cycling. Common flanking sequences on each primer pair permit attachment of barcode indexes and sequencing adaptors. This allows for the attachment of platform-specific adaptors and barcodes pre target enrichment.
  • the multiplex amplicon harvests were recovered for each DNA template, approximately 150! was used to generate barcode and adapter products specific to the MiSeq platform (Alumina, San Diego, CA). Amplicon harvest volume was adjusted to 20 ⁇ using PGR certified water.
  • barcode-fusion PG reactions were prepared using CS tagged primer pairs, for instance, a reaction with A_BC6_CSi and CS2__ . P1. This strategy permitted amplicon sequencing in both orientations.
  • 10 ⁇ of the Fluidigm IFC harvest was added to 86 ⁇ of a Herculase II Fusion PGR mix, as per manufacturer's instructions (Agilent
  • Cycling conditions were as follows: an initial incubation of 98°C for 30 sec, followed by two cycles of 98°C for 30 sec, 54°C for 30 sec and 72°C for 30 sec; after the last cycle reactions were held at 72°C for 2 min, and then at 4°C.
  • Fluidigm sequencing primers FLi and FL2 were diluted to 0.5UM final in a 20ul volume and spiked into the appropriate Miseq cartridge positions ie FLi in positions #12 for Readi and #14 for Read2, FL2 in position #13 for Index Read.
  • the MiSeq cartridge was loaded into Miseq and the run was set up following Illumina MiSeq sequencing standard procedures.
  • Familial hypercholesterolaemia is a common Mendelian disorder associated with early onset coronary heart disease that can be treated by cholesterol lowering drugs. The majority of cases in the UK are currently without a molecular diagnosis partly due to the cost and time associated with standard screening techniques. This study tested the sensitivity and specificity of two next-generation sequencing (NGS) protocols for genetic diagnosis of FH. Methods :
  • LDLR LDLR insertions/deletions in LDLR.
  • One sample was homozygous for LDLR .Gln384Pro and one sample was a compound heterozygote with one missense mutation and one large deletion in LDLR.
  • the remaining 5isamples in the validation cohort had negative molecular diagnoses.
  • 29 were processed using the SureSelect Target Enrichment System alone, 42 using the Access Array System micro fluidic platform alone and the remaining 33 using both platforms.
  • 84 consecutive patients referred for molecular testing by the Hammersmith Hospital lipid clinic over a period of one year were studied by the PCR-based Access Array System microfluidic platform.
  • GTTGTTGTCCAAGCATTCGTT were designed to amplify exons 4 to 7 of LDLR.
  • the genomic co-ordinates of the 18 targeted genes were d etermined using the March 2006 build (NCBl36/hgi8) of the human genome in the Ensembl genome browser 12 .
  • the density of bait tiling was 5-fold and the baits were allowed to overlap into repeat regions by sobp.
  • the total targeted DNA length was 399kb.
  • All libraries were generated from sheared DNA (Covaris, Woburn, MA, USA) with an average insert size of 200bp following the SureSelect Target Enrichment System XT (Agilent, Santa Clara, CA, USA) protocol for Alumina multiplexed sequencing version 1.2. After dilution to 2nM, up to 30 libraries were pooled and sequenced on one lane on the HiSeq2000 platform (Alumina, San Diego, CA, USA) to generate 2xioobp paired-end reads.
  • the design included 43 amplicons covering all exons of LDLR, with the majority of the coding sequence covered by more than one overlapping fragment. Amplicons were also designed to cover exons 2, 4, 7, and 9 of PCSK9 containing the most common gain-of- function pathogenic mutations; APOB (one amplicon covering the most common familial defective apolipoprotein B-100 mutation p.Arg3527Gln); APOE (one amplicon covering the APOE E2 variant site, rs74i2); and SLC01B1 (one amplicon covering 1-84149056, the myopathy-associated variant). The average amplicon length was i84b with 57% GC content. Primer sequences are shown in Table Si.
  • Copy number variant analysis from NGS data was performed for the samples sequenced using targeted capture.
  • a read-depth based method 17 as implemented in R package ExomeDepth, was used to identify deletions and duplications spanning at least one exon.
  • Each sequencing batch of samples was processed separately in order to increase the quality of a reference set for each sample and therefore to maximise the power to detect copy number variants.
