WO2022082199A1 - Procédé de détection de la sclérose latérale amyotrophique - Google Patents

Procédé de détection de la sclérose latérale amyotrophique Download PDF

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WO2022082199A1
WO2022082199A1 PCT/US2021/071865 US2021071865W WO2022082199A1 WO 2022082199 A1 WO2022082199 A1 WO 2022082199A1 US 2021071865 W US2021071865 W US 2021071865W WO 2022082199 A1 WO2022082199 A1 WO 2022082199A1
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als
mutations
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genes
lateral sclerosis
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Sean James MILLER
Brian G. Williams
Robert Logan
Nicolas A. SCHCOLNICOV
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Pluripotent Diagnostics Corp.
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to methods for detecting amyotrophic lateral sclerosis.
  • Each method comprises sequencing 16 target genes or 23 target genomic loci from a biological sample of a subject, and identifying one or more mutations such as single nucleotide polymorphisms or insertions/deletions , if present, in the 16 target genes or 23 target genomic loci.
  • ALS Amyotrophic lateral sclerosis
  • ALS cases can be grouped by two categories: familial ALS (fALS), where the patient has a genetically related family member also affected, and sporadic ALS (sALS), where the patient has no family history of ALS 9 . Historically, 5-10% of cases are fALS, and the other 90-95% cases are sALS. In the past ten years, the C9ORF72 hexanucleotide repeat expansion, has been identified as the most prevalent genomic mutation found in the ALS disease population 13 . C9ORF72 repeat expansions can be found in up to 34% of fALS and 5% of sALS cases.
  • FIG. 1 Distribution of SNPs present only in the 338 ALS sample. rs767982303 and rs760890146 (SNIP IDs) are each in greater than 25% of the ALS population. The dots represent percentage of the ALS sample for each selected SNP with the 68.5% Confidence Level (CL) Clopper-Pearson interval on the true binomial proportion. The grey area represents the range of the possible percentage in the healthy population, with a 95% CL Clopper-Pearson interval.
  • CL Confidence Level
  • FIG. 2 SNPs that are not mutated in the control sample. The number of ALS cases out of the overall 338 patient cohort, percent of total ALS cases with the 99% CL Clopper- Pearson interval, and p-value.
  • FIG. 3 Distribution of mutated genes found only in the 338 ALS sample. Dots represent percentage of the ALS group for each selected gene. The grey area represents an upper-bound on the potential false-positive percentage in the healthy population. This upper bound is set via the 99% CL Clopper-Pearson interval on the binomial proportion. MIR7155 mutations are detected in 51% of the ALS cohort.
  • FIG. 4 16 genes that are not mutated in the control sample. The number of ALS cases out of the 338-patient cohort, number of unique SNPs, percent of total ALS cases, and p-value with the 99% CL Clopper-Pearson interval are shown, respectively.
  • FIGs. 5A-5B Classifier Analysis using candidate ALS-only mutated genes. Selecting patients with three or more genes mutated of the 16 candidate genes yields a falsepositive rate less than 0.1% and false-negative rate less than 59% at 99% CL. 52% of the ALS cases have at least three of the 16 candidate genes mutated.
  • (5B) The percentage of ALS cases with at least the given number of genes mutated from the candidate list (light). The maximum false positive rate at 99% CL (dark).
  • FIG. 6 Distribution of candidate ALS-only mutated genes and probability of having ALS based on number of mutations. The distribution of the number of genes out of the top 22 candidates found in each of the 713 ALS cases is shown in grey. The probability of having ALS and the probability of not having ALS is represented is shown. DETAILED DESCRIPTION OF THE INVENTION
  • locus is a specific, fixed position on a chromosome where a particular gene or genetic marker is located.
  • a “single nucleotide polymorphism” is a germline substitution of a single nucleotide at a specific position in the genome. For example, at a specific base position in the human genome, the G nucleotide may appear in most individuals, but in a minority of individuals, the position is occupied by an A. This means that there is a SNP at this specific position, and the two possible nucleotide variations - G or A - are the alleles for this specific position.
