WO2010147714A1 - Biomarqueurs associés à l'autisme et utilisations de ces derniers - Google Patents

Biomarqueurs associés à l'autisme et utilisations de ces derniers Download PDF

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WO2010147714A1
WO2010147714A1 PCT/US2010/034254 US2010034254W WO2010147714A1 WO 2010147714 A1 WO2010147714 A1 WO 2010147714A1 US 2010034254 W US2010034254 W US 2010034254W WO 2010147714 A1 WO2010147714 A1 WO 2010147714A1
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nucleotide
carbohydrate
seq
nucleic acid
autism
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PCT/US2010/034254
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English (en)
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W. Ian Lipkin
Mady Hornig
Brent L. Williams
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The Trustees Of Columbia University In The City Of New York
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Priority to EP10789901A priority Critical patent/EP2443259A4/fr
Publication of WO2010147714A1 publication Critical patent/WO2010147714A1/fr
Priority to US13/328,982 priority patent/US9050276B2/en
Priority to US14/691,498 priority patent/US20150329909A1/en

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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
    • C12Q2600/158—Expression markers

Definitions

  • Autistic disorder is one of five pervasive developmental disorders defined in the
  • Autistic disorder is a developmental disorder of the human brain that manifests during infancy or childhood and is characterized by behavioral and social abnormalities that appear to be developmentally based (for example, impairments in social interaction and communication).
  • autism interferes with imagination and the ability to reason. Autism is frequently associated with other disorders such as attention deficit/hyperactivity disorder (AD/HD) and can be associated with psychiatric symptoms such as anxiety and depression.
  • AD/HD attention deficit/hyperactivity disorder
  • autism diagnoses have increased by 300% to 500% in the United States and many other countries. A means of prevention and treatment is needed for this health
  • PDDs Pervasive developmental disorders
  • ASDs Autism Deformation Disorders
  • PDD Proliferative Disorders
  • ASP Asperger's Disorder
  • MeCP2 DNA methylation binding protein gene
  • GI gastrointestinal
  • the invention is based, at least in part, on the finding that decreased levels in sucrase isomaltase, maltase glucoamylase, lactase, GLUT2, and SGLTl can serve as markers for human Autism Spectrum Disorders. Accordingly, in one aspect, the invention provides a method for detecting the presence of or a predisposition to autism or an autism spectrum disorder (ASD) in a human subject or a child of a human subject. The method comprises: (1) obtaining a biological sample from a human subject; and (2) detecting whether or not there is an alteration in the expression of a carbohydrate metabolic enzyme protein or a carbohydrate transporter protein in the subject as compared to a non-autistic subject.
  • ASSD autism spectrum disorder
  • the carbohydrate metabolic enzyme comprises sucrase isomaltase, maltase glucoamylase, lactase, or a combination thereof.
  • the carbohydrate transporter comprises GLUT2, SGLTl , or a combination thereof.
  • the method further comprises detecting a decrease in Bacteriodetes, an increase in the Firmicute/Bacteroidete ratios, an increase in cumulative levels of Firmicutes and Proteobacteria, an increase in Beta-
  • the detecting comprises detecting whether there is an alteration in the gene locus that encodes the carbohydrate metabolic enzyme protein or the carbohydrate transporter protein. In a further embodiment, the detecting comprises detecting whether expression of the carbohydrate metabolic enzyme protein or the carbohydrate transporter protein is reduced. In some embodiments, the detecting comprises detecting in the sample whether there is a reduction in the mRNA expression of the carbohydrate metabolic enzyme protein or the carbohydrate transporter protein. In some embodiments of the invention, the subject is a human embryo, a human fetus, or an unborn human child.
  • the sample comprises blood, serum, sputum, lacrimal secretions, semen, vaginal secretions, fetal tissue, skin tissue, small intestine tissue (e.g., the ileum), large intestine tissue (e.g., the cecum), muscle tissue, amniotic fluid, or a combination thereof.
  • blood serum, sputum, lacrimal secretions, semen, vaginal secretions, fetal tissue, skin tissue, small intestine tissue (e.g., the ileum), large intestine tissue (e.g., the cecum), muscle tissue, amniotic fluid, or a combination thereof.
  • An aspect of the invention provides a method for treating or preventing autism or an autism spectrum disorder in a subject in need thereof.
  • the method comprises administering to the subject a therapeutic amount of a pharmaceutical composition comprising a functional carbohydrate metabolic enzyme molecule or a carbohydrate transporter molecule, thereby treating or preventing autism or an autism spectrum disorder.
  • the administering comprises a subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; an infusion; oral, nasal, or topical delivery; or a combination of the delivery modes described.
  • the administering comprises delivery of a carbohydrate metabolic enzyme molecule or a carbohydrate transporter molecule to the alimentary canal or intestine of the subject.
  • the administering comprises feeding the human subject or child thereof a therapeutically effective amount of the carbohydrate metabolic enzyme molecule or a carbohydrate transporter molecule. In further embodiments, the administering occurs daily, weekly, twice weekly, monthly, twice monthly, or yearly.
  • the invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising: a carbohydrate metabolic enzyme molecule or a carbohydrate transporter molecule molecule; and a pharmaceutically acceptable carrier.
  • the composition comprises a nucleic acid molecule having at least about 80% identity to SEQ ID NO: 11, 12, 13, or 14. In one embodiment, the composition comprises a nucleic acid molecule having at least about 85% identity to SEQ ID NO: 11, 12, 13, or 14. In one embodiment, the composition comprises a nucleic acid molecule having at least about 90% identity to SEQ ID NO: 11, 12, 13, or 14. In one embodiment, the composition comprises a nucleic acid molecule having at least about 95% identity to SEQ ID NO: 11, 12, 13, or 14.
  • the composition comprises a nucleic acid molecule having at least about 98% identity to SEQ ID NO: 11, 12, 13, or 14. In one embodiment, the composition comprises a nucleic acid molecule having at least about 99% identity to SEQ ID NO: 11, 12, 13, or 14. In one embodiment, the composition is SEQ ID NO: 11, 12, 13, or 14.
  • An aspect of the invention provides for a diagnostic kit for detecting the presence of
  • the kit comprises a nucleic acid molecule that specifically hybridizes to or a primer combination that amplifies a Sutterella sp. 16S nucleic acid sequence.
  • the nucleic acid molecule comprises a nucleic acid primer or nucleic acid probe.
  • the 16S nucleic acid sequence comprises at least about 80% of SEQ ID NO: 59 or SEQ ID NO: 60.
  • the 16S nucleic acid sequence comprises at least about 85% of SEQ ID NO: 59 or SEQ ID NO: 60.
  • the 16S nucleic acid sequence comprises at least about 90% of SEQ ID NO: 59 or SEQ ID NO: 60.
  • the 16S nucleic acid sequence comprises at least about 95% of SEQ ID NO: 59 or SEQ ID NO: 60. In another embodiment, the 16S nucleic acid sequence comprises at least about 98% of SEQ ID NO: 59 or SEQ ID NO: 60. In some embodiments, the 16S nucleic acid sequence comprises at least about 99% of SEQ ID NO: 59 or SEQ ID NO: 60. In further embodiments, the 16S nucleic acid sequence is SEQ ID NO: 59 or SEQ ID NO: 60. In one embodiment, the probe comprises a nucleotide sequence having SEQ ID NOS: 13 or 14 in Table 1, or the italicized nucleotide of sequence SEQ ID NO: 19.
  • the probe comprises at least 10 consecutive nucleotide bases comprising SEQ ID NO: 19, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or T nucleotide, and wherein H is an A nucleotide and/or T nucleotide and/or C nucleotide.
  • the probe comprises a reverse complement of SEQ ID NOS: 11, 12, 15,
  • the primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 11, 12, 15, 16, 17, or 18, wherein, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or T nucleotide, and wherein H is an A nucleotide and/or T nucleotide and/or C nucleotide.
  • the sample is from a human or non-human animal.
  • the sample comprises intestinal tissue (e.g., the small intestine or large intestine), feces, blood, skin, or a combination of the mentioned tissues.
  • An aspect of the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence of or a predisposition to autism or an autism spectrum disorder (ASD).
  • the kit comprising a nucleic acid primer that specifically hybridizes to an autism biomarker, wherein the primer will prime a polymerase reaction only when an autism biomarker is present.
  • the primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 11, 12, 15, 16, 17, or 18, wherein, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or T nucleotide, and wherein H is an A nucleotide and/or T nucleotide and/or C nucleotide.
  • the autism biomarker is a carbohydrate trasporter molecule, a carbohydrate metabolic enzyme molecule, or a gastrointestinal Sutterella sp. bacterium.
  • the carbohydrate trasporter molecule is GLUT2 or SGLTl .
  • the carbohydrate metabolic enzyme molecule is SI, MGAM, or LCT.
  • the sample is from a human or non-human animal.
  • the sample comprises intestinal tissue (e.g., the small intestine or large intestine), feces, blood, skin, or a combination of the mentioned tissues.
  • An aspect of the invention provides for a method of treating or preventing a disease associated with elevated levels of Beta-proteobacteria.
  • the method of the invention comprises administering to a subject in need thereof a therapeutic amount of an antimicrobial composition
  • the antimicrobial composition is an antibiotic, a probiotic agent, or a combination thereof.
  • the disease is ASD, autism, or a gastrointestinal disease.
  • the gastrointestinal disease is diarrhea, inflammatory bowel disease, antimicrobial- associated colitis, or irritable bowel syndrome.
  • the diarrhea or inflammatory bowel diseases is ulcerative colitis or Crohn's disease.
  • the antibiotic comprises lincosamides, chloramphenicols, tetracyclines, aminoglycosides, beta- lactams, vancomycins, bacitracins, macrolides, amphotericins, sulfonamides, methenamin, nitrofurantoin, phenazopyridine, trimethoprim; rifampicins, metronidazoles, cefazolins, lincomycin, spectinomycin, mupirocins, quinolones, novobiocins, polymixins, gramicidins, antipseudomonals, or a combination of the stated antibiotics.
  • the probiotic agent comprises Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Sutterella, Bilophila, Campylobacter, Wolinella, Butyrovibrio, Megamonas, Desulfomonas, Desulfovibrio, Bifidobacterium, Lactobacillus, Eubacterium, Actinomyces, Eggerthella, Coriobacterium, Propionibacterium, other genera of non-sporeforming anaerobic gram-positive bacilli, Bacillus, Peptostreptococcus, newly created genera originally classified as Peptostreptococcus, Peptococcus, Acidaminococcus, Ruminococcus, Megasphaera, Gaffkya, Coprococcus, Veillonella, Sarcina, Clostridium, Aerococcus, Streptococcus, Enterococcus, Pediococcus, Micrococcus, Staphylococc
  • An aspect of the invention provides for a method of detecting a Sutterella sp. in a sample.
  • the method comprises: (a) selecting a Sutterlla ⁇ .-specific primer pair, wherein the primer pair mediates amplification of a polynucleotide amplicon of a selected, known length from a nucleic acid of a Sutterlla sp.; contacting a nucleic acid from the sample with the Sutterlla ⁇ .-specific primer pair in a reaction mixture under conditions that promote amplification of a polynucleotide amplicon, wherein the primer pair will prime a polymerase reaction only when the nucleic acid of a Sutterlla sp.
  • the sample comprises intestinal tissue (e.g., the small intestine or large intestine), feces, blood, skin, or a combination of the listed tissues.
  • the primer pair comprises a forward primer and a reverse primer.
  • the forward primer comprises SEQ ID NO: 11 or 17, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or T nucleotide, and wherein H is an A nucleotide and/or T nucleotide and/or C nucleotide.
  • the reverse primer comprises SEQ ID NO: 12 or 18, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or T nucleotide, and wherein H is an A nucleotide and/or T nucleotide and/or C nucleotide.
  • the forward primer comprises at least 10 consecutive nucleotide bases comprising SEQ ID NO: 17 or 19, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or T nucleotide, and wherein H is an A nucleotide and/or T nucleotide and/or C.
  • the reverse primer comprises at least 10 consecutive nucleotide bases comprising SEQ ID NO: 18 or 19, wherein S is a G nucleotide and/or a C nucleotide, wherein Y is a C nucleotide and/or T nucleotide, wherein R is an A nucleotide and/or G nucleotide, wherein W is an A nucleotide and/or T nucleotide, and wherein H is an A nucleotide and/or T nucleotide and/or C nucleotide, wherein B is a T nucleotide, C nucleotide, or G nucleotide, wherein V is an A nucleotide, G nucleotide, or C nucleotide; wherein D is an A nucleotide, G nucleotide, or T nucleotide; and wherein K is a G nucleotide or T nucleotide.
  • FIG. 1 is a schematic depicting carbohydrate metabolizing enzymes (e.g., sucrase isomaltase, maltase glucoamylase, and lactase) and carbohydrate transporter proteins (e.g., GLUT2 and SGLTl) involved in carbohydrate metabolism, uptake, and absorption in the enterocytes of the ileum.
  • carbohydrate metabolizing enzymes e.g., sucrase isomaltase, maltase glucoamylase, and lactase
  • carbohydrate transporter proteins e.g., GLUT2 and SGLTl
  • FIG. 2 shows bar graphs depicting that carbohydrate metabolizing enzyme mRNAs are reduced in the ileum of ASD subjects. Graphs are shown for sucrase isomaltase (left), maltase glucoamylase (center), and lactase (right).
  • FIG. 3 shows bar graphs depicting that carbohydrate transporter mRNAs are reduced in the ileum of ASD subjects. Graphs are shown for SGLTl (Top) and GLUT2 (Bottom).