  • Read depth was assessed for each exon in the target region and the ratio of expected and observed read count was obtained, as well as a Bayes factor for the copy number variant calls, as implemented in the method.
  • MLPA was performed using the kit LDLR- P062 (MRC-Holland) following the manufacturer's protocol.
  • the novel exon 16 deletion was confirmed by PCR.
  • the previously-described large deletions and duplications are detailed in Tosi et alfi .
  • the sensitivity of an assay was defined as the percentage of pathogenic mutations correctly identified with respect to previous or new Sanger sequencing and MLPA.
  • the specificity is defined as the percentage of mutation negative samples correctly identified as negative with respect to previous or new Sanger sequencing and MLPA.
  • Genotypes of the SLC01B1 myopathy-associated variant 1-84149056 were scored from the Access Array System microf!uidic platform and SureSelect Targeted Enrichment System data in all patients, and any history of adverse effects was obtained by review of medical records. Side effects were defined biochemically (transaminase or creatine kinase levels more than three times the upper limit of a normal range) or
  • Hybridisation-based capture is known to target GC-rich regions poorly; however, all regions containing known FH-causing mutations were covered sufficiently (>25x) for confident variant calling,
  • the initial analysis of the sequencing results was carried out blinded to the gene and mutation details for each sample. All twenty heterozygous and one homozygous short pathogenic mutations, including point mutations and insertions/deletions of less than 15 base pairs, were detected (Table 1). In addition one compound heterozygote and six large
  • LDLR low-density lipoprotein
  • p.Asp227Glu two were large deletions (deletion of exon 16 and deletion of promoter/ exon 1).
  • a novel variant was identified in the LDLR promoter (c. -227G>T, GERP score 3.29, located in the highly-conserved footprint 1 (FPi) site) and two rare variants were identified in APOB in patients without a previous molecular diagnosis.
  • DNA samples from 53 previously characterized patients including 40 with point mutations (39 heterozygotes and one homozygote), 6 with insertions/deletions (all heterozygotes), 6 with large deletions or duplications (all heterozygotes), and one compound heterozygote with one missense mutation and one large deletion in LDLR (Table 1) were amplified together with 22 patients that had previously screened negative.
  • the median coverage per sample was 572X (min 461, max 625). All amplicons except A POE (see Methods) amplified with a mean coverage of 506X.
  • the coverage for individual amplicons is listed in Table S2. Overall, 90% of bases were covered more than 25-fold.
  • Asp227Glu was identified in a sample that previously had no molecular diagnosis. No pathogenic mutations were found in remaining samples that were negative on previous screening. Large deletions could not be detected with the PCR-based Access Array System because no reduction in coverage was observed within deleted regions. The overall sensitivity of this assay compared to SureSelect Target Enrichment System was therefore 82% (47/57).
  • the prospective cohort included six patients with definite FH as defined by the Simon Broome criteria, 65 with possible FH and 13 hypercholesterolaemic patients not fulfilling Simon Broome criteria for FH.
  • the highest defection rate of clearly pathogenic mutations was in the group of patients with a definite FH diagnosis (4/6, 67%) followed by the group with a diagnosis of possible FH (17/65, 26%),
  • One mutation (1/13, 8%) was identified amongst the 13 hypercholesterolaemic patients that did not fulfill FH diagnostic criteria.
  • the SureSelect Target Enrichment System assay included 13 genes that reside on cholesterol metabolism pathways. Rare variants identified in these genes, particularly in hypercholesterolaemic individuals who screened negative for mutations in LDLR, PCSK9 and APOB, are potentially responsible for patients' raised cholesterol. Rare variants (MAF ⁇ o.oi) identified in such individuals are listed in Table S3. In patients with no previously known molecular diagnosis, seven rare non-synonymous variants were found, of which six were not predicted to be functionally significant by SIFT and PolyPhen. The single variant, that was most likely to be of functional significance and therefore potentially pathogenic, was p.ValSogMet in SR EBFL However, this was excluded from further analysis as it did not segregate with the phenotype in the family (data not shown).