  • ALS Amyotrophic Lateral Sclerosis
  • the present invention identifies a set of mutations in genomic-coding regions that are present in ALS patients but not in healthy control samples.
  • the present invention provides methods to detect and diagnose ALS before clinical and pathological onset, which is imperative to prolonging patient lifespan, understanding the pathobiology, and designing therapies for early intervention.
  • the inventors compute and analyze large datasets of genomes of over 1,500 ALS disease patients and healthy controls.
  • the inventors unravel mutations such as single nucleotide polymorphisms (SNPs) and Indels (insertions and deletions) in gene-coding and inter-genic regions that are associated with ALS disease diagnosis and always absent in healthy control patients.
  • SNPs single nucleotide polymorphisms
  • Indels insertions and deletions
  • the inventors have analyzed nextgeneration genomic sequencing data from two cohorts of ALS and healthy controls from the Answer ALS Consortium. In doing so, the inventors discover mutations in protein-coding genes that have not been associated with ALS previously.
  • the present invention is directed to methods for detecting amyotrophic lateral sclerosis in a subject by detecting one or more mutations in specific genes or gene loci.
  • the inventors have discovered that 16 target genes of the human genome, MIR7155, NPM1P49, RP11-20B24.3, HNRNPA1P44, OXR1, H2AFZP1, TAB3P1, RPL5P35, ZNF92P2, CIR1P3, GNAI2, CCDC42, RP11-370110.6, ADIPOR1P1, KIAA1841, and AC008074.4, are important for detecting ALS.
  • the invention provides a method for detecting ALS in a subject, comprising obtaining a biological sample from a subject, and from the sample, detecting one or more mutations in 16 target genes selected from the groups consisting of: MIR7155, NPM1P49, RP11-20B24.3, HNRNPA1P44, OXR1, H2AFZP1, TAB3P1, RPL5P35, ZNF92P2, CIR1P3, GNAI2, CCDC42, RP11-370110.6, ADIPOR1P1, KIAA1841, and AC008074.4.
  • the method comprising the steps of: (a) sequencing 16 target genes from a biological sample of a human subject, wherein the target genes are MIR7155, NPM1P49, RP11-20B24.3, HNRNPA1P44, OXR1, H2AFZP1, TAB3P1, RPL5P35, ZNF92P2, CIR1P3, GNAI2, CCDC42, RP11-370110.6, ADIPOR1P1, KIAA1841, and AC008074.4, (b) comparing each of the DNA sequences of the 16 target genes with its corresponding normal genes, (c) identifying one or more mutations such as SNPs, if present, in each of the DNA sequences of the 16 target genes, and (d) detecting amyotrophic lateral sclerosis in the subject if at least one of the 16 target genes has one or more mutations. With at least one target gene found mutated, 67% to 80%, at 99% CL (C-P), of ALS can be detected, with at least one target
  • ALS is detected in the subject if at least two of the 16 target genes have one or more mutations. With at least two target genes mutated, 50% to 64%, at 99% CL, of ALS can be detected, with a false positive rate less than 0.9% at 99% CL.
  • ALS is detected in the subject if at least three of the 16 target genes have one or more mutations. With at least three target genes mutated, 45% to 59%, at 99% CL, of ALS can be detected, with a false positive rate less than 0.09% at 99% CL.
  • the DNA is first extracted from a biological sample of a human subject.
  • the biological sample is blood (such as peripheral whole blood), a tissue sample (such as fibroblast (skin) biopsy, or a mucosal sample), or any cell derived from the patient of a human subject.
  • Method for extracting DNA from a biological sample is well-known to a person skilled in the art. For Example, see protocols for extracting DNAs from blood from Thermo Fisher product sheet catalog CS11040.