  • FIG. 4 shows graphs depicting that mRNA for ileal inflammatory markers are increased in the ileum of ASD subjects. Graphs are shown for ClQA (Top Left), Resistin (Top Right), and ILl 7F (Bottom Left and Right).
  • FIG. 5 shows bar graphs depicting the differences in bacteria phylum found in the ileum of ASD subjects. Changes at the phylum level were observed. Bar graphs show a decrease in Bacteroidetes (left) and increase in Firmicute/Bacteroidete ratios in ileum of AUT-GI children.
  • FIG. 6 is a bar graph depicting the copy number of bacteroidetes found in the ileum of ASD subjects. Real-time PCR confirmed a decrease in Bacteroidete. Bacteroidete 16S rDNA copies (Normalized to Total Bacterial 16S rDNA).
  • FIG. 7 is a schematic summarizing the interplay between expression levels of carbohydrate metabolic enzymes (e.g., sucrase isomaltase, maltase glucoamylase, and lactase), carbohydrate transporters (e.g., GLUT2 and SGLTl) and the population of bacteria in the ileum of ASD subjects.
  • carbohydrate metabolic enzymes e.g., sucrase isomaltase, maltase glucoamylase, and lactase
  • carbohydrate transporters e.g., GLUT2 and SGLTl
  • FIGS. 8A-B are bar graphs showing the abundance of Sutler ella sp. in the ileum
  • FIG. 8A cecum
  • FIG. 8B cecum
  • FIGS. 8C-D are bar graphs showing the abundance of Sutler ella sp. sequences in the ileum (FIG. 8C) and cecum (FIG. 8D) of autism and control patients.
  • FIGS. 8E-F are bar graphs showing the abundance of Sutterella sp. sequences comprising the Beta-proteobacteria sequences in the ileum (FIG. 8C) and cecum (FIG. 8D) of autism and control patients.
  • FIG. 9 is a photograph of an agarose gel showing the results of classical PCR experiments for the detection of Sutterella.
  • FIG. 1OA is an amplification plot of Sutterella sp. through cycles of Real-time PCR experiments.
  • FIG. 1OB is a standard curve graph showing the copy number of Sutterella sp. from
  • FIG. 11 is a photograph of an agarose gel showing the results of Sutterella detection in the ileum and cecum of patients using the V6-V8 Sutterella sp. -specific PCR.
  • FIGS. 12A-B are bar graphs showing the copy number of Sutterella sp. in the ileum
  • FIG. 12A cecum
  • FIG. 12B cecum
  • FIGS. 12C-D are bar graphs showing the copy number of Sutterella sp. in the ileum (FIG. 12C) and cecum (FIG. 12D) of autism and control patients using the V6-V8 Sutterella s/7. -specific PCR.
  • FIG. 13 is a sequence alignment for the V6-V8 region of Sutterella sp. obtained from biological samples of Autism patients 1, 3, 10, 11, and 12 (SEQ ID NO: 59), and Autism patients 5 and 7 (SEQ ID NO: 60).
  • FIG. 14 depicts Sutterella sp. sequence clustering from the Operational Taxonomic
  • FIG. 15A is a schematic depicting Sutterella sp. treeing analysis of the V6-V8 sequences.
  • FIG. 15B is a schematic depicting Sutterella sp. treeing analysis of the V2 sequence.
  • FIG. 16 shows graphs that show quantitative real-time PCR analysis of disaccharidases, hexose transporters, villin and CDX2.
  • FIG. 17 shows graphs depicting pyrosequencing analysis of intestinal microbiota.
  • FIGS. 17A-B Phylum-level comparison of the average relative abundance of bacterial taxa in ileal (FIG. 17A) and cecal (FIG. 17B) biopsies from AUT-GI and Control-GI patients.
  • FIGGS. 17A-B Phylum-level comparison of the average relative abundance of bacterial taxa in ileal (FIG. 17A) and cecal (FIG. 17B) biopsies from AUT-GI and Control-GI patients.
  • FIGS. 17C-D Box-and-whisker plot displaying the distribution of Bacteroidetes as a
  • FIGGS. 17G-H Heatmaps displaying abundance distributions (% of total sequence reads per patient) of Bacteroidetes classified at the family level in ileal (FIG. 17G) and cecal (FIG. 17H) biopsies from AUT-GI and Control-GI children (Bottom row displays cumulative levels of all family members by patient). *,p ⁇ 0.05, **,p ⁇ 0.01.
  • FIG. 18 shows graphs of Firmicute abundance in AUT-GI and Control-GI children.
  • FIG. 18D Mann- Whitney; biopsies from AUT-GI and Control-GI children.
  • FIGS. 18E-18F Heatmaps displaying abundance distribution (% of total sequence reads per patient) of family members in the class Clostridia in ileum (FIG. 18E) and cecum (FIG. 18F) of AUT-GI and Control-Gi children (Bottom row displays cumulative levels of all family members by patient).
  • FIGGS. 181-18 J Heatmaps displaying abundance distribution (% of total sequence reads per patient) of family members in the class Clostridia in ileum (FIG. 18E) and cecum (FIG. 18F) of AUT-GI
  • FIG. 19 shows graphs of the abundance of Proteobacteria in AUT-GI and Control-
  • FIG. 20 shows schematics depicting factors that mediate GI disease in AUT-GI children.
  • FIG. 20A Schematic representation of enterocyte-mediated digestion of disaccharides and absorption/transport of monosaccharides in the small intestine.
  • Disaccharidase enzymes (SI, MGAM, and LCT) in the enterocyte brush border break down disaccharides into their component monosaccharides.
  • the monosaccharides, glucose and galactose are transported from the small intestinal lumen into the enterocyte by the sodium-dependent transporter SGLTl .
  • the facilitative transporter On the basolateral enterocyte membrane, the facilitative transporter, GLUT2, transports glucose, galactose and fructose out of the enterocyte and into the circulation, thus regulating postprandial blood glucose levels.
  • GLUT2 may also be transiently inserted into the apical enterocyte membrane, contributing a diffusive component to monosaccharide absorption in certain circumstances (Kellet et al., 2008).
  • the expression levels of disaccharidases and hexose transporters may be controlled by the transcription factor CDX2. (FIG. 20B) In the normal small intestine, where expression of disaccharidases and hexose transporters are high, the majority, if not all, of disaccharides are efficiently digested and monosaccharides are absorbed
  • ASD-GI This specifically manifests as an increase in Firmicute/Bacteroidete ratios, cumulative levels of Firmicutes and Proteobacteria, and in levels of Betaproteobacteria in both the ileum and cecum.
  • the level of dysbiosis in the ileum and cecum may thus be controlled by the degree and type of deficiency of carbohydrate metabolism and transport in the small intestine.
  • malabsorbed monosaccharides can lead to osmotic diarrhea; non-absorbed sugars may also serve as substrates for intestinal microflora, that produce fatty acids and gases (methane, hydrogen, and carbon dioxide) and promote additional GI symptoms of bloating and flatulence.
  • dysbiosis may manifest in changes in SCFAs that can reduce colonic pH, further inhibiting the growth of Bacteroidetes.
  • Disruption of symbiotic relationships between the host and the intestinal microbial ecosystem as a result of dysbiosis may also play a fundamental role in development, distribution, activation and differentiation of immune cells within the intestine (Abt and Artis, 2009; Mazmanian et al., 2008), thus providing a framework for understanding previous reports of inflammatory indices in the AUT-GI intestine.
  • FIG. 21 depicts lactase genotyping.
  • FIG. 21A Representative agarose gel banding patterns observed for LCT- 13910 and LCT-22018 polymorphisms.
  • FIG. 21C Box-and-whisker plot displaying the distribution of LCT mRNA expression in all individuals (AUT-GI and Control-GI) with the homozygous adult-type hypolactasia
  • FIG. 23 shows graphs of the diversity of AUT-GI and Control-GI phylotypes.
  • FIGS. 23A-23B Rarefaction curves assessing the completeness of sampling from pyrosequencing data obtained for individual AUT-GI (red) and Control-GI (blue) subjects' ileal (FIG. 23A) and cecal (FIG. 23B) biopsies.
  • the y-axis indicates the number of OTUs detected (defined at 97% threshold for sequence similarity), the x-axis the number of sequences sampled.
  • FIGS. 23C-23D Rarefaction curves to estimate phylotype diversity, using the Shannon Diversity Index, from pyrosequencing data obtained for individual AUT-GI (red) and Control-GI (blue) subjects' ileal (FIG. 23C) and cecal (FIG. 23D) biopsies.
  • FIG. 24 shows graphs depicting the distribution of pyrosequencing reads by patient.
  • FIGS. 24A-24B Phylum level distribution of bacteria by patient obtained from 16S rRNA gene barcoded pyrosequencing for ilea (FIG. 24A) and ceca (FIG. 24B).
  • FIGGS. 24C-D Distribution of low abundance bacterial phyla obtained by barcoded pyroseqeuncing. By-patient distribution of low abundance bacterial phyla in ilea (FIG. 24C) and ceca (FIG. 24D) from AUT-GI (patients 1-15) and Control-GI (patients 16-22).
  • FIG. 25 shows the OTU analysis of Bacteroidete phylotypes. (FIGS. 25A-25B)
  • FIG. 25B biopsies from AUT-GI and Control-GI children (Bottom row displays cumulative levels of all 12 OTUs by patient).
  • FIG. 25E Greengenes- or microbial blast(*)-derived classification of representative sequences obtained from each Bacteroidete OTU. Color code denotes the family-level, Ribosomal Database-derived taxonomic classification of each OTU sequence.
  • FIG. 26 shows graphs depicting order-level analysis of Firmicute/Bacteroidete ratio and confirmation by real-time PCR.
  • FIG. 27 shows graphs of the abundance of Firmicutes assayed by pyrosequencing and real-time PCR.
  • FIG. 28 shows genus-level distribution of members of the families
  • FIGS. 28A-28B Heatmap representation of the individual patient distributions of Ruminococcaceae and Lachnospiraceae genus members in ileal (FIG. 28A) and cecal (FIG. 28B) biopsies from AUT-GI (Patients 1-15) and Control-GI
  • FIG. 29 shows graphs depicting increases in inflammatory markers, such as ClQ,
  • Resistin CD163, Tweak, IL17F, and nNOS. These inflammatory markers may also serve as biomarkers for diagnosis of human Autism Spectrum Disorders, as well as for detecting the presence of or a predisposition to autism or an autism spectrum disorder.
  • Autism one of the ASDs, is mostly diagnosed clinically using behavioral criteria because few specific biological markers are known for diagnosing the disease.
  • Autism is a neuropsychiatric developmental disorder characterized by impaired verbal communication, nonverbal communication, and reciprocal social interaction. It is also characterized by restricted and stereotyped patterns of interests and activities, as well as the presence of developmental abnormalities by 3 years of age (Bailey et al, (1996) J Child Psychol Psychiatry 37(1):89-126).
  • Autism-associated disorders, diseases or pathologies can comprise any metabolic, immune or systemic disorders; gastrointestinal disorders; epilepsy; congenital malformations or genetic syndromes; anxiety, depression, or AD/HD; or speech delay and motor in-coordination.
  • ASD Autism spectrum disorders
  • Macroscopic and histological observations in ASD include findings of ileo-colonic lymphoid nodular hyperplasia (LNH), enterocolitis, gastritis and esophagitis (Wakefield et al., 2000; Wakefield et al., 2005; Furlano et al., 2001; Torrente et al., 2002; Horvath et al., 1999).
  • Associated changes in intestinal inflammatory parameters include higher densities of lymphocyte populations, aberrant cytokine profiles, and deposition of immunoglobulin (IgG) and complement CIq on the basolateral enterocyte membrane (Furlano et al., 2001; Ashwood and Wakefield, 2006).
  • Functional disturbances include increased intestinal permeability (D'Eufemia et al., 1996), compromised sulphoconjugation of phenolic compounds (O'Reilly and Waring, 1993; Alberti et al., 1999), deficient enzymatic activity of disaccharidases
  • the gastrointestinal tract is exposed to an onslaught of foreign material in the form of food, xenobiotics, and microbes.
  • the intestinal muco-epithelial layer must maximize nutritional uptake of dietary components while maintaining a barrier to toxins and infectious agents. Although some aspects of these functions are host-encoded, others are acquired through symbiotic relationships with microbial flora. Dietary carbohydrates enter the intestine as monosaccharides (glucose, fructose, and galactose), disaccharides (lactose, sucrose, maltose), or complex polysaccharides.
  • carbohydrates are further digested by disaccharidases expressed by absorptive enterocytes in the brush border of the small intestine and transported as monosaccharides across the intestinal epithelium.
  • disaccharidases expressed by absorptive enterocytes in the brush border of the small intestine and transported as monosaccharides across the intestinal epithelium.
  • humans lack the glycoside hydrolases and polysaccharide lyases necessary for cleavage of glycosidic linkages present in plant cell wall polysaccharides, oligosaccharides, storage polysaccharides, and resistant starches. Intestinal bacteria encoding these enzymes expand our capacity to extract energy from dietary polysaccharides (Sonnenburg et al., 2008; Flint et al., 2008).
  • bacteria produce short- chain fatty acids (butyrate, acetate, and propionate) that serve as energy substrates for colonocytes, modulate colonic pH, regulate colonic cell proliferation and differentiation, and contribute to hepatic gluconeogenesis and cholesterol synthesis (Wong et al., 2006; Jacobs et al., 2009).
  • Oxida and Shanahan 2006; Macpherson and Harris, 2004.
  • the present invention provides the discovery and the identification of GLUT2 as well SGLTl as biomarkers for human Autism Spectrum Disorders.