  • the single variant that was not detected by the PCR-based Access Array System protocol was an nbp deletion
  • LDLR coding sequence three mutations in the LDLR coding sequence (one single nucleotide variant, p.Asp227Giu, and two large deletions) and one LDLR promoter variant (e.-227G>T) were identified in patients who were previously classified as mutation negative.
  • the SureSelect Target Enrichment System protocol allows for comprehensive co verage of targeted regions (current custom design up to 24Mb) while the Access Array System protocol is limited to 480 amplicons with a maximum length of 40obp when sequenced using the latest MlSeq system. Sequence capture also allows the detection of all types of variants including large deletions and duplications, which was not possible in this study using the PCR-based Access Array System.
  • the Access Array System is considerably cheaper to run 19 > 22 with reagent costs approximately 10-fold lower than for the SureSelect Target Enrichment System protocol 22 and, in addition, the library preparation turnaround time is shorter. In our hands 96 samples can be processed within a day using the Access Array System protocol compared to at least three days needed for the SureSelect Target Enrichment System in-solution capture.
  • the LDLR promoter variant c.-227G>T has not previously been reported in FH patients, or in other populations, but was investigated previously as part of a study delineating the conserved FPi site.
  • a luciferase assay showed that the c.-227G>T variant had around 75% transcription levels compared with the wild-type site 26 . Whilst a 25% reduction is not a definitive decrease in promoter activity, it cannot be excluded that this change is sufficient to cause raised cholesterol levels in this patient.
  • Family members were unavailable for segregation analysis and we therefore classify this variant as being of uncertain significance pending further functional and segregation data.
  • the remaining two novel variants in A FOB, rs.1.2714097 and ⁇ 72653087 were located outside LDLR binding sites, regions currently not associated with
  • hypercholesterolaeniia and their pathogenicity therefore also remains to be elucidated.
  • the types of mutation identified here reflect the distribution of variants published in the LDLR locus specific database ⁇ with exonic substitutions (56%) being the most common followed by short insertions deletions (28%), Most of the mutations identified here were unique with only 20% common among UK FH patients which would be identified using the Elucigene FH20 commercial ARMS kit. In our prospective study, we did not identify any mutations in PCSK9 or large rearrangements of LDLR. These variants are generally rare among patients with FH " and we therefore consider it unlikely that our results are biased in anyway.
  • next-generation assays is flexible and can be readily extended to include full coding regions of APOB and PCSK9 as well as coding regions of other medically relevant genes, such as APOE and SLC01B1 that currently need to be genotyped separately.
  • Wierzbicki AS Humphries SE, Minhas R. Familial hypercholesterolaemia: summary of NICE guidance. BMJ. 2008:337:31095.
  • Soutar AK Naoumova RP. Mechanisms of Disease: genetic causes of familial hypercholesterolemia. Nat Clin Pract Cardiovasc Med. 2007;4(4):214-225. . Tosi I, Toledo- Leiva P, Neuwirth C, Naoumova RP, Soutar AK. Genetic defects causing familial hypercholesterolaemia: Identification of deletions and duplications in the LDL-receptor gene and summary of all mutations found in patients attending the Hammersmith Hospital Lipid Clinic. A therosclerosis. 2007;l94(l): 102-111.
  • cytoplasmic tails is required for coated pit-mediated internalization of the low density lipoprotein receptor. Journal of Biological Chem istry . February 25, 1990 I990;205(6):3116-3123.

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Abstract

La présente invention concerne un procédé de collecte d'informations utiles pour aider au diagnostic et au traitement de l'hypercholestérolémie familiale (HF), le procédé comprenant la fourniture d'un échantillon d'ADN du sujet, le dosage dans ledit ADN (i) de la mutation dans un ou plusieurs gènes indiquant un diagnostic de HF ; (ii) de la présence d'un ou plusieurs SNP indiquant un risque accru de cholestérol élevé ; et (iii) de la mutation dans un gène indiquant une probabilité de toxicité des statines ; le dosage dudit ADN comprenant l'étape de mise en contact dudit échantillon d'ADN avec une ou plusieurs amorces d'amplification et/ou de séquençage du (des) segment(s) pertinent(s) dudit ADN caractérisé en ce que chacune desdites étapes de dosage (i), (ii) et (iii) sont réalisées en un seul cycle de dosage. L'invention concerne en outre un procédé de traitement, et des amorces.
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