  • the DNA extracted from the biological sample of the human subject is then performed target-specific amplification and target-specific sequencing to sequence each of the 16 target genes: MIR7155, NPM1P49, RP11-20B24.3, HNRNPA1P44, OXR1, H2AFZP1, TAB3P1, RPL5P35, ZNF92P2, CIR1P3, GNAI2, CCDC42, RP11-370110.6, ADIPORIP 1, KIAA1841, and AC008074.4.
  • Whole genome sequencing which is a genomic technique for sequencing all the protein-coding regions of genes in a genome, is not performed in this method.
  • each of the DNA sequences of the 16 specific target genes is compared with its corresponding reference gene sequence.
  • Targeted gene data are processed through an automated pipeline to perform read alignment and mutation analysis including variants such as SNPs, indels and substitutions in either introns, exons or both.
  • paired- end 150bp reads are aligned to the GRCh38 human reference using the Burrows-Wheeler Aligner (BWA-MEM) and processed using the GATK best-practices workflow that includes marking of duplicate reads by the use of Picard tools, local realignment around indels, and base quality score recalibration (BQSR) via Genome Analysis Toolkit (GATK).
  • BWA-MEM Burrows-Wheeler Aligner
  • GATK Genome Analysis Toolkit
  • step (c) single nucleotide variant analysis is performed to identify one or more mutations, if present, in each of the DNA sequences of the 16 target genes.
  • Variant discovery is a two-step process. HaplotypeCaller is run on each sample separately in gVCF mode (GATK v3.5). This produces an intermediate file format called gVCF (genomic VCF). For projects with large number of samples, gVCFs are combined by batches into merged gVCFs. gVCFs are then run through a joint genotyping step (GATK v3.5) to produce a multi-sample VCF. Variant filtration is performed using Variant Quality Score Recalibration (VQSR) which identifies annotation profiles of variants that are likely to be real, and assigns a score (VQSLOD) to each variant.
  • VQSR Variant Quality Score Recalibration
  • Variant effects annotation is performed using SnpEff (PMID: 22728672), bcftools (http://github.com/samtools/bcftools) and in-house software.
  • Other functional annotations include variant frequencies in different populations from 1000 Genomes project (PMID:20981092), Exome Aggregation Consortium - ExAC(http://biorxiv.org/content/early/2015/10/30/030338), dbSNP147 (PMID: 11125122); cross-species conservation scores from PhyloP (PMID: 15965027), Genomic Evolutionary Rate Profiling (GERP; PMID: 21152010), PhastCons (PMID: 21278375); functional prediction scores from Polyphen2 (PMID: 20354512) and SIFT (PMID: 19561590); Clinvar(http://www.ncbi.
  • Variant discovery for example, is described in the following references: “A framework for variation discovery and genotyping using next-generation DNA sequencing data” DePristo M, et al, 2011 NATURE GENETICS 43:491-498; and “From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline.” Van der Auwera G, et al., Curr Protoc Bioinformatics. 2013; 43: 1-33.
  • step (d) ALS is detected in the subject, if at least one of the 16 target genes has one or more mutations, preferably at least two of the 16 target genes have one or more mutations, and more preferably at least three of the 16 target genes have one or more mutations.
  • the present invention provides a method to detect and diagnose ALS before clinical- and pathological-onset, which is imperative to prolonging patient lifespan, understanding the pathobiology, and designing therapies for early intervention.
  • ALS is a devastating neurodegenerative disorder, with no cures or genetic diagnostics.
  • the present method detects 45%-59% of the ALS-only population, at 99% CL, with the 16 target genomic signatures, when at least 3 of the 16 target genes contain a mutation.
  • the present method provides use of genetic screening in early ALS diagnosis and therapeutic intervention.
  • this applications show that the detection of single mutations can identify up to 59% of the ALS population with genes that are never found mutated in the healthy control sample.
  • This application illustrates two novel mutations in gene-coding regions of the genome that are never present in the healthy group yet are found in over 25% of the ALS cohort.