  • the present invention provides for methods to use genes encoding carbohydrate metabolic enzyme molecules (such as sucrase isomaltase, maltase glucoamylase, and lactase) or carbohydrate transporter molecules, or a combination of the two, and corresponding expression products for the diagnosis, prevention and treatment of autism and autism spectrum disorders.
  • the methods of the invention are useful in various subjects, such as humans, including adults, children, and developing human fetuses at the prenatal stage.
  • the GLUT2 gene locus can comprise all GLUT2 sequences or products in a cell or organism, including GLUT2 coding sequences, GLUT2 non-coding sequences (e.g., introns), GLUT2 regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer, terminator).
  • a GLUT2 gene also known as SLC2A2, encodes the glucose transporter 2 isoform.
  • GLUT2 mediates the bidirectional transport of glucose.
  • the GLUT2 gene also encompasses its variants, analogs and fragments thereof, including alleles thereof (e.g., germline mutations) which are related to susceptibility to autism and/or autism spectrum disorders.
  • the SGLTl gene locus can comprise all SGLTl sequences or products in a cell or organism, including SGLTl coding sequences, SGLTl non-coding sequences (e.g., introns), SGLTl regulatory sequences controlling transcription and/or translation (e.g., promoter, enhancer, terminator).
  • a SGLTl gene also known as SLC5A1, encodes the sodium/glucose co -transporter
  • the sodium dependent glucose transporter is an integral plasma membrane glycoprotein of the intestine.
  • SGLTl mediates glucose and galactose uptake from the intestinal lumen. Mutations in this gene have been associated with glucose-galactose malabsorption.
  • the SGLTl gene also encompasses its variants, analogs and fragments thereof, including alleles thereof (e.g., germline mutations) which are related to susceptibility to autism and/or autism spectrum disorders.
  • carbohydrate transport activity means the ability of a polypeptide to bind a carbohydrate, such as glucose, to a transporter protein, and subsequently facilitate uptake of the carbohydrate from the serum or extracellular millieu into a cell (e.g., a liver cell, or pancreatic ⁇ -cell).
  • Glucose transport activity can be measured as described by Hissin et al., 1982, J. Clin. Invest. 70(4): 780-90.
  • the carbohydrate transport activity is glucose transport activity, and the activity can be measured by determining glucose transport activity as described in Hissin as well as the ability to decrease extracellular or serum glucose levels.
  • Non- limiting examples of a carbohydrate transporter include GLUTl, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUTlO, GLUTl 1, GLUT12, and HMIT (see Scheepers et al., JPEN J Parenter Enteral Nutr. 2004 Sep-Oct;28(5):364-71).
  • a sucrase isomaltase (SI) gene encodes a sucrase-isomaltase protein, which is a glucosidase enzyme, that is expressed in the intestinal brush border.
  • the encoded protein is synthesized as a precursor protein that is cleaved by pancreatic proteases into two enzymatic subunits, sucrase and isomaltase. The two subunits heterodimerize to form the sucrose- isomaltase complex, which is essential for the digestion of dietary carbohydrates including starch, sucrose and isomaltose. Mutations in this gene are the cause of congenital sucrase- isomaltase deficiency.
  • the SI gene also encompasses its variants,
  • a maltase glucoamylase (MGAM) gene encodes a maltase-glucoamylase enzyme.
  • the MGAM gene also encompasses its variants, analogs and fragments thereof, including alleles thereof (e.g., germline mutations) which are related to susceptibility to autism and/or autism spectrum disorders.
  • a lactase (LCT) gene encodes a glycosyl hydrolase of family 1.
  • the protein is integral to plasma membrane and has both phlorizin hydrolase activity and lactase activity.
  • carbohydrate metabolic enzyme activity includes “sucrase isomaltase activity”, “maltase glucoamylase activity”, “lactase activity”, “sucrase activity”, “maltase activity”, “trehalase activity”, “amylase activity”, “cellulase activity”, “glucosidase activity”, “pullulanase activity”, “galactosidase activity”, “alpha-Mannosidase acivity”, “glucuronidase activity”, “hyaluronidase activity”, “glycosylase activity”, “fucosidase activity”, “hexosaminidase activity”, “iduronidase activity”, or “maltase-glucoamylase activity”.
  • “Sucrase isomaltase activity” means the ability of a polypeptide to catalyze the hydrolysis of sucrose to fructose and glucose and to enzymatically digest polysaccharides at the alpha 1-6 linkages. Sucrase and isomaltase activities can be measured as described by Dahlqvist, A. (1964) Anal. Biochem. 7, 18-25 and and the enzyme assays described by Goda et al., Biochem J. 1988 February 15; 250(1): 41 ⁇ -6.
  • “Maltase glucoamylase activity” means the ability of a polypeptide to enzymatically digest starch, releasing malstose and free glucose, as well as to catalyze the hydrolysis of the disaccharide maltose.
  • Maltase and glucoamylase activities can be measured as described by Dahlqvist A. Specificity of the human intestinal disaccharidases and implications for hereditary disaccharide intolerance. J Clin Invest. 1962;41 :463-9; Dahlqvist A. Assay of intestinal disaccharidases. Scand J Clin Lab Invest. 1984;44: 169-72; and Quezada-Calvillo et al., J. Nutr. 137: 1725-1733, July 2007.
  • “Lactase activity” means the ability of a polypeptide to hydro lyze lactose to galactose and glucose. Lactase activity can be measured as described by
  • SEQ ID NO: 1 is the human wild type amino acid sequence corresponding to the
  • GLUT2 enzyme (residues 1-524) having GenBank Accession No. NP_000331 :
  • SEQ ID NO: 2 is the human wild type nucleic acid sequence corresponding to the
  • GLUT2 enzyme (bps 1-3439) having GenBank Accession No. NM_000340:
  • SEQ ID NO: 3 is the human wild type amino acid sequence corresponding to the
  • SGLTl enzyme (residues 1-664) having GenBank Accession No. NP 000334:
  • SEQ ID NO: 4 is the human wild type nucleic acid sequence corresponding to the
  • SEQ ID NO: 5 is the human wild type amino acid sequence corresponding to the sucrase isomaltase (SI) enzyme (residues 1-1827) having GenBank Accession No. NP_001032:
  • SEQ ID NO: 6 is the human wild type nucleic acid sequence corresponding to the sucrase isomaltase (SI) enzyme (bps 1-6023) having GenBank Accession No. NM_001041 :
  • gaacaattcc caacagaggg aatttgtgca cagagaggct gctgctggag gccgtggaat
  • SEQ ID NO: 7 is the human wild type amino acid sequence corresponding to the maltase glucoamylase (MGAM) enzyme (residues 1-1857) having GenBank Accession No. NP 004659:
  • SEQ ID NO: 8 is the human wild type nucleic acid sequence corresponding to the maltase glucoamylase (MGAM) enzyme (bps 1-6513) having GenBank Accession No. NM 004668:
  • gaggtgaata tttcacaatc aacctacaag gaccccaata atttagcatt taatgagatt
  • SEQ ID NO: 9 is the human wild type amino acid sequence corresponding to the lactase (LCT) enzyme (residues 1-1927) having GenBank Accession No. NP 002290: i MELSWHWFI ALLSFSCWGS DWESDRNFIS TAGPLTNDLL HNLSGLLGDQ SSNFVAGDKD
  • SEQ ID NO: 10 is the human wild type nucleic acid sequence corresponding to the lactase (LCT) enzyme (bps 1-6274) having GenBank Accession No. NM_002299:
  • a "carbohydrate transporter molecule” means a nucleic acid which encodes a polypeptide that exhibits carbohydrate transporter activity, or a polypeptide or peptidomimetic that exhibits carbohydrate transporter activity.
  • a carbohydrate transporter molecule can include the human GLUT2 protein (e.g., having the amino acid sequence shown in SEQ ID NO: 1), or a variant thereof, such as a fragment thereof, that exhibits carbohydrate transporter activity.
  • a carbohydrate transporter molecule can include the human SGLTl protein (e.g., having the amino acid sequence shown in SEQ ID NO: 3), or a variant thereof, such as a fragment thereof, that exhibits carbohydrate transporter activity.
  • the nucleic acid can be any type of nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA.
  • a carbohydrate transporter molecule can comprise a recombinant nucleic acid encoding human GLUT2 protein or human SGLTl protein.
  • a carbohydrate transporter molecule can comprise a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences).
  • a carbohydrate transporter molecule can be double-stranded.
  • a carbohydrate transporter molecule can be single-stranded.
  • the carbohydrate transporter molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art.
  • a nucleic acid that is a carbohydrate transporter molecule can be obtained by screening DNA libraries, or by amplification from a natural source.
  • the carbohydrate transporter molecules of the invention can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof.
  • Non-limiting examples of a carbohydrate transporter molecule, that is a nucleic acid is the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • Another example of a carbohydrate transporter molecule is a fragment of a nucleic acid having the sequence shown in SEQ ID NO: 2 or SEQ ID NO:4, wherein the fragment is exhibits carbohydrate transporter activity.
  • a "carbohydrate metabolic enzyme molecule” means a nucleic acid which encodes a polypeptide that exhibits carbohydrate metabolic enzyme activity, or a polypeptide or peptidomimetic that exhibits carbohydrate metabolic enzyme activity.
  • a carbohydrate metabolic enzyme molecule can include the human sucrase-isomaltase (SI) protein (e.g., having the amino acid sequence shown in SEQ ID NO: 5), or a variant thereof, such as a fragment thereof, that exhibits carbohydrate metabolic enzyme activity.
  • SI human sucrase-isomaltase
  • a carbohydrate metabolic enzyme molecule can include the human maltase-glucoamylase protein (e.g., having the amino acid sequence shown in SEQ ID NO: 7), or a variant thereof, such as a fragment thereof, that exhibits carbohydrate metabolic enzyme activity.
  • a carbohydrate metabolic enzyme molecule can include the human lactase protein (e.g., having the amino acid sequence shown in SEQ ID NO: 9), or a variant thereof, such as a fragment thereof, that exhibits carbohydrate metabolic enzyme activity.
  • the nucleic acid can be any type of nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semisynthetic DNA, as well as any form of corresponding RNA.
  • a carbohydrate metabolic enzyme molecule can comprise a recombinant nucleic acid encoding human sucrase-
  • a carbohydrate metabolic enzyme molecule can comprise a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences).
  • a carbohydrate metabolic enzyme molecule can be double-stranded.
  • a carbohydrate metabolic enzyme molecule can be single-stranded.
  • the carbohydrate metabolic enzyme molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art.
  • a nucleic acid that is a carbohydrate metabolic enzyme molecule can be obtained by screening DNA libraries, or by amplification from a natural source.
  • the carbohydrate metabolic enzyme molecules of the invention can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof.
  • a non- limiting example of a carbohydrate metabolic enzyme, that is a nucleic acid is the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 6, 8, or 10.
  • Another example of a carbohydrate metabolic enzyme molecule is a fragment of a nucleic acid having the sequence shown in SEQ ID NO: 6, 8, or 10, wherein the fragment is exhibits carbohydrate metabolic enzyme activity.
  • a carbohydrate transporter molecule encompass es orthologs of human GLUT2 and SGLTl.
  • a carbohydrate metabolic enzyme molecule encompass orthologs of human sucrase-isomaltase (SI), human maltase- glucoamylase, and human lactase.
  • SI human sucrase-isomaltase
  • a carbohydrate transporter molecule or a carbohydrate metabolic enzyme molecule encompass the orthologs in mouse, rat, non-human primates, canines, goat, rabbit, porcine, feline, and horses.
  • a carbohydrate transporter molecule or a carbohydrate metabolic enzyme molecule can comprise a nucleic acid sequence homologous to the human nucleic acid that encodes a human GLUT2 and SGLTl protein, or human sucrase-isomaltase (SI), human maltase-glucoamylase, and human lactase protein, respectively, wherein the nucleic acid is found in a different species and wherein that homolog encodes a protein with a glucose transporter function similar to a carbohydrate transporter molecule or an enzymatic function similar to a carbohydrate metabolic enzyme molecule.
  • SI human sucrase-isomaltase
  • a carbohydrate transporter molecule of this invention also encompasses variants of the human nucleic acid encoding the GLUT2 or SGLTl proteins that exhibit carbohydrate transporter activity, or variants of the human GLUT2 or SGLTl proteins that exhibit carbohydrate transporter activity.
  • a carbohydrate transporter molecule of this invention also includes a fragment of the human GLUT2 or SGLTl nucleic acid which encodes a polypeptide that exhibits carbohydrate transporter activity.
  • a carbohydrate transporter molecule of this invention encompasses a fragment of the human GLUT2 or SGLTl protein that exhibits carbohydrate transporter activity.
  • a carbohydrate metabolic enzyme molecule of this invention also encompasses variants of the human nucleic acid encoding the sucrase-isomaltase (SI), human maltase- glucoamylase, and human lactase proteins that exhibit carbohydrate metabolic enzyme activity, or variants of the human sucrase-isomaltase (SI), human maltase-glucoamylase, and human lactase proteins that exhibit carbohydrate metabolic enzyme activity.
  • a carbohydrate metabolic enzyme molecule of this invention also includes a fragment of the human sucrase-isomaltase (SI), human maltase-glucoamylase, and human lactase nucleic acid which encodes a polypeptide that exhibits carbohydrate metabolic enzyme activity.
  • a carbohydrate metabolic enzyme molecule of this invention encompasses a fragment of the human sucrase-isomaltase (SI), human maltase-glucoamylase, and human lactase protein that exhibits carbohydrate metabolic enzyme activity.
  • a carbohydrate transporter molecule is a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NO: 2, wherein the variant has a nucleotide sequence identity to SEQ ID NO: 2 of at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with SEQ ID NO: 2.
