Utilization of the Biolog Platform to Understand Neurodevelopmental Disorders Utilization of the...

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  • Slide 1
  • Utilization of the Biolog Platform to Understand Neurodevelopmental Disorders Utilization of the Biolog Platform to Understand Neurodevelopmental Disorders Luigi Boccuto, MD JC Self Research Institute Greenwood Genetic Center
  • Slide 2
  • BIOLOG PLATES 96-well plates 50 l/well of cell suspension (20,000 cells/well) Incubate 40-48 hours, add Redox Dye Mix (tetrazolium) and incubate for 24 hours Plate substrates: 1. Carbon energy sources (sugars, polysaccharides, nucleotides, and carboxylic acids) 2-4. Amino acids and dipeptides 5. Ions 6-8. Hormones and metabolic effectors
  • Slide 3
  • BIOLOG studies (in bold significant results) ASDs (151 cases) NLGN4 (2) SHANK3 (3) FMR1 (4) MECP2 (2) ZNF711 (1) Other (128) XLID (60 cases) MED12 (3 FG and 1 Lujan) UPF3B (3) SMS (8) FMR1 (8+4, no ASDs) ACSL4 (1) SLC6A8 (1) SLC9A6 (1) MECP2-L1CAM dup (2) ZNF711 (1) ATRX (1) RSK2 (1) FGD1 (14) L1CAM (1) MCT8 (1) Schizophrenia (10 cases) Autosomal ID (5 cases) ZBTB20 (2) ST3GAL5 (1) Angelman (2)
  • Slide 4
  • Autism Spectrum Disorders (ASDs) ASDs include a series of conditions characterized by delays or abnormal functioning before the age of 3 in one or more of the following domains: social interaction; communication; restricted, repetitive, and stereotyped patterns of behavior, interests, and activities. The main disorders of the spectrum (DSM-IV-TR) are: Autistic disorder Asperger syndrome Pervasive developmental disorder not otherwise specified (PDD-NOS). Prevalence: 1/88 (US Center for Disease Control and Prevention, 2012). ASD research funding in US (2010): $408.5 M, (+84% from 2008).
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  • G1G2G3G4G5G6G7G8G9G10 Substrate 15627 (NLGN4 A289T) 16309 (SHANK3 c.1304+48 C>T + microdel 18) 17702 (NLGN4 S259P) CMS 13961 (twin) CMS 13963 (twin) CMS 17711CMS 4613 12718 (SHANK3 c.1304+48 C>T + EGR2 SNPs) 16453 (SHANK3 c.1304+48 C>T) CMS 6693 (t 14;15) P value Trp-Arg0.1230.2230.1830.1880.2360.2960.2490.2760.2380.2770.00014 Trp-Glu0.1330.2770.2030.2250.2710.3230.3170.4370.2510.290.00028 L-Tryptophan0.2150.3780.3050.3030.3770.2930.440.3440.3940.4470.00028 Trp-Gly0.1390.280.2120.2220.2360.2580.2660.3890.3630.3370.00042 Trp-Trp0.1950.2970.3020.250.3370.2960.350.4330.4080.3120.00049 Ala-Trp0.1790.2690.2190.240.3110.3260.3250.3190.2880.3330.00066 Asp-Trp0.1380.2560.1990.2190.250.290.2680.270.2210.4020.00071 Phe-Trp0.190.2290.2620.2390.2970.2610.2740.40.3380.320.00189 Met-Trp0.180.3020.3230.3350.4170.3020.3710.3250.3830.4360.00210 Glu-Trp0.1480.2860.2470.2960.3480.3010.3590.320.2550.550.00305 Trp-Asp0.1240.2570.1710.1970.2730.3460.2450.3960.2590.280.00353 Leu-Trp0.1710.3260.2050.3010.4180.2680.3220.3390.4720.3590.00459 Trp-Ala0.1580.2730.2220.2350.330.3140.3450.4380.3470.4160.00466 Trp-Tyr0.1320.3030.2110.2630.3360.2540.3090.4080.3420.3180.00673 Trp-Val0.1590.3090.1850.2740.3380.2480.3360.4120.2860.3480.00804 Ile-Trp0.1660.3110.2460.2930.3480.3160.3730.3630.3550.4730.00961 Arg-Trp0.1270.1810.140.1690.2180.3420.2050.2490.3450.270.01054 