  • the inventors have discovered that 22 target genes of the human genome, AL033528.3, THRAP3, AC106707.1, LIPH, AC007690.1, FAM184B, AC096747.1-NDUFB5P1, NDUFS4, RPL5P16-AC008885.1, SLF1, TNRC18, AC023095.1, TRPM3, AL161629.1, NCS1, TXNP1-INPP5F, CCDC59, ATP10A, COX5A, RN7SL33P, TOP2A, and ZC3H7B, which do not mutate in a normal subject, are important for detecting ALS.
  • the invention provides a method for detecting ALS in a subject, comprising obtaining a biological sample from a subject, and from the sample, detecting one or more mutations in 22 target genes selected from the groups consisting of: AL033528.3, THRAP3, AC106707.1, LIPH, AC007690.1, FAM184B, AC096747.1-NDUFB5P1, NDUFS4, RPL5P16- AC008885.1, SLF1, TNRC18, AC023095.1, TRPM3, AL161629.1, NCS1, TXNP1-INPP5F, CCDC59, ATP10A, COX5A, RN7SL33P, TOP2A, and ZC3H7B, and detecting amyotrophic lateral sclerosis in the subject if the 22 genes has one or more mutations.
  • 22 target genes selected from the groups consisting of: AL033528.3, THRAP3, AC106707.1, LIPH, AC007690.1, FAM184B, AC096747.1-
  • the invention also provides a method for detecting ALS in a subject, comprising obtaining a biological sample from a subject, and from the sample, detecting one or more mutations in 23 genomic loci selected from the groups consisting of: chrl :25854953 (chromosome 1 at nucleotide position 25854953), chrl :3624870, chr3: 158557839, chr3: 185543848, chr3: 186923875, chr4: 17685198, chr4: 180358067, chr5:53655366, chr5: 82813472, chr5:94666955, chr7:5338617, chr8: 62196626, chr9:71428255, chr9: 89866631, chr9: 130224292, chrlO: 119712877, chrlO: 119712899, chrl2
  • the method comprises the step of: (a) amplifying DNA extracted from a biological sample of a subject by target-specific polymerase chain reaction to amplify specific genomic loci comprising 23 specific chromosome positions of chrl :25854953, chrl :3624870, chr3: 158557839, chr3:185543848, chr3: 186923875, chr4: 17685198, chr4: 180358067, chr5:53655366, chr5: 82813472, chr5:94666955, chr7:5338617, chr8: 62196626, chr9:71428255, chr9: 89866631, chr9: 130224292, chrlO: 119712877, chrlO: 119712899, chrl2:82295320, chrl5:25687571,
  • Whole genome sequencing which is a genomic technique for sequencing all the protein-coding regions of genes in a genome, is not performed in this method.
  • step (a) of the method the DNA is first extracted from a biological sample of a human subject, as described in the first method.
  • the DNA extracted from the biological sample of the human subject is then performed target-specific amplification to amplify the 23 loci of the 22 genes.
  • Table 1 shows the 22 genes that frequently has at least one mutation in ALS patients and the position of the mutation in terms of nucleotide position on a chromosome.
  • Gene TXNP1-INPP5F has two mutated loci chrlO: 119712877 and chrlO: 119712899 in ALS patients.
  • PCR polymerase chain reaction
  • the forward and reverse primer are designed to be 30-400 bases away from the target site, e,g, 40- 250 bases, 40-200 bases, 40-150 bases, or 40-100 bases.
  • Table 1 illustrates one design of the forward primer and reverse primer for each of the 23 target loci.
  • the primer design shown in Table 1 is an example, and the present invention is not limited to such specific primer sequences.
  • the two loci of chrlO: 119712877 and chrlO: 119712899 of Gene TXNP1-INPP5F are only 22 bases apart from each other and therefore one set of forward and reverse primers can conveniently amplify both loci.
  • step (b) the amplified DNA is purified, and sequenced according to methods known to a person skilled in the art.