  • a carbohydrate transporter molecule is a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NO: 4, wherein the variant has a nucleotide sequence identity to SEQ ID NO: 4 of at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 91%,
  • USlDOCS 7494238v2 - 34 - at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with SEQ ID NO: 4.
  • a carbohydrate metabolic enzyme molecule is a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NO: 6, wherein the variant has a nucleotide sequence identity to SEQ ID NO: 6 of at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with SEQ ID NO: 6.
  • a carbohydrate metabolic enzyme molecule is a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NO: 8, wherein the variant has a nucleotide sequence identity to SEQ ID NO: 8 of at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with SEQ ID NO: 8.
  • a carbohydrate metabolic enzyme molecule is a nucleic acid variant of the nucleic acid having the sequence shown in SEQ ID NO: 10, wherein the variant has a nucleotide sequence identity to SEQ ID NO: 10 of at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% with SEQ ID NO: 10.
  • a carbohydrate transporter molecule encompasses any portion of at least about 8 consecutive nucleotides of SEQ ID NO: 2 or 4.
  • the fragment can comprise at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides of SEQ ID NO: 2 or 4. Fragments include all possible nucleotide lengths between about 8 and 100 nucleotides, for example, lengths between about 15 and 100, or between about 20 and 100.
  • a carbohydrate metabolic enzyme molecule encompasses any portion of at least about 8 consecutive nucleotides of SEQ ID NO: 6, 8, or 10.
  • the fragment can comprise at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides of SEQ ID NO: 6, 8, or 10. Fragments include all possible nucleotide lengths between about 8 and 100 nucleotides, for example, lengths between about 15 and 100, or between about 20 and 100.
  • the invention further provides for nucleic acids that are complementary to a nucleic acid encoding GLUT2, SGLTl, sucrase-isomaltase (SI), human maltase-glucoamylase, or human lactase proteins.
  • Such complementary nucleic acids can comprise nucleic acid sequences, which hybridize to a nucleic acid sequence encoding a GLUT2, SGLTl, sucrase-isomaltase (SI), maltase-glucoamylase, or lactase protein under stringent hybridization conditions.
  • Non-limiting examples of stringent hybridization conditions include temperatures above 30 0 C, above 35°C, in excess of 42°C, and/or salinity of less than about 500 mM, or less than 200 mM.
  • Hybridization conditions can be adjusted by the skilled artisan via modifying the temperature, salinity and/or the concentration of other reagents such as SDS or SSC.
  • a carbohydrate transporter molecule comprises a protein or polypeptide encoded by a carbohydrate transporter nucleic acid sequence, such as the sequence shown in SEQ ID NO: 2 or 4.
  • the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids.
  • An example of a carbohydrate transporter molecule is the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 3.
  • a carbohydrate metabolic enzyme molecule comprises a protein or polypeptide encoded by a carbohydrate metabolic enzyme nucleic acid sequence, such as the sequence shown in SEQ ID NO: 6, 8, or 10.
  • polypeptide in another embodiment, can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids.
  • a carbohydrate transporter molecule is the polypeptide having the amino acid sequence shown in SEQ ID NO: 5, 7, or 9.
  • a carbohydrate transporter molecule can be a fragment of a carbohydrate transporter protein, such as GLUT2 or SGLTl.
  • the carbohydrate transporter molecule can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 1 or 3.
  • the fragment can comprise at least about 10 amino acids, a least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, a least about 50 amino acids, at least about 60 amino acids, or at least about 75 amino acids of SEQ ID NO: 1 or 3.
  • a carbohydrate metabolic enzyme molecule can be a fragment of a carbohydrate metabolic enzyme protein, such as sucrase-isomaltase (SI), maltase-glucoamylase, or lactase.
  • a carbohydrate metabolic enzyme molecule can encompass any combination of carbohydrate metabolic enzyme protein, such as sucrase-isomaltase (SI), maltase-glucoamylase, or lactase.
  • SI sucrase-isomaltase
  • maltase-glucoamylase such as lactase
  • lactase lactase
  • the fragment can comprise at least about 10 amino acids, a least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, a least about 50 amino acids, at least about 60 amino acids, or at least about 75 amino acids of SEQ ID NO: 5, 7, or 9.
  • Fragments include all possible amino acid lengths between about 8 and 100 about amino acids, for example, lengths between about 10 and 100 amino acids, between about 15 and 100 amino acids, between about 20 and 100 amino acids, between about 35 and 100 amino acids, between about 40 and 100 amino acids, between about 50 and 100 amino acids, between about 70 and 100 amino acids, between about 75 and 100 amino acids, or between about 80 and 100 amino acids.
  • the carbohydrate transporter molecule of the invention includes variants of the human GLUT2 or SGLTl protein (having the amino acid sequence shown in SEQ ID NO: 1 and 3, respectively).
  • variants can include those having at least from about 46% to about 50% identity to SEQ ID NO: 1 or 3, or having at least from about 50.1% to about 55% identity to SEQ ID NO: 1 or 3, or having at least from about 55.1% to about 60% identity to SEQ ID NO: 1 or 3, or having from at least about 60.1% to about 65% identity to SEQ ID NO: 1 or 3, or having from about 65.1% to about 70% identity to SEQ ID NO: 1 or 3, or having at least from about 70.1% to about 75% identity to SEQ ID NO: 1 or 3, or having at least from about 75.1% to about 80% identity to SEQ ID NO: 1 or 3, or having at least from about 80.1% to about 85% identity to SEQ ID NO: 1 or 3, or having at least from about 85.1% to about 90% identity to SEQ ID
  • the carbohydrate metabolic enzyme molecule of the invention includes variants of the human sucrase-isomaltase (SI), maltase-glucoamylase, or lactase protein (having the amino acid sequence shown in SEQ ID NO: 5, 7, and 9, respectively).
  • SI human sucrase-isomaltase
  • maltase-glucoamylase or lactase protein (having the amino acid sequence shown in SEQ ID NO: 5, 7, and 9, respectively).
  • Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NO: 5, 7, or 9, or having at least from about 50.1% to about 55% identity to SEQ ID NO: 5, 7, or 9, or having at least from about 55.1% to about 60% identity to SEQ ID NO: 5, 7, or 9, or having from at least about 60.1% to about 65% identity to SEQ ID NO: 5, 7, or 9, or having from about 65.1% to about 70% identity to SEQ ID NO: 5, 7, or 9, or having at least from about 70.1% to
  • the carbohydrate transporter molecule of the invention encompasses a peptidomimetic which exhibits carbohydrate transporter activity. In another embodiment, the carbohydrate transporter molecule of the invention encompasses a peptidomimetic which exhibits carbohydrate transporter activity. In another embodiment, the carbohydrate metabolic enzyme molecule of the invention encompasses a peptidomimetic which exhibits carbohydrate metabolic enzyme activity. In another embodiment, the carbohydrate metabolic enzyme molecule of the invention encompasses a peptidomimetic which exhibits carbohydrate metabolic enzyme activity.
  • a peptidomimetic is a small protein-like chain designed to mimic a peptide that can arise from modification of an existing peptide in order to protect that molecule from enzyme degradation and increase its stability, and/or alter the molecule's properties (for example modifications that change the molecule's stability or biological activity). These modifications involve changes to the peptide that can not occur naturally (such as altered backbones and the incorporation of non- natural amino acids). Drug- like compounds may be able to be developed from existing peptides.
  • a peptidomimetic can be a peptide, partial peptide or non-peptide molecule that mimics the tertiary binding structure or activity of a selected native peptide or protein functional domain (e.g., binding motif or active site). These peptide mimetics include recombinantly or chemically modified peptides.
  • a carbohydrate transporter molecule comprising SEQ ID NO:
  • SEQ ID NO: 3 variants of each, or fragments thereof, can be modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains.
  • a carbohydrate metabolic enzyme molecule comprising SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, variants of , or fragments thereof, can be modified to produce peptide mimetics by replacement of one or more naturally occurring side chains of the 20 genetically encoded amino
  • USlDOCS 7494238v2 - 38 - acids with other side chains.
  • This can occur, for instance, with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4, 5-, 6-, to 7-membered heterocyclics.
  • proline analogs can be made in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members.
  • Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.
  • Heterocyclic groups can contain one or more nitrogen, oxygen, and/or sulphur heteroatoms.
  • groups include the furazanyl, ifuryl, imidazolidinyl imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g. 1- piperazinyl), piperidyl (e.g.
  • These heterocyclic groups can be substituted or unsubstituted.
  • the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.
  • Peptidomimetics may also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties.
  • peptidomimetics can be designed and directed to amino acid sequences encoded by a carbohydrate transporter molecule comprising SEQ ID NO: 1 or 3.
  • peptidomimetics can be designed and directed to amino acid sequences encoded by a carbohydrate metabolic enzyme molecule comprising SEQ ID NO: 5, 7, or 9.
  • peptide mimetics with the same or similar desired biological activity as the corresponding native but with more favorable activity than the peptide with respect to solubility, stability, and/or susceptibility to hydrolysis or proteolysis (see, e.g., Morgan & Gainor, Ann. Rep. Med. Chem. 24,243-252, 1989).
  • Certain peptido mimetic compounds are based upon the amino acid sequence of the peptides of the invention.
  • Peptidomimetic compounds can be synthetic compounds having a three-dimensional structure (i.e. a peptide motif) based upon the three-dimensional structure of a selected peptide.
  • the peptide motif provides the peptidomimetic compound with the desired biological activity, wherein the binding activity of the mimetic compound is not substantially reduced, and is often the same as or greater than the activity of the native peptide on which the mimetic is modeled.
  • Peptidomimetic compounds can have additional characteristics that enhance their therapeutic
  • the invention provides diagnosis methods based on monitoring a gene encoding a carbohydrate metabolic enzyme molecule (such as sucrase isomaltase, maltase glucoamylase, or lactase) or a carbohydrate transporter molecule (such as GLUT2 or SGLTl).
  • a carbohydrate metabolic enzyme molecule such as sucrase isomaltase, maltase glucoamylase, or lactase
  • a carbohydrate transporter molecule such as GLUT2 or SGLTl
  • diagnosis includes the detection, typing, monitoring, dosing, comparison, at various stages, including early, pre-symptomatic stages, and late stages, in adults, children, and unborn human children. Diagnosis can include the assessment of a predisposition or risk of development, the prognosis, or the characterization of a subject to define most appropriate treatment (pharmacogenetics).
  • the invention provides diagnostic methods to determine whether an individual is at risk of developing autism or an autism spectrum disorder (ASD), or suffers from autism or an ASD, wherein the disease reflects an alteration in the expression of a gene encoding a carbohydrate metabolic enzyme molecule (such as sucrase isomaltase, maltase glucoamylase, or lactase) or a carbohydrate transporter molecule (such as GLUT2 or SGLTl).
  • Subjects diagnosed with autism, as well as ASD can display some core symptoms in the areas of a) social interactions and relationships, b) verbal and non-verbal communication, and c) physical activity, play, physical behavior.
  • symptoms related to social interactions and relationships can include but are not limited to the inability to establish friendships with children the same age, lack of empathy, and the inability to develop nonverbal communicative skills (for example, eye- to-eye gazing, facial expressions, and body posture).
  • symptoms related to verbal and nonverbal communication comprises delay in learning to talk, inability to learn to talk, failure to initiate or maintain a conversation, failure to interpret or understand implied meaning of words, and repetitive use of language.
  • symptoms related to physical activity, play, physical behavior include, but are not limited to unusual focus on pieces or parts of an
  • USlDOCS 7494238v2 - 40 - object such as a toy, a preoccupation with certain topics, a need for routines and rituals, and stereotyped behaviors (for example, body rocking and hand flapping).
  • a method of detecting the presence of or a predisposition to autism or an autism spectrum disorder in a subject is provided.
  • the subject can be a human or a child thereof.
  • the subject can also be a human embryo, a human fetus, or an unborn human child.
  • the method can comprise detecting in a sample from the subject the presence of an alteration in the expression of a gene of a carbohydrate metabolic enzyme molecule (such as sucrase isomaltase, maltase glucoamylase, or lactase) or a carbohydrate transporter molecule (such as GLUT2 or SGLTl).
  • a carbohydrate metabolic enzyme molecule such as sucrase isomaltase, maltase glucoamylase, or lactase
  • a carbohydrate transporter molecule such as GLUT2 or SGLTl
  • the detecting comprises detecting whether there is an alteration in the gene locus encoding a carbohydrate metabolic enzyme molecule (such as sucrase isomaltase, maltase glucoamylase, or lactase) or a carbohydrate transporter molecule (such as GLUT2 or SGLTl). In a further embodiment, the detecting comprises detecting whether expression of a carbohydrate metabolic enzyme molecule (such as sucrase isomaltase, maltase glucoamylase, or lactase) or a carbohydrate transporter molecule (such as GLUT2 or SGLTl) is reduced.
  • a carbohydrate metabolic enzyme molecule such as sucrase isomaltase, maltase glucoamylase, or lactase
  • a carbohydrate transporter molecule such as GLUT2 or SGLTl
  • the detecting comprises detecting in the sample whether there is a reduction in an mRNA encoding a carbohydrate metabolic enzyme molecule or a carbohydrate transporter molecule, or a reduction in either the carbohydrate metabolic enzyme protein or a carbohydrate transporter protein, or a combination thereof.
  • the presence of such an alteration is indicative of the presence or predisposition to autism or an autism spectrum disorder.
  • the presence of an alteration in a gene encoding a carbohydrate metabolic enzyme molecule or a carbohydrate transporter molecule in the sample is detected through the genotyping of a sample, for example via gene sequencing, selective hybridization, amplification, gene expression analysis, or a combination thereof.
