Microbiome markers and therapies for autism spectrum disorders

Abstract

The present disclosure provides for characterization of normal flora and identifying biomarkers in the gut of healthy, neurotypical subjects. Aspect of the disclosure provide for the characterization of the gut microbiome in ADS subjects, characterized by reduced richness and significant loss of the ‘ Prevotella -like enterotype’ compared to neurotypical subjects. The relative abundance of genera Prevotella, Coprococcus , Prevotellaceae and Veillonellaceae are significantly lower in autistic children than in neurotypical children. Further, Prevotella , is one of the three main classifiers for the human enterotypes, along with Bacteroides and Ruminococcus . These three core genera are among main contributors in the principle component analysis. ‘ Prevotella -like enterotype’ was absent in the autistic group, while neurotypical samples showed an even distribution among the three enterotypes. The present disclosure provides for an understanding the association between gut microbiota, health, and disease states, and provides for potential diagnostic and therapeutic targets.

1 . An assay comprising:
 (a) subjecting nucleic acid extracted from a test sample of a human subject to a genotyping assay that detects at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria, the test sample including microbiota from a gut of the subject;   (b) determining a relative abundance of the at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria that is below a predetermined abundance; and   (c) selecting, when the relative abundance is below the predetermined abundance, a treatment regimen that comprises at least one of:
 (i) modifying microbiota of the gut of the subject using at least one of a prebiotic, probiotic, or pharmaceutical, or 
 (ii) applying a therapeutic regimen for treating autism spectrum disorders. 
   
     
     
         2 . The assay of  claim 1 , wherein the predetermined abundance is a neurotypical amount. 
     
     
         3 . The assay of  claim 1 , wherein the modifying microbiota includes at least one of applying a prebiotic mixture or applying an effective amount of microbiota affecting drugs. 
     
     
         4 . The assay of  claim 1 , wherein the genotyping assay includes at least one of qPCR, RT-qPCR, clone libraries, DGGE, T-RFLP, ARISA, microarrays, FIFH, dot-blot hybridization, and any other DNA hybridization methods that will detect a specific sequence in  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae. 
     
     
         5 . The assay of  claim 1 , wherein the assay detects at least two of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae. 
     
     
         6 . The assay of  claim 1 , wherein the genotyping assay comprises contacting the nucleic acid with at least one primer that specifically binds to 16S rRNA nucleic acid of at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae. 
     
     
         7 . An assay comprising:
 (a) Subjecting protein extracted from a test sample of a human subject to a protein assay that determines at least one protein indicative of at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria, the test sample including microbiota from a gut of the subject;   (b) determining a relative abundance of the at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria that is below a predetermined abundance; and   (c) selecting, when the relative abundance is below the predetermined abundance, a treatment regimen that comprises at least one of:
 (i) modifying microbiota of a gastrointestinal tract of the subject, or 
 (ii) applying a therapeutic regimen for treating autism spectrum disorders. 
   
     
     
         8 . The assay of  claim 7 , wherein the protein assay includes at least one of 2-Dimensional Gel Electrophoresis, 2-Dimensional Difference Gel Electrophoresis (2D-DIGE), MALDI TOF-MS, (2D-) LC-ESI-MS/MS, AQUA and iTRAQ. 
     
     
         9 . A method of selecting a treatment regimen for a human subject, comprising:
 (a) subjecting a test sample from the human subject, including microbiota from a gut of the subject, to at least one of:
 (i) nucleic acid extraction, or 
 (ii) protein extraction; 
   (b) detecting, using at least one of the extracted nucleic acid or protein, a relative abundance of at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria in the gut of the subject;   (c) comparing the detected relative abundance of the at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria to a predetermined abundance of the at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria;   (d) selecting, when the relative abundance is below the predetermined abundance, the treatment regimen comprising at least one of:
 (i) modifying microbiota of a gastrointestinal tract of the subject, or 
 (ii) applying a therapeutic regimen for treating autism spectrum disorders. 
   
     
     
         10 . A method of increasing balance of a microbiome of a gut of a human subject having autism spectrum disorder (ASD) comprising:
 (a) determining a relative abundance of at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria in the gut of the subject   (b) administering, when the relative abundance is below a predetermined amount, at least one of a prebiotic, probiotic, or pharmaceutical capable of modifying the relative abundance of the at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria in the gut.   
     
     
         11 . A method of treating a human subject with autism spectrum disorder gut-related symptoms, comprising:
 administering, to the subject, a prebiotic to stimulate growth of at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria in a gut of the subject,   wherein a relative abundance of the at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria in the gut of the subject is below a predetermined abundance.   
     
     
         12 . A method of selecting a human subject with autism spectrum disorders (ASD) or gut-related symptoms for inclusion in or exclusion from a clinical trial, comprising:
 (a) subjecting a test sample from a human subject, including microbiota from a gut of the subject, to at least one of:
 (i) nucleic acid extraction, or 
 (ii) protein extraction; 
   (b) detecting, using at least one of the extracted nucleic acid or protein, a relative abundance of at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria in the gut of the subject;   (c) selecting the subject for inclusion in the clinical trial when the relative abundance of the at least one of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria is below a first predetermined abundance, the first predetermined abundance being an upper-limit for indicating at least one of ASD or gut-related symptoms, and selecting the subject for exclusion from the clinical trial when the relative abundance is above a second predetermined abundance, the second predetermined abundance being a lower limit for indicating at least one of a neurotypical subject or no gut-related symptoms.   
     
     
         13 - 14 . (canceled) 
     
     
         15 . A method of detecting, in a stool sample, the relative abundance of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria, that are indicative of autism spectrum disorders (ASD), comprising:
 determining the relative abundance of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae bacteria in a test stool sample;   wherein a decreased relative abundance of  Prevotella, Coprococcus , Prevotellaceae or Veillonellaceae bacteria relative to a neurotypical population of said bacteria relative to a neurotypical population of said bacterium, is indicative of pure gut health and/or ASD-gut related problems.   
     
     
         16 . A method of detecting, in a stool sample, the relative abundance of  Prevotella -genus, that is indicative of ASD-gut related problems, comprising:
 determining the relative abundance of  Prevotella -genus bacteria in a test stool sample;   wherein a decreased relative abundance of  Prevotella -like enterotype bacteria relative to a neurotypical relative abundance of said bacteria is indicative of ASD.   
     
     
         17 . The method of  claim 1  or  2 , wherein the determining comprises obtaining nucleic acids from said stool sample and sequencing with any deep sequencing technique (ie. 454 pyrosequencing, illumine, ion torrent) the nucleic acids. 
     
     
         18 . The method of  claim 3 , wherein the nucleic acids to be sequenced are bacterial 16S rRNA genes. 
     
     
         19 . An assay for the detection of relative abundance  Prevotella -genus in a biological sample, wherein the absence of  Prevotella -genus relative to a healthy relative abundance of said bacteria is indicative of poor gut health. 
     
     
         20 . A method for quantifying the population of  Prevotella, Coprococcus , Prevotellaceae, or Veillonellaceae indicative of ASD comprising:
 contacting a stool specimen of a subject with a reagent that detects said bacteria and detecting a lower relative abundance of said bacteria compared with the relative abundance of said bacteria in a neurotypical sample is indicative of ASD.   
     
     
         21 - 27 . (canceled)