  • DNA purification is a step that removes everything that is not the amplicon from the PCR product, this includes unused primers, nucleotides, enzymes, and other impurities.
  • Sequencing includes library preparation and the act of DNA sequencing itself, done by a sequencing system. Library preparation typically consists of fragmenting the DNA sample and adding sequencing adapters to the fragments that are needed for the sequencing step (next generation sequencing). The act of sequencing itself includes reading the nucleotides in the DNA sample and saving them sequentially into a digital file.
  • the specific protocol for DNA purification and DNA sequencing may differ depending on a number of factors, including the method used for DNA amplification and the sequencing system used.
  • the amplified DNA can be purified and sequenced by using QIAquickPCR Purification Kit for DNA purification, following QIAquick® Spin Handbook protocol; TruSeq DNA LT kit (see product sheet of TruSeq DNA Library Prep Kits®, Illumina) for library preparation, following the protocol available at Ilumina's website TruSeq® DNA Sample Preparation Guide; and sequencing done by Illumina MiSeq system (see MiSeqTM System specification sheet, Illumina).
  • step (c) the amplified DNA sequences of (b) is analyzed and compared with its corresponding DNA sequence of the normal genomic loci. See description in the first method.
  • step (d) single nucleotide variant analysis is performed to identify one or more mutations, if present, in each of the DNA sequences of the 23 target loci. See description in the first method.
  • step (e) ALS is detected in the subject, if at least one of the 23 target loci has single mutation, preferably at least two of the 23 target loci have mutations, and more preferably at least three of the 23 target loci have mutations.
  • the present method detects over 30% of the ALS-only population, at 99% CL, with the 23 target genomic signatures, when at least 1 of the 23 target genes contain a mutation.
  • the present method provides use of genetic screening in early ALS diagnosis and therapeutic intervention.
  • the inventors show that at least two genes must be mutated in the list of 23 top candidates to achieve 35.7-44.9% accuracy at detection of ALS.
  • the following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.
  • Clopper-Pearson Interval Bounds are set on the true fractions of either population with a given feature(s). The number of people within a sample that are positive for the feature-of- interest will have a binomial distribution. Clopper-Pearson intervals on the binomial proportion are calculated for true population proportions 11 .
  • Fisher's Exact Test The probability (p-value) of the null hypothesis, that a mutation is present in the ALS population in the same proportion as in the control population 12 . This test statistic is ideal for this study because it is the exact probability that the two proportions are equal and can still be calculated in a reasonable amount of time due to the sample size limits. T -tests are approximations of this probability which converge to the exact value in the limit of large sample size.
  • Row- wise Conditional Percentage To quantify how often a pair of our top 16 genes is mutated in the same patient, we calculated a conditional probability considering the independent probabilities of each mutation occurring on its own. For every possible ordered pairing of two genes (240 combinations), we counted the number of cases which have both gene mutations and divided by the total number of cases where the first gene was present. This metric is visually represented as a matrix, with each row and column representing a particular mutated gene from the set of 16, and each element representing the conditional probability of the column and row mutation happening in the same patient, adjusted for the baseline prevalence of the row mutation. The probability is converted into a percentage and can provide insights into how often two gene mutations co-occur in each patient.
  • Answer ALS Data were provided by the Answer ALS consortium.
  • C9ORF72 hexanucleotide-repeats are the most prevalent ALS mutation known to date, effecting 5-10% of all cases, and up to 34% of familial (fALS) 13 .
  • fALS familial
  • rs767982303 and rs760890146 were each found in 25% of the total ALS population yet are absent in controls (FIG2. 1 and 2). rs767982303 (located on the 0XR1 gene) and rs760890146 (located on the NPM1P49 gene) both lead to an acceptor variant mutation. Other top SNPs-of-interest and their significance are illustrated.
  • FIGs 5A and 5B We propose a simple classifier that requires at least three of the 16 genes to be mutated. A conservative upper limit on the rate in the healthy population of having a gene mutation for each of these top 16 genes is estimated to be less than 10% (at 99% CL) using the Clopper-Pearson interval since each gene was not found in 53 control patients 11 .