  • the sample can comprise blood, serum, sputum, lacrimal secretions, semen, vaginal secretions, fetal tissue, skin tissue, ileum tissue, cecum tissue, muscle tissue, amniotic fluid, or a combination thereof.
  • the invention also provides a method for treating or preventing autism or an autism spectrum disorder in a subject.
  • the method comprises (1) detecting the presence of an alteration in a carbohydrate transporter gene or a carbohydrate metabolic enzyme in a sample from the subject, where the presence of the alteration is indicative of autism or an
  • the carbohydrate transporter gene can be a GLUT2 gene or a SGLTl gene.
  • the carbohydrate metabolic enzyme gene can be a sucrase isomaltase gene, a maltase glucoamylase gene, or a lactase gene.
  • the therapeutic treatment can be a drug administration (for example, a pharmaceutical composition comprising a functional carbohydrate transporter molecule or a functional carbohydrate metabolic enzyme molecule).
  • the molecule comprises a carbohydrate transporter polypeptide comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and exhibits the function of restoring functional carbohydrate transporter expression in deficient individuals, thus restoring the capacity for carbohydrate transport.
  • the molecule comprises a carbohydrate metabolic enzyme polypeptide comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the amino acid sequence of SEQ ID NO: 5, 7, or 9, and exhibits the function of restoring functional carbohydrate metabolic enzyme expression in deficient individuals, thus restoring the capacity for carbohydrate metabolism.
  • the molecule comprises a nucleic acid encoding a carbohydrate transporter polypeptide comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleic acid sequence of SEQ ID NO: 2 or 4 and encodes a polypeptide with the function of restoring functional carbohydrate transporter expression in deficient individuals, thus restoring the capacity for carbohydrate transport.
  • the molecule comprises a nucleic acid encoding a carbohydrate metabolic enzyme polypeptide comprising at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleic acid sequence of SEQ ID NO: 6, 8, or 10, and encodes a polypeptide with the function of restoring functional carbohydrate metabolic enzyme expression in deficient individuals, thus restoring the capacity for carbohydrate metabolism.
  • the alteration can be determined at the DNA, RNA or polypeptide level of the carbohydrate transporter or carbohydrate metabolic enzyme.
  • the detection can also be determined by performing an oligonucleotide ligation assay, a confirmation based assay, a hybridization assay, a sequencing assay, an allele-specific amplification assay, a microsequencing assay, a melting curve analysis, a denaturing high performance liquid chromatography (DHPLC) assay (for example, see Jones et al, (2000) Hum Genet., 106(6):663- 8), or a combination thereof.
  • DPLC denaturing high performance liquid chromatography
  • the detection is performed by sequencing all or part of a carbohydrate transporter or carbohydrate metabolic enzyme gene or by selective hybridization or amplification of all or part of a carbohydrate transporter or carbohydrate metabolic enzyme gene.
  • a carbohydrate transporter or carbohydrate metabolic enzyme gene specific amplification can be carried out before the alteration identification step.
  • SGLTl are located
  • a carbohydrate metabolic enzyme gene locus e.g., where SI, MGAM, or LCT are located
  • Deletions can affect smaller regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions can occur as well. Rearrangement includes inversion of sequences.
  • the carbohydrate transporter gene locus alteration or carbohydrate metabolic enzyme gene locus alteration can result in amino acid substitutions, RNA splicing or processing, product instability, the creation of stop codons, frame-shift mutations, and/or truncated polypeptide production.
  • the alteration can result in the production of a carbohydrate transporter polypeptide or a carbohydrate metabolic enzyme with altered function, stability, targeting or structure.
  • the alteration can also cause a reduction in protein expression.
  • the alteration in a carbohydrate transporter gene locus can comprise a point mutation, a deletion, or an insertion in the carbohydrate transporter gene or corresponding expression product.
  • the alteration in a carbohydrate metabolic enzyme gene locus can comprise
  • USlDOCS 7494238v2 - 43 - a point mutation, a deletion, or an insertion in the carbohydrate metabolic enzyme gene or corresponding expression product.
  • the alteration can be a deletion or partial deletion of a carbohydrate transporter gene or a carbohydrate metabolic enzyme gene.
  • the alteration can be determined at the level of the DNA, RNA, or polypeptide of a carbohydrate transporter or a carbohydrate metabolic enzyme.
  • the method can comprise detecting the presence of an altered RNA expression of a carbohydrate transporter or a carbohydrate metabolic enzyme.
  • Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, or the presence of an altered quantity of RNA. These can be detected by various techniques known in the art, including by sequencing all or part of the RNA of a carbohydrate transporter or a carbohydrate metabolic enzyme, or by selective hybridization or selective amplification of all or part of the RNA.
  • the method can comprise detecting the presence of an altered polypeptide expression of a carbohydrate transporter or a carbohydrate metabolic enzyme.
  • Altered polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of carbohydrate transporter polypeptide or carbohydrate metabolic enzyme polypeptide, or the presence of an altered tissue distribution. These can be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies).
  • RNA expression, or sequence can be detected or quantify altered gene expression, which include, but are not limited to, hybridization, sequencing, amplification, and/or binding to specific ligands (such as antibodies).
  • Other suitable methods include allele-specific oligonucleotide (ASO), oligonucleotide ligation, allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, denaturing HLPC, melting curve analysis, heteroduplex analysis, RNase protection, chemical or enzymatic mismatch cleavage, ELISA, radioimmunoassays (RIA) and immuno-enzymatic assays (IEMA).
  • ASO allele-specific oligonucleotide
  • ligation for DNAs
  • SSCA single-stranded conformation analysis
  • FISH fluorescent
  • Some of these approaches are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments can then be sequenced to
  • USlDOCS 7494238v2 - 44 - confirm the alteration.
  • Some other approaches are based on specific hybridization between nucleic acids from the subject and a probe specific for wild type or altered gene or RNA.
  • the probe can be in suspension or immobilized on a substrate.
  • the probe can be labeled to facilitate detection of hybrids.
  • Some of these approaches are suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand specific for the polypeptide, for example, the use of a specific antibody.
  • Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on the complete gene or on specific domains thereof, such as those known or suspected to carry deleterious mutations or other alterations.
  • Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction.
  • Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele-specific PCR, or PCR-SSCP. Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction.
  • nucleic acid primers useful for amplifying sequences from the gene or locus of a carbohydrate transporter (such as GLUT2 or SGLTl) or a carbohydrate metabolic enzyme (such as SI, MGAM, or LCT) are able to specifically hybridize with a portion of the gene locus that flanks a target region of the locus, wherein the target region is altered in certain subjects having autism or an autism spectrum disorder.
  • amplification comprises using forward and reverse RT-PCR primers comprising nucleotide sequences of SEQ ID NOS: 26, 27, 29, 30, 32, 33, 35, 36, 38, or 39.
  • the invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of a coding sequence (e.g., gene or RNA)of a carbohydrate transporter (such as GLUT2 or SGLTl) or a carbohydrate metabolic enzyme (such as SI, MGAM, or LCT) that is altered in certain subjects having autism or an coding sequence (e.g., gene or RNA)of a carbohydrate transporter (such as GLUT2 or SGLTl) or a carbohydrate metabolic enzyme (such as SI, MGAM, or LCT) that is altered in certain subjects having autism or an coding sequence (e.g., gene or RNA)of a carbohydrate transporter (such as GLUT2 or SGLTl) or a carbohydrate metabolic enzyme (such as SI, MGAM, or LCT) that is altered in certain subjects having autism or an coding sequence (e.g., gene or RNA)of a carbohydrate transporter (such as
  • Primers of the invention can thus be specific for altered sequences in a gene or RNA of a carbohydrate transporter or a carbohydrate metabolic enzyme. By using such primers, the detection of an amplification product indicates the presence of an alteration in the gene or the absence of such gene.
  • Examples of primers of this invention can be single-stranded nucleic acid molecules of about 5 to 60 nucleotides in length, or about 8 to about 25 nucleotides in length.
  • the sequence can be derived directly from the sequence of the carbohydrate transporter or the carbohydrate metabolic enzyme gene (e.g., GLUT2 or SGLTl, and SI, MGAM, or LCT, respectively).
  • the primer can be an isolated nucleic acid comprising a nucleotide sequence of SEQ ID NOS: 26, 27, 29, 30, 32, 33, 35, 36, 38, or 39.
  • a nucleic acid primer or a pair of nucleic acid primers as described above can be used in a method for detecting the presence of or a predisposition to autism or an autism spectrum disorder in a subject.
  • Amplification methods include, e.g., polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560, 1989; Landegren, Science 241:1077, 1988; Barringer, Gene 89:117, 1990); transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad. Sci.
  • LCR ligase chain reaction
  • Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s).
  • a detection technique involves the use of a nucleic acid probe specific for wild type or altered gene or RNA, followed by the detection of the presence of
  • USlDOCS 7494238v2 - 46 - a hybrid can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies).
  • the probe can be labeled to facilitate detection of hybrids.
  • the probe according to the invention can comprise a nucleic acid having SEQ ID NOS: 28, 31, 34, 37, or 40.
  • a sample from the subject can be contacted with a nucleic acid probe specific for a wild type carbohydrate transporter or carbohydrate metabolic enzyme gene or an altered carbohydrate transporter or carbohydrate metabolic enzyme gene, and the formation of a hybrid can be subsequently assessed.
  • the method comprises contacting simultaneously the sample with a set of probes that are specific, respectively, for the wild type carbohydrate transporter or carbohydrate metabolic enzyme gene and for various altered forms thereof.
  • a set of probes that are specific, respectively, for the wild type carbohydrate transporter or carbohydrate metabolic enzyme gene and for various altered forms thereof.
  • a probe can be a polynucleotide sequence which is complementary to and specifically hybridizes with a, or a target portion of a, carbohydrate transporter or carbohydrate metabolic enzyme gene or RNA, and that is suitable for detecting polynucleotide polymorphisms associated with alleles of such, which predispose to or are associated with autism or an autism spectrum disorder.
  • Useful probes are those that are complementary to the carbohydrate transporter or carbohydrate metabolic enzyme gene, RNA, or target portion thereof. Probes can comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance between 10 and 800, between 15 and 700, or between 20 and 500.
  • a useful probe of the invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridize to a region of a UGT2B17 gene or RNA that carries an alteration.
  • the sequence of the probes can be derived from the sequences of the carbohydrate transporter or carbohydrate metabolic enzyme genes provided herein. Nucleotide substitutions can be performed, as well as chemical modifications of the probe. Such chemical modifications can be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Some examples of labels include, without limitation, radioactivity, fluorescence, luminescence, and enzymatic labeling.
  • alteration in a carbohydrate transporter or carbohydrate metabolic enzyme gene locus or in carbohydrate transporter or carbohydrate metabolic enzyme expression can also be detected by screening for alteration(s) in corresponding polypeptide sequence or expression levels.
  • Different types of ligands can be used, such as specific antibodies.
  • the sample is contacted with an antibody specific for a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide and the formation of an immune complex is subsequently determined.
  • Various methods for detecting an immune complex can be used, such as ELISA, radioimmunoassays (RIA) and immuno- enzymatic assays (IEMA).
  • an antibody can be a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab'2, or CDR regions. Derivatives include single-chain antibodies, humanized antibodies, or poly-functional antibodies.
  • An antibody specific for a carbohydrate transporter or a carbohydrate metabolic enzyme polypeptide can be an antibody that selectively binds a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide, respectively, namely, an antibody raised against a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide or an epitope- containing fragment thereof.
  • the method comprises contacting a sample from the subject with an antibody specific for a wild type or an altered form of a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide, and determining the presence of an immune complex.
  • the sample can be contacted to a support coated with antibody specific for the wild type or altered form of a carbohydrate transporter or
  • the sample can be contacted simultaneously, or in parallel, or sequentially, with various antibodies specific for different forms of a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide, such as a wild type and various altered forms thereof.
  • the invention also provides for a diagnostic kit comprising products and reagents for detecting in a sample from a subject the presence of an alteration in a carbohydrate transporter gene (e.g., GLUT2 or SGLTl) or a carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or LCT), or a carbohydrate transporter polypeptide or carbohydrate metabolic enzyme polypeptide; alteration in the expression of a carbohydrate transporter gene (e.g., GLUT2 or SGLTl) or carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or LCT), or a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide; and/or an alteration in carbohydrate transporter or carbohydrate metabolic enzyme activity.
  • a carbohydrate transporter gene e.g., GLUT2 or SGLTl
  • a carbohydrate metabolic enzyme gene e.g., SI, MGAM, or LCT
  • the kit can be useful for determining whether a sample from a subject exhibits reduced carbohydrate transporter or carbohydrate metabolic enzyme expression or exhibits a gene deletion of a carbohydrate transporter (e.g., GLUT2 or SGLTl) or carbohydrate metabolic enzyme (e.g., SI, MGAM, or LCT).
  • a carbohydrate transporter e.g., GLUT2 or SGLTl
  • carbohydrate metabolic enzyme e.g., SI, MGAM, or LCT
  • the diagnostic kit according to the present invention comprises any primer, any pair of primers, any nucleic acid probe and/or any ligand, (for example, an antibody directed to a carbohydrate transporter or carbohydrate metabolic enzyme).
  • the diagnostic kit according to the present invention can further comprise reagents and/or protocols for performing a hybridization, amplification or antigen-antibody immune reaction.
  • the kit can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from a carbohydrate transporter (e.g., GLUT2 or SGLTl) or carbohydrate metabolic enzyme (e.g., SI, MGAM, or LCT).
  • a carbohydrate transporter e.g., GLUT2 or SGLTl
  • carbohydrate metabolic enzyme e.g., SI, MGAM, or LCT
  • the primer can comprise a nucleotide sequence of SEQ ID NOS: 26, 27, 29, 30, 32, 33, 35, 36, 38, or 39.
  • the diagnosis methods can be performed in vitro, ex vivo, or in vivo. These methods utilize a sample from the subject in order to assess the status of a carbohydrate transporter gene locus or a carbohydrate metabolic enzyme gene locus.
  • the sample can be any biological sample derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include, but are not limited to, fluids, tissues, cell samples, organs, or tissue biopsies. Non-limiting examples of samples include blood, plasma, saliva, urine, or
  • USlDOCS 7494238v2 - 49 - seminal fluid Pre-natal diagnosis can also be performed by testing fetal cells or placental cells, for instance. Screening of parental samples can also be used to determine risk/likelihood of offspring possessing the germline mutation.
  • the sample can be collected according to conventional techniques and used directly for diagnosis or stored.
  • the sample can be treated prior to performing the method, in order to render or improve availability of nucleic acids or polypeptides for testing. Treatments include, for instance, lysis (e.g., mechanical, physical, or chemical), centrifugation.
  • the nucleic acids and/or polypeptides can be pre-purified or enriched by conventional techniques, and/or reduced in complexity.
  • Nucleic acids and polypeptides can also be treated with enzymes or other chemical or physical treatments to produce fragments thereof.
  • the sample is contacted with reagents, such as probes, primers, or ligands, in order to assess the presence of an altered carbohydrate transporter gene locus or carbohydrate metabolic enzyme gene locus.
  • Contacting can be performed in any suitable device, such as a plate, tube, well, or glass.
  • the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array.
  • the substrate can be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, or polymers.
  • the substrate can be of various forms and sizes, such as a slide, a membrane, a bead, a column, or a gel.
  • the contacting can be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.
  • Identifying an altered polypeptide, RNA or DNA of a carbohydrate transporter (e.g., GLUT2 or SGLTl) or a carbohydrate metabolic enzyme (e.g., SI, MGAM, or LCT) in the sample is indicative of the presence of an altered carbohydrate transporter or carbohydrate metabolic enzyme gene in the subject, which can be correlated to the presence, predisposition or stage of progression of autism or an autism spectrum disorder.
  • an individual having a germ line mutation in a carbohydrate transporter gene e.g., GLUT 2 or SGLTl
  • a carbohydrate metabolic enzyme gene e.g., SI, MGAM, or LCT
  • the determination of the presence of an altered gene locus in a subject also allows the design of appropriate therapeutic intervention, which is more effective and customized. Also, this determination at the pre-symptomatic level allows a preventive regimen to be applied.
  • An aspect of the invention provides for a new PCR strategy for the identification, quantitation, and taxonomic classification of Sutterella bacterial colonization from biological samples.
  • intestinal biopsies of children with autism accompanied by gastrointestinal (GI) complaints showed highly significant elevation of intestinal levels of Sutterella bacteria.
  • Sutterella sp. sequences have been identified in intestinal biopsies and fecal samples from individuals with Crohn's disease and ulcerative colitis (A2, A3). Sutterella sp. have also been found in canine faeces and the cecal microbiota of domestic and wild turkeys (A4, A5). However, little is known about the pathogenic potential of Sutterella sp. According to the Sutterella s 1 /?.
  • Sutterella detection can be achieved in a mammal, such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, a turkey, or a human.
  • Sutterella bacterial infections have been associated with ASD in addition to Crohn's disease and ulcerative colitis.
  • Bacterial infections are also associated with various intestinal diseases, such as irritable bowel syndrome (IBS).
  • IBS irritable bowel syndrome
  • Over 40 million people in the U.S. suffer from irritable bowel syndrome (IBS), a type of inflammatory bowel disease.
  • IBS though not fatal, has a huge impact on quality-of-life. After the common cold, IBS is the second most common reason for missed work and is estimated to generate $30B in healthcare costs.
  • Few simple molecular diagnostic tests for IBS/IBD are presently available. Diagnosis usually relies upon symptom analysis and/or invasive colonoscopy procedures.
  • the IBD/IBS diagnostics market has significant growth potential.
  • An aspect of the invention provides for a PCR assay that allows for rapid identification, quantification, classification, and diagnosis of Sutterella sp. in biological or industrial samples. This would allow for specific therapies to be implemented in subjects in need (e.g., ASD patients, IB patients, intestinal disease patients, etc.) following identification of Sutterella in infections. Directed administration of antimicrobial agents (e.g., antibiotics) that limit the growth of Sutterella could be fascilitated rapidly following identification of Sutterella species.
  • An antibiotic refers to any compound known to one of ordinary skill in the art that will inhibit the growth of, or kill, bacteria.
  • antibiotics include lincosamides (clindomycin); chloramphenicols; tetracyclines (such as Tetracycline, Chlortetracycline, Demeclocycline, Methacycline, Doxycycline, Minocycline); aminoglycosides (such as Gentamicin, Tobramycin, Netilmicin, Amikacin, Kanamycin, Streptomycin, Neomycin); beta-lactams (such as penicillins, cephalosporins, Imipenem, Aztreonam); vancomycins; bacitracins; macrolides (erythromycins), amphotericins; sulfonamides (such as Sulfanilamide, Sulfamethoxazole, Sulfacetamide, Sulfadiazine, Sulfisoxazole, Sulfacytine, Sulfadoxine, Mafenide, p-Aminobenzoic Acid, Trimethoprim
  • antibiotics can be obtained commercially, e.g., from Daiichi Sankyo, Inc. (Parsipanny, NJ), Merck (Whitehouse Station, NJ), Pfizer (New York, NY), Glaxo Smith Kline (Research Triangle Park, NC), Johnson & Johnson (New Brunswick, NJ), AstraZeneca (Wilmington, DE), Novartis (East Hanover, NJ), and Sanofi-Aventis (Bridgewater, NJ).
  • the antibiotic used will depend on the type of bacterial infection.
  • the invention provides for a method of detecting Sutterella sp. DNA from biological or industrial sources, e.g. intestinal tissue, feces, blood, or skin.
  • the invention provides for Sutterella diagnostics to detect Sutterella sp. in samples from children with autism as well as patients with intestinal disease, e.g. irritable bowel syndrome (IBS).
  • IBS irritable bowel syndrome
  • the invention provides for PCR-based methods of
  • primers having SEQ ID NOS: 11, 12, 15, or 16 can be used for detecting Sutterella sp. DNA.
  • SEQ ID NOS: 17 and 18 can also be used for detecting Sutterella sp. DNA.
  • primers having SEQ ID NOS: 11, 12, and 15-18 may be used to assess the presence or absence of Sutterella species.
  • degenerate bases may be used to increase coverage of Sutterella species (for example, where S- can be a G nucleotide and/or a C nucleotide; where Y can be a C nucleotide and/or T nucleotide; where R can be an A nucleotide and/or G nucleotide; where Wean be an A nucleotide and/or T nucleotide; where H can be an A nucleotide and/or T nucleotide and/or C nucleotide; where B_ can be a T nucleotide, C nucleotide, or G nucleotide; where V can be an A nucleotide, G nucleotide, or C nucleotide; where D can be an A nucleotide, G nucleotide, or T nucleotide; where K can be a G nucleotide or T nucleotide
  • USlDOCS 7494238v2 - 53 - detection and quantitation The reverse complement of SEQ ID NOS: 11, 12, or 15-19 can also be used to detect the opposite DNA strand of Sutterella species 16S rRNA genes.
  • the invention can be used to detect Sutterella sp. 16S rRNA sequences in small amounts of DNA from any biological or industrial source. These sources include, but are not limited to human or animal intestinal tissue, feces, blood, or skin (swabs or tissue). Based on these findings, the invention can be used to detect, quantitate, and classify Sutterella sp. in biological samples from children with Autism. In one embodiment, the invention can be used to detect Sutterella sp. in biological samples from individuals with various forms of intestinal disease. Intestinal diseases include, but are not limited to, Crohn's disease and Ulcerative colitis. In one embodiment, detection of Sutterella sp. can occur in biological samples from any undiagnosed infection below or above the diaphragm.
  • the invention will allow for large cohort investigations of Sutterella sp. in the aforementioned, and as yet to be determined, diseases in order to establish an association between Sutterella sp. and disease manifestation.
  • the presence and quantity of Sutterella sp. in intestinal tissues can be investigated following any number of experimental manipulations.
  • Experimental manipulations include, but are not limited to, responses to chemicals (i.e. antibiotics), changes in diet, pathogen exposure (i.e. pathogenic viruses, bacteria, fungi), or probiotic usage.
  • the rapid identification of Sutterella sp. in human and animal samples facilitated by this invention may lead to rapid diagnosis and directed antimicrobial treatment of infections caused by Sutterella sp.
  • the invention encompasses an altered or mutated genes of a carbohydrate transporter or carbohydrate metabolic enzyme, or a fragment thereof.
  • the invention also encompasses nucleic acid molecules encoding an altered or mutated polypeptide of s carbohydrate transporter or carbohydrate metabolic enzyme, or a fragment thereof.
  • the alteration or mutation of the nucleotide or amino acid sequence modifies the carbohydrate transporter or carbohydrate metabolic enzyme activity, respectively.
  • the invention provides for a vector that comprises a nucleic acid encoding a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide (for example, a nucleic acid comprising SEQ ID NO: 2 or 4, and a nucleic acid comprising SEQ ID NO: 6, 8, or 10, respectively) or mutant thereof.
  • a nucleic acid encoding a carbohydrate transporter or carbohydrate metabolic enzyme polypeptide (for example, a nucleic acid comprising SEQ ID NO: 2 or 4, and a nucleic acid comprising SEQ ID NO: 6, 8, or 10, respectively) or mutant thereof.
  • USlDOCS 7494238v2 - 54 - can be a cloning vector or an expression vector, i.e., a vector comprising regulatory sequences causing resulting in the expression of carbohydrate transporter or carbohydrate metabolic enzyme polypeptides from the vector in a competent host cell.
  • These vectors can be used to express polypeptides, or mutants thereof, of carbohydrate transporters or carbohydrate metabolic enzymes in vitro, ex vivo, or in vivo, to create transgenic or Knock-Out non-human animals, to amplify the nucleic acids, or to express antisense RNAs.
  • nucleic acids used to practice the invention can be produced or isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly.
  • Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity.
  • Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams, J. Am. Chem. Soc.
  • the invention provides oligonucleotides comprising sequences of the invention, e.g., subsequences of the exemplary sequences of the invention.
  • Oligonucleotides can include, e.g., single stranded poly-deoxynucleotides or two complementary polydeoxynucleotide strands which can be chemically synthesized.
  • nucleic acids such as, subcloning, labeling probes (for example, random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, and hybridization are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2 ND ED.), VOIS. 1-3, Cold Spring Harbor Laboratory, 1989; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York, 1997; LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:
  • Nucleic acids, vectors, or polypeptides can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, for example, analytical biochemical methods such as radiography, electrophoresis, NMR, spectrophotometry, capillary electrophoresis, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and hyperdiffusion chromatography; various immunological methods, such as immuno-electrophoresis, Southern analysis, Northern analysis, dot-blot analysis, fluid or gel precipitation reactions, immunodiffusion, quadrature radioimmunoassay (RIAs), enzyme- linked immunosorbent assays (ELISAs), immuno-fluorescent assays, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.
  • analytical biochemical methods such as radiography, electrophoresis, NMR,
  • Obtaining and manipulating nucleic acids used to practice the methods of the invention can be done by cloning from genomic samples, and, if desired, screening and re- cloning inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
  • Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld, Nat. Genet.
  • MACs mammalian artificial chromosomes
  • yeast artificial chromosomes YAC
  • bacterial artificial chromosomes BAC
  • Pl artificial chromosomes see, e.g., Woon, Genomics 50:306-316, 1998
  • Pl -derived vectors PACs
  • cosmids recombinant viruses, phages or plasmids
  • the vectors of this invention can comprise a coding sequence for a carbohydrate transporter molecule or a carbohydrate metabolic enzyme molecule that is operably linked to regulatory sequences, e.g., a promoter, or a polyA tail. Operably linked indicates that the coding and regulatory sequences are functionally associated so that the regulatory sequences cause expression (e.g., transcription) of the coding sequences.
  • the vectors can further comprise one or several origins of replication and/or selectable markers.
  • the promoter region can be homologous or heterologous with respect to the coding sequence, and can provide for ubiquitous, constitutive, regulated and/or tissue specific expression, in any appropriate host cell, including for in vivo use.
  • promoters include bacterial promoters (T7, pTAC, Trp promoter), viral promoters (LTR, TK, CMV-IE), mammalian gene promoters (albumin, PGK), etc.
  • the vector can be a plasmid, a virus, a cosmid, a phage, a BAC, a YAC.
  • Plasmid vectors can be prepared from commercially available vectors such as pBluescript, pUC, or pBR,.
  • Viral vectors can be produced from baculoviruses, retroviruses, adenoviruses, or AAVs, according to recombinant DNA techniques known in the art.
  • a recombinant virus can encode a polypeptide of a carbohydrate transporter or carbohydrate metabolic enzyme of the invention.
  • the recombinant virus is useful if replication-defective, for example, if selected from El- and/or E4-defective adenoviruses, Gag-, pol- and/or env-defective retroviruses and Rep- and/or Cap-defective AAVs.
  • Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, or 293 cells. Detailed protocols for producing such replication-defective recombinant viruses can be found for instance in WO95/14785, WO96/22378, U.S.