  • 16 mutations has a false-positive rate less than 0.1% (1/1000), meaning the specificity is greater than 99.9% at 99% CL.
  • the sensitivity of this classifier is 52% ⁇ 7% at 99% CL, identifying just over half of the ALS sample.
  • Example 1 demonstrates SNPs in coding-regions or entire genes that are associated in a majority of the ALS population.
  • the Answer ALS consortium utilized the latest next-generation sequencing technology and annotation with the highest quality control and protocols to allow us to perform unbiased genetic analyses on protein-coding genes and other genomic areas of interest. We are the first to report on this novel genomic database using these statistical and computational methods.
  • OXR1 is an essential member of the antioxidant defense mechanisms in the cell.
  • microRNA MIR7155
  • Answer ALS Data were provided by the Answer ALS consortium and Alzheimer’s Disease Neuroimaging Initiative.
  • C9ORF72 hexanucleotide-repeats are the most prevalent ALS mutation know to data, affecting 5-10% of all cases and up to 34% of familial (fALS).
  • fALS familial
  • Table 2 shows the 22 genes that are not mutated in the control sample. The gene names, the number of ALS cases out of the 713-patient cohort, percent of total ALS cases with the 99% CL Clopper-Pearson interval are shown, and p-value, respectively.
  • Table 2 shows the sensitivity and specificity of combined loci in detecting ALS. The sensitivity of any number of combination of mutations and specificity are shown.
  • Diagnostic testing based on novel gene sequence identification could serve as an early disease detection tool.
  • FIG. 6 illustrates distribution of candidate ALS-only mutated genes and probability of having ALS or not having ALS based on the number of positive results or negative results on mutations.
  • the distribution of numbers of variants found out of the 23 genomic loci in the 713 ALS cases is shown in grey.
  • the diamond plus represents the probability of having ALS, which shows an increasing probability with increasing positive numbers of variants.
  • the star represents the probability of not having ALS, which shows a decreasing probability base with increasing positive numbers of variants.
  • ALS A clinical and comprehensive multi- omics signature for ALS employing induced pluripotent stem cell derived motor neurons from 1000 sporadic and familial ALS patients nationwide. Annals of Neurology 80, S243- S243 (2016).

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Abstract

Les inventeurs ont identifié 23 loci génomiques et établi qu'une majorité de patients atteints de sclérose latérale amyotrophique (SLA) présentent des mutations dans au moins un des loci cibles. La présente invention concerne un procédé pour détecter la SLA chez un sujet, comprenant les étape suivantes : (A) amplification d'ADN extrait d'un échantillon biologique d'un sujet par une réaction en chaîne par polymérase spécifique à une cible pour amplifier des loci génomiques spécifiques comprenant 23 positions chromosomiques spécifiques de chrl:25854953, chrl:3624870, chr3:158557839, chr3:185543848, chr3: 186923875, chr4: 17685198, chr4: 180358067, chr5:53655366, chr5: 82813472, chr5:94666955, chr7:5338617, chr8: 62196626, chr9:71428255, chr9:89866631, chr9: 130224292, chrlO: 119712877, chrlO: 119712899, chrl2:82295320, chrl5:25687571, chrl 5:74926032, chrl7:2562894, chrl7:40390624, et chr22:41330858 ; (b) purification, et séquençage de l'ADN amplifié ; (c) analyse de chacune des séquences d'ADN amplifiées et comparaison avec sa séquence d'ADN correspondante des loci génomiques normaux, (d) identification d'une ou plusieurs mutations, si elles sont présentes, aux 23 positions chromosomiques, et (e) détection de la SLA chez le sujet si les 23 positions chromosomiques présentent une ou plusieurs mutations.
PCT/US2021/071865 2020-10-16 2021-10-14 Procédé de détection de la sclérose latérale amyotrophique WO2022082199A1 (fr)

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