  • the invention provides for a recombinant host cell comprising a recombinant carbohydrate transporter gene (e.g., GLUT2 or SGLTl) or a carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or LCT), or a recombinant vector as described herein.
  • Suitable host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, or plant cells). Specific examples include E.
  • the invention provides a method for producing a recombinant host cell expressing a polypeptide of a carbohydrate transporter or carbohydrate metabolic enzyme.
  • the method can entail (a) introducing in vitro or ex vivo into a competent host cell a recombinant nucleic acid or a vector as described herein, (b) culturing in vitro or ex vivo the recombinant host cells obtained, and (c) optionally, selecting the cells which express the polypeptide of a carbohydrate transporter or carbohydrate metabolic enzyme.
  • USlDOCS 7494238v2 - 57 - recombinant host cells can be used for the production of carbohydrate transporter or carbohydrate metabolic enzyme polypeptides, as well as for screening of active molecules, as described below. Such cells can also be used as a model system to study autism. These cells can be maintained in suitable culture media, such as HAM, DMEM, or RPMI, in any appropriate culture device (plate, flask, dish, tube, or pouch).
  • suitable culture media such as HAM, DMEM, or RPMI
  • a carbohydrate transporter molecule e.g., GLUT2 or SGLTl
  • carbohydrate metabolic enzyme molecule e.g., SI, MGAM, or LCT
  • a carbohydrate transporter or carbohydrate metabolic enzyme molecule of the invention can be administered once or twice daily to a subject in need thereof for a period of from about two to about twenty-eight days, or
  • USlDOCS 7494238v2 - 58 - from about seven to about ten days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.
  • the carbohydrate transporter or carbohydrate metabolic enzyme molecule of the invention can be co-administrated with another therapeutic, such as an anti-depressant, an antipsychotic, a benzodiazepine drug, or a combination thereof.
  • the effective amount of the carbohydrate transporter or carbohydrate metabolic enzyme molecule administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.
  • the carbohydrate transporter or carbohydrate metabolic enzyme molecules of the invention can be administered to a subject by any means suitable for delivering the carbohydrate transporter or carbohydrate metabolic enzyme molecules to cells of the subject, such as ileum cell or cecum cells.
  • carbohydrate transporter or carbohydrate metabolic enzyme molecules can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.
  • compositions of this invention can be formulated and administered to reduce the symptoms associated with autism or an ASD by any means that produces contact of the active ingredient with the agent's site of action in the body of an animal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration.
  • USlDOCS 7494238v2 - 59 - and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (1985), the entire disclosure of which is herein incorporated by reference.
  • an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.
  • compositions of the invention can comprise a carbohydrate transporter or carbohydrate metabolic enzyme molecule (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier.
  • the pharmaceutical formulations of the invention can also comprise the carbohydrate transporter or carbohydrate metabolic enzyme molecules of the invention which are encapsulated by liposomes and a pharmaceutically-acceptable carrier.
  • Useful pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, or hyaluronic acid.
  • compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTP A-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • solid pharmaceutical compositions of the invention conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of
  • Solid formulations can be used for enteral (oral) administration. They can be formulated as, e.g., pills, tablets, powders or capsules.
  • conventional nontoxic solid carriers can be used which include, e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, or magnesium carbonate.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10% to 95% of active ingredient (e.g., peptide).
  • a non- solid formulation can also be used for enteral administration.
  • the carrier can be selected from various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, or sesame oil.
  • suitable pharmaceutical excipients include e.g., starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.
  • Nucleic acids, peptides, or polypeptides of the invention when administered orally, can be protected from digestion. This can be accomplished either by complexing the nucleic acid, peptide or polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the nucleic acid, peptide or polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are well known in the art, see, e.g., Fix, Pharm Res. 13: 1760-1764, 1996; Samanen, J. Pharm. Pharmacol. 48: 119- 135, 1996; U.S. Pat. No.
  • the carbohydrate transporter molecule e.g., GLUT2 or SGLTl
  • carbohydrate metabolic enzyme molecule e.g., SI, MGAM, or LCT
  • the therapeutic compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or
  • USlDOCS 7494238v2 - 61 - hydroxypropyl methylcellulose fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • the tablets can be coated by methods well known in the art.
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils
  • preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid.
  • the preparations can
  • Preparations for oral administration can be suitably formulated to give controlled release of the active agent.
  • the therapeutic compositions can take the form of tablets or lozenges formulated in a conventional manner.
  • the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflate or can be formulated
  • the therapeutic compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra- tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
  • the carbohydrate transporter or carbohydrate metabolic enzyme molecules of the invention can be administered by injection, in
  • the therapeutic compositions can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the therapeutic compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents can be used to facilitate permeation.
  • Transmucosal administration can be through nasal sprays or using suppositories.
  • the compositions of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • a wash solution can be used locally to treat an injury or inflammation to accelerate healing.
  • the therapeutic compositions are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • a composition of the present invention can also be formulated as a sustained and/or timed release formulation.
  • sustained and/or timed release formulations can be made by
  • USlDOCS 7494238v2 - 63 - sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference.
  • compositions of the present invention can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions.
  • Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention.
  • Single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gel- caps, caplets, or powders, that are adapted for sustained release are encompassed by the present invention.
  • the carbohydrate transporter or carbohydrate metabolic enzyme molecules can be administered to the subject either as RNA, in conjunction with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences which expresses the gene product.
  • a delivery reagent e.g., a recombinant plasmid or viral vector
  • Suitable delivery reagents for administration of the carbohydrate transporter or carbohydrate metabolic enzyme molecules include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • the dosage administered can be a therapeutically effective amount of the composition sufficient to result in amelioration of symptoms of autism or an autism spectrum disorder in a subject, and can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.
  • an effective enzyme unit of amount of SI, MGAM, and/or LCT can be administered to a subject in need thereof.
  • the enzyme unit (U) is a unit for the amount of a particular enzyme.
  • One U is defined as the amount of the enzyme that catalyzes the conversion of 1 micro mole of substrate per minute.
  • the therapeutically effective amount of the administered carbohydrate enzyme is at least about 1 U, at least about 10 U, at least about 20 U, at least about 25 U, at least about 50 U, at least about 100 U, at least about 150 U, at least about 200 U, at least about 250 U, at least about 300 U, at least about 350 U, at least about 400 U, at least about 450 U, at least about 500 U, at least about 550 U, at least about 600 U, at least about 650 U, at least about 700 U, at least about 750 U, at least about 800 U, at least about 850 U, at least about 900 U, at least about 950 U, at least about 1000 U, at least about 1250 U, at least about 1500 U, at least about 1750 U, at least about 2000 U, at least about 2250 U, at least about 2500 U, at least about 2750 U, at least about 3000 U, at least about 3
  • the effective amount of the administered carboydrate transporter molecule is at least about 0.01 ⁇ g/kg body weight, at least about 0.025 ⁇ g/kg body weight, at least about 0.05 ⁇ g/kg body weight, at least about 0.075 ⁇ g/kg body weight, at least about 0.1 ⁇ g/kg body weight, at least about 0.25 ⁇ g/kg body weight, at least about 0.5 ⁇ g/kg body weight, at least about 0.75 ⁇ g/kg body weight, at least about 1 ⁇ g/kg body weight, at least about 5 ⁇ g/kg body weight, at least about 10 ⁇ g/kg body weight, at least about 25 ⁇ g/kg body weight, at least about 50 ⁇ g/kg body weight, at least about 75 ⁇ g/kg body weight, at least about 100 ⁇ g/kg body weight, at least about 150 ⁇ g/kg body weight, at least about 200 ⁇ g/kg body weight, at least about 0.01 ⁇ g/kg body weight, at least about 0.025 ⁇ g/kg body
  • Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or
  • USlDOCS 7494238v2 - 65 - experimental animals e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Therapeutic agents that exhibit large therapeutic indices are useful.
  • Therapeutic compositions that exhibit some toxic side effects can be used.
  • a therapeutically effective dose of carbohydrate transporter or carbohydrate metabolic enzyme molecules can depend upon a number of factors known to those or ordinary skill in the art.
  • the dose(s) of the carbohydrate transporter or carbohydrate metabolic enzyme molecules can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the carbohydrate transporter or carbohydrate metabolic enzyme molecules to have upon the nucleic acid or polypeptide of the invention. These amounts can be readily determined by a skilled artisan.
  • the invention provides methods for treating or preventing autism or an autism spectrum disorder in a subject.
  • the method can comprise administering to the subject a functional (e.g., wild-type) carbohydrate transporter molecule (e.g., GLUT2 or SGLTl) or carbohydrate metabolic enzyme molecule (e.g., SI, MGAM, or LCT), which can be a polypeptide or a nucleic acid.
  • a functional e.g., wild-type carbohydrate transporter molecule
  • carbohydrate metabolic enzyme molecule e.g., SI, MGAM, or LCT
  • Various approaches can be carried out to restore the carbohydrate transporter or carbohydrate metabolic enzyme activity or function in a subject, such as those carrying an altered gene locus comprising a carbohydrate transporter gene (e.g., GLUT2 or SGLTl) or a carbohydrate metabolic enzyme gene (e.g., SI, MGAM, or LCT).
  • a carbohydrate transporter gene e.g., GLUT2 or SGLTl
  • a carbohydrate metabolic enzyme gene e.g., SI, MGAM, or LCT.
  • Supplying wild-type function of the carbohydrate transporter or carbohydrate metabolic enzyme to such subjects can suppress phenotypic expression of autism or an autism spectrum disorders in a pathological cell or organism.
  • Increasing carbohydrate transporter or carbohydrate metabolic enzyme activity can be accomplished through gene or protein therapy as discussed later herein.
  • a nucleic acid encoding a carbohydrate transporter or carbohydrate metabolic enzyme or a functional part thereof can be introduced into the cells of a subject in one embodiment of the invention.
  • the wild-type carbohydrate transporter gene or carbohydrate metabolic enzyme gene (or a functional part thereof) can also be introduced into the cells of the subject in need thereof using a vector as described herein.
  • the vector can be a viral vector or a plasmid.
  • the gene can also be introduced as naked DNA.
  • the gene can be provided so as to integrate into the genome of the recipient host cells, or to remain extra-chromosomal. Integration can occur randomly or at precisely defined sites, such as through homologous recombination.
  • a functional copy of the carbohydrate transporter gene or a carbohydrate metabolic enzyme gene can be inserted in replacement of an altered version in a cell, through homologous recombination.
  • Further techniques include gene gun, liposome - mediated transfection, or cationic lipid-mediated transfection.
  • Gene therapy can be accomplished by direct gene injection, or by administering ex vivo prepared genetically modified cells expressing a functional polypeptide.
  • nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).
  • viral vectors e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus
  • physical DNA transfer methods e.g., liposomes or chemical treatments.
  • Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (see, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)).
  • a nucleic acid or a gene encoding a polypeptide of the invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).
  • Cells may also be cultured ex vivo in the presence of therapeutic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
  • Nucleic acids can be inserted into vectors and used as gene therapy vectors.
  • a number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40
  • Non- limiting examples of in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993), incorporated entirely by reference).
  • viral typically retroviral
  • viral coat protein-liposome mediated transfection Dzau et al., Trends in Biotechnology 11:205-210 (1993), incorporated entirely by reference.
  • naked DNA vaccines are generally known in the art; see Brower, Nature Biotechnology, 16: 1304-1305 (1998), which is incorporated by reference in its entirety.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No.
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • Protein replacement therapy can increase the amount of protein by exogenously introducing wild-type or biologically functional protein by way of infusion.
  • a replacement polypeptide can be synthesized according to known chemical techniques or may be produced and purified via known molecular biological techniques. Protein replacement therapy has been developed for various disorders.
  • a wild-type protein can be purified from a recombinant cellular expression system (e.g., mammalian cells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.; U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No.
  • a polypeptide encoded by a carbohydrate transporter gene e.g., GLUT2 or SGLTl
  • a carbohydrate metabolic enzyme gene for example, SI, MGAM, or LCT
  • the polypeptide may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see is Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71 :105 (1989)).
  • a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in
  • Example 1 Identification of carbohydrate transporters and carbohydrate metabolic enzymes as biomarkers in a subset of Autism Spectrum Disorders (ASD)
  • ASSD Autism Spectrum Disorders
  • Gastrointestinal disturbances complicate clinical management in some children with autism.
  • Reports of ileo-colonic lymphoid nodular hyperplasia and deficiencies in disaccharidase enzymatic activity led us to survey intestinal gene expression and microflora in children with autism and gastrointestinal disease (AUT-GI) or gastrointestinal disease alone (Control-GI).
  • AUT-GI ileal transcripts for the disaccharidases sucrase isomaltase, maltase glucoamylase, and lactase, and the monosaccharide transporters, sodium-dependent glucose co- transporter, and glucose transporter 2 were significantly decreased.
  • Deficiencies in intestinal disaccharidase and/or glucoamylase activity are reported in over half of autistic children with gastrointestinal disturbances (AUT-GI) (Horvath et al., 1999).
  • AUT-GI gastrointestinal disturbances
  • Tables 5A-C are summary tables for gene expression and bacterial assays. Increases or decreases in AUT-GI children in both gene expression and bacterial parameters were determined for each individual based on the levels of each parameter in the Control-GI group. The values for a given parameter in the AUT-GI children that exceeded the 75* (arrow pointing up) percentile or were below the 25* percentile (arrow pointing down) for the corresponding parameter in the Control-GI children were scored as an increase or decrease, respectively. Values that were also above the 90 th or below the 10 th percentiles of Control-GI children are indicated by double arrows.
  • Table 5A Summary tables for gene expression and bacterial assays.
  • SGLTl located on the luminal membrane of enterocytes, is responsible for the active transport of glucose and galactose from the intestinal lumen into enterocytes.
  • GLUT2 transports glucose, galactose and fructose across the basolateral membrane into the circulation and may also translocate to the apical membrane (Kellett et al., 2008).
  • Realtime PCR revealed a decrease in SGLTl mRNA (FIG.
  • Betaproteo AUT -0 63* -0 60* -0.56 * -0.44f -0.60* -0.45t -0.70" Heum Control -0 75f -0 82 * -0.54 -0.61 -0.57 -0.39 -0.61
  • Betaprote ⁇ AUT -Q.S6 * -0.58 * -0.64 * -0.511 -D.er -Q.61 " -0.85"" Cecum Control -0,43 -0,43 0.14 -0,0Q 0.14 0.14 ⁇ Q.D0
  • CDX2 a member of the caudal-related homeobox transcription factor family, regulates expression of SI, LCT, GLUT2, SGLTl and villin (Suh and Traber, 1996; Troelsen et al, 1997; Uesaka et al, 2004; Balakrishnan et al, 2008; and Yamamichi et al, 2009).
  • ileal and cecal biopsies from AUT-GI and Control-GI children were analyzed by bacterial 16S rRNA gene pyrosquencing (See also Methods and FIGS. 23A-23D).
  • Bacteroidetes and Firmicutes were the most prevalent taxa present in the ileal and cecal tissues of AUT-GI children, with the exception of the ileal samples of patients 2, 15, and 19 and cecal samples of patient 15, wherein levels of Proteobacteria exceeded those of Firmicutes and/or Bacteroidetes (FIGS. 17A-B and FIGS. 24A-B).
  • FIGS. 17A-B and FIGS. 24A-24D Other phyla identified at lower levels included Verrucomicrobia, Actinobacteria, Fusobacteria, Lentisphaerae, TM7, and Cyanobacteria, as well as unclassified bacterial sequences (FIGS. 17A-B and FIGS. 24A-24D).
  • Real-time PCR using Bacteroidete-specific primers confirmed decreases in Bacteroidetes in AUT-GI ilea (FIG.
  • OTU Operaational Taxonomic Unit
  • Lachnospiraceae members of the genus Lachnopsiraceae Incertae Sedis, Unclassified Lachnospiraceae, and to a lesser extent Bryantella (cecum only) contributed to the overall trend toward increased Clostridia in ASD-GI patients (FIGS. 28 A-B).
  • Betaproteobacteria were above the 75* percentile of Control-GI children in 53.3% of AUT-GI ilea and 66.7% of AUT-GI ceca (Table 5B).
  • Family-level analysis revealed that members of the families Alcaligenaceae and Incertae Sedis 5 (patient 2 only) contributed substantively to the observed increases in Beta-
  • ASD Alzheimer's disease
  • GI disturbances in ASD found low activities of at least one disaccharidase or glucoamylase in duodenum in 58% of children examined (21 of 36) (Horvath et al, 1999).
  • 93.3% of AUT-GI children had decreased mRNA levels for at least one of the three disaccharidases (SI, MGAM, or LCT).
  • SI, MGAM, or LCT three disaccharidases
  • SGLTl important hexose transporters
  • ASD Abnormalities in glucose metabolism and homeostasis have been documented in ASD: recovery of blood glucose levels was delayed in ASD children following insulin-induced hypoglycemia (Maher et al., 1975). Brain glucose metabolism is decreased in ASD by positron emission tomography (Toal et al., 2005; Haznedar et al., 2000; Haznedar et al., 2006). A reduced capacity to digest carbohydrates and absorb glucose due to deficient expression of disaccharidases and hexose transporters could provide a mechanistic explanation for these previous observations in ASD.
  • a direct role for Bacteroidetes in carbohydrate metabolism is also evident.
  • B. thetaiotaomicron encodes in its genome an expansive number of genes dedicated to polysaccharide acquisition and processing, including 236 glycoside hydrolases and 15 polysaccharide lyases (Flint et al., 2008).
  • deficient digestion and absorption of di- and monosaccharides in the small intestine may alter the milieu of growth substrates in the ileum and cecum.
  • the growth advantages that Bacteroidetes enjoy in the healthy intestine as a result of their expansive capacity to thrive on polysaccharides may be compromised in AUT-GI children as bacterial species better suited for growth on undigested and unabsorbed carbohydrates flourish.
  • polysaccharide A a single molecule from another Bacteroidete member, Bacteroides fragilis, protects germ-free mice from Helicobacter hepaticus- and chemically-induced colitis by correcting defects in T-cell development, suppressing production of IL-17 and TNF-alpha, and inducing IL-10 (Mazmanian et al., 2008).
  • PSA polysaccharide A
  • Bacteroides fragilis protects germ-free mice from Helicobacter hepaticus- and chemically-induced colitis by correcting defects in T-cell development, suppressing production of IL-17 and TNF-alpha, and inducing IL-10 (Mazmanian et al., 2008).
  • PSA polysaccharide A
  • Bacteroidete members play in the maintenance of intestinal homeostasis, including maturation of epithelium; regulation of intestinal gene expression, including carbohydrate metabolizing genes and transporters; metabolism of polysaccharides in the colon; and development of
  • ob/ob mice diet-induced obese mice, and in obese humans, the decrease in Bacteroidetes is accompanied by an increase in Firmicutes (Turnbaugh et al., 2008; Ley et al., 2005; Ley et al., 2006).
  • the increased Firmicute/Bacteroidete ratio in obesity is hypothesized to increase the capacity to harvest energy from the diet (Turnbaugh et al., 2006).
  • RNA and DNA extraction were extracted sequentially from individual ileal and cecal biopsies (total of 176 biopsies: 88 ileal and 88 cecal biopsies; 4 biopsies per patient per region; 15 AUT-GI patients and 7 Control-GI patients) in TRIzol using standard protocols. RNA and DNA concentrations and integrity were determined using a Nanodrop ND- 1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE) and Bioanalyzer (Agilent Technologies, Foster City, CA) and stored at -8O 0 C.
  • PCR standards for determining copy numbers of target transcripts were generated from amplicons cloned into the vector pGEM-T easy (Promega Corporation, Madison, WI). Linearized plasmids were quantitated by UV spectroscopy and 10- fold serial dilutions (ranging from 5 x 10 to 5 x 10 copies) were created in water containing
  • USlDOCS 7494238v2 - 83 - yeast tRNA (1 ng/ ⁇ l). Unpooled RNA from individual ileal biopsies were used for real time PCR assays; each individual biopsy was assayed in duplicate. cDNA was synthesized using Taqman reverse transcription reagents (Applied Biosystems) from 2 ⁇ g unpooled RNA per 100 ⁇ l reaction. Each 25- ⁇ l amplification reaction contained 10 ⁇ l template cDNA, 12.5 ⁇ l Taqman Universal PCR Master Mix (Applied Biosystems), 300 nM gene-specific primers and 200 nM gene-specific probe (Table 2).
  • the thermal cycling profile using a ABI StepOnePlus Real-time PCR System consisted of: Stage 1, one cycle at 5O 0 C for 2 min; Stage 2, 1 cycle at 95° C for 10 min; Stage 3, 45 cycles at 95° C for 15 s and 60° C for 1 min (1 min 30 s for LCT).
  • GAPDH and B-actin mRNA were amplified in duplicate reactions by real-time PCR from the same reverse transcription reaction as was performed for the gene of interest.
  • the mean concentration of GAPDH or Beta-actin in each sample was used to control for integrity of input RNA and to normalize values of target gene expression to those of the housekeeping gene expression.
  • Genotyping primers for C/T- 13910 and G/A-22018 polymorphisms are as follows: C/T-13910For (5'-GGATGCACTGC TGTGATGAG-3'[SEQ ID NO: 20]), C/T-13910Rev (5 '-CCCACTGACCTATCCTCGTG-S' [SEQ ID NO: 21]), G/A-22018For (5 '-AACAGGCACGTGGAGGAGTT-S' [SEQ ID NO: 22]), and G/A-22018Rev (5'-CCCACCTCAGCCTCTTGAGT-S' [SEQ ID NO: 23]).
  • Each 50- ⁇ l amplification reaction contained 500 ng genomic DNA, 400 nM forward and reverse primers, and 25 ⁇ l High Fidelity PCR master mix. Thermal cycling consisted of 1 cycle at 94 0 C for 4 min followed by 40 cycles at 94 0 C for 1 min, 6O 0 C for 1 min, and 72 0 C for 1 min. PCR reactions for C/T-13910 were directly digested with the restriction enzyme BsmFI at 65 0 C for 5 hrs. PCR
  • BsmFI digestion of the C/T-13910 amplicons generates two fragments (351bp and 97bp) for the hypolactasia genotype (C/C), four fragments (351bp, 253bp, 98bp, and 97bp) for the heterozygous genotype (C/T), and three fragments (253bp, 98bp, and 97bp) for the normal homozygous allele (T/T).
  • Hhal digestion of the G/A-22018 amplicons generates two fragments (284bp and 184bp) for the hypolactasia genotype (G/G), three fragments (448bp, 284bp, and 184bp) for the heterozygous genotype (G/ A), and a single fragment (448bp) for the normal homozygous allele (A/A).
  • PCR amplification of bacterial 16S rRNA gene and bar coded 454 pyrosequencing of intestinal microbiota For DNA samples from 88 ileal biopsies (4 biopsies per patient; 15 AUT-GI patients, 7 Control-GI patients) and 88 cecal biopsies from the same patients, PCR was carried out using bacterial 16S rRNA gene-specific (V2-region), barcoded primers as previously described (Hamady et al., 2008).
  • Composite primers were as follows: (For) 5'- GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-S ' [SEP ID NO: 24], (Rev) 5 '-GCCTCCCTCGCGCCATCAGNNNNNNCATGCTGCCTCCCGTAGGAGT-S' [SEQ ID NO: 25].
  • Underlined sequences in the Forward and Reverse primers represent the 454 Life Sciences® primer B and primer A, respectively.
  • Bold sequences in the forward and reverse primers represent the broadly-conserved bacterial primer 27F and 338R, respectively.
  • NNNNNNNN represents the eight-base barcode, which was unique for each patient.
  • PCR reactions consisted of 8 ⁇ l 2.5X 5 PRIME HotMaster Mix (5 PRIME Inc., Gaithersburg, MD), 6 ⁇ l of 4 ⁇ M forward and reverse primer mix, and 200 ng DNA in a 20- ⁇ l reaction volume. Thermal cycling consisted of one cycle at 95° C for 2 min; and 30 cycles at 95° C for 20 seconds, 52° C for 20 seconds, and 65° C for 1 min. Each of 4 biopsies per patient was amplified in triplicate, with a single, distinct barcode applied per patient. Ileal and cecal biopsies were assayed separately.
  • USlDOCS 7494238v2 - 85 - Equimolar ratios were combined to create two master DNA pools, one for ileum and one for cecum, with a final concentration of 25 ng/ ⁇ l. Master pools were sent for unidirectional pyrosequencing with primer A at 454 Life Sciences (Branford, CT) on a GS FLX sequencer.
  • Table 4 Real-time PCR primers and probes used for gene expression and bacterial quantitative analysis.
  • CDX2 44 For S ' -GGCAGCCAAGTGAAAACC AG-3 ' 112
  • GAPDH 50 For. 5 ' -CCTGTTCGACAGTCAGCCG-Cf 1Q0
  • a representative amplicon with high homology to Bacteroides intestinalis (Accession #: NZ_ABJL02000007) 16S rRNA gene was used with total Bacteria primers. Cloned sequences were classified using the RDP Seqmatch tool and confirmed by the Microbes BLAST database. Plasmids were linearized with the Sphl restriction enzyme and ten-fold serial dilutions of plasmid standards were created ranging from 5X10 to 5x10 copies for Bacteroidetes, Firmicutes and total Bacteria. Amplification and detection of DNA by real-time PCR were performed with the ABI StepOnePlus Real-time PCR System (Applied Biosystems).
  • Each 25- ⁇ l amplification reaction mixture contained 50 ng DNA, 12.5 ⁇ l SYBR Green Master Mix (Applied Biosystems), and 300 nM bacteria-specific (Bacteroidete, Firmicute or total Bacteria) primers. DNA from each of 88 ileal biopsies (4 biopsies per patient) and 88 cecal biopsies (4 biopsies per patient) was assayed in duplicate.
  • the final results were expressed as the mean number of Bacteroidete or Firmicute 16S rRNA gene copies normalized to 16S rRNA gene copies obtained using total Bacterial primers.
  • Eight water/reagent controls were included for all amplifications. The average copy number for water/reagent controls (background) was subtracted from each ileal and cecal amplification prior to normalization.
  • All water controls contained undetectable levels of amplification.
  • For the Firmicute assay average amplification signal from water samples were minimal, 12.03 +/- 15.0 copies.
  • OTUs Orthogonal Taxonomic Units
  • Finegold SM Molitoris D
  • Song Y et al. Gastrointestinal microflora studies in late-onset autism. Clin Infect Dis 2002;35:S6-S16.
  • Kalhan SC Kilic I. Carbohydrate as nutrient in the infant and child: range of acceptable intake. Eur J Clin Nutr 1999;53 Suppl l:S94-100.
  • Nehlig A Cerebral energy metabolism, glucose transport and blood flow: changes with maturation and adaptation to hypoglycaemia. Diabetes Metab 1997;23: 18-29.

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Abstract

L'invention porte sur des biomarqueurs de l'autisme humain et sur des procédés qui permettent de traiter, prévenir et diagnostiquer l'autisme humain et les troubles liés à l'autisme.
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