Trp-Leu0.1950.3310.2270.2840.3840.2420.2970.3610.4640.3350.01984 Lys-Lys0.0960.0930.0760.1020.1150.2430.1020.2070.1920.2730.02204 Leu-Ile0.1670.1160.0810.2070.2660.4080.2340.3060.3710.4030.02210 Trp-Phe0.1810.3550.2930.3080.4180.2530.30.4140.3690.2930.02273 Ile-Tyr0.0990.1640.10.1510.1850.3560.1870.2680.190.3090.02423 His-Trp0.1280.2410.1780.2260.2470.3040.2290.2220.2550.3180.02525 Gly-Trp0.1380.2980.2530.3160.3490.3630.3570.2760.3330.3090.02582 Trp-Ser0.1660.3170.2320.2550.2980.2260.3230.3720.3980.3110.02632 a-Keto-Butyric Acid0.1130.1140.1110.1590.0940.1060.1570.1890.2260.4220.02740 Mono Methyl Succinate1.2361.542.1161.2861.6640.7141.0481.951.5451.4120.03126 Ile-Gln0.3850.80.5440.6250.9770.4260.70.8490.7920.8350.03223 L-Lactic Acid (DL)0.4640.5720.8670.6660.9220.7060.3180.5880.8230.7970.03291 D,L-a-Glycerol Phosphate 0.4080.3280.5160.2520.2360.4090.2470.6240.370.4960.03566 His-Glu0.0820.0930.0720.0960.1050.2330.1160.1560.1770.20.03605 Tyr-Gly0.1020.1380.1030.1340.1550.2350.1470.3080.2690.2610.03918 His-Val0.080.0830.0720.1010.090.280.0860.1990.1260.260.04165 a-Methyl-D-Mannoside0.1180.1720.1490.2160.4010.5240.2210.7190.4260.2490.04571 Significant metabolites in 10 non-syndromic ASD patients vs. 10 controls
  • Slide 6
  • Substrate Control Average GreenAverageOrangeAverage Purple Average Z Average P-Value Glycogen1.4891.1641.1610.9111.4020.026949 L-Lactic Acid (DL)0.4790.6950.7690.5560.6570.006211 Succinamic Acid1.6261.2981.4220.8121.6510.023753 Mono Methyl Succinate1.8841.4751.6710.9921.8190.011704 b-Hydroxy-Butyric Acid0.3590.5290.5570.3790.4120.023910 Hexanoic Acid0.3380.4890.5540.4150.3900.011992 L-Tryptophan0.6310.3650.3750.2410.3770.001461 Ala-Arg0.1940.2730.3140.2220.2110.018475 Ala-Trp0.4720.2920.2980.2030.2990.001675 Arg-Trp0.3570.2350.2380.1650.2320.001373 Asp-Trp0.4490.2640.3140.1660.2420.001115 Glu-Trp0.5320.3290.3420.1780.3300.000942 Gly-Trp0.4620.3170.2970.1840.2990.006416 Gly-Tyr0.2770.2010.2310.1460.1890.015515 His-Trp0.3790.2470.2540.1870.2490.004921 His-Tyr0.2380.1710.2410.1150.1610.031716 Ile-Gln1.0210.7280.9790.9040.6110.029556 Ile-Trp0.5380.3420.3970.2780.3240.006989 Ile-Tyr0.3310.2120.3130.2160.2090.018489 Leu-Phe0.1900.2410.3710.3380.1890.039625 Leu-Trp0.4980.3340.3710.3720.3200.002536 Met-Trp0.5280.3550.3590.2480.3480.001670 Met-Tyr0.3100.2250.2750.1680.2150.021180 Phe-Trp0.3950.2910.3370.2030.2880.000191 Pro-Trp0.5000.4060.3670.3110.3370.021670 Ser-Tyr0.3010.2140.2660.1850.2120.013745 Trp-Ala0.4860.3240.3210.2230.3090.001209 Trp-Arg0.4020.2410.2900.1580.2310.000173 Trp-Asp0.4120.2690.3180.1410.2250.000740 Trp-Glu0.4940.2880.3520.1620.2550.000132 Trp-Gly0.4720.2850.3170.1900.2680.000299 Trp-Leu0.4470.3250.3090.2120.3140.001800 Trp-Lys0.3990.3100.2760.1630.2790.014358 Trp-Phe0.4200.3340.3120.2310.2950.000590 Trp-Ser0.5010.3040.2970.2360.2910.004266 Trp-Trp0.5050.3320.3200.2190.3410.000153 Trp-Tyr0.4720.3050.2950.1790.2870.001164 Trp-Val0.4810.3040.3440.1950.3100.003150 Tyr-Ala0.3140.2100.2700.1570.2000.036694 Tyr-Gly0.2870.1940.2440.0880.1760.005801 Tyr-Ile0.3470.2300.2950.1550.2000.019385 Tyr-Phe0.2520.2000.2390.1310.1450.027475 Tyr-Trp0.3360.2530.2780.1570.2310.002382 Tyr-Tyr0.3280.2330.2250.1240.1850.003382 Tyr-Val0.2930.2100.2420.1160.1540.014950 Val-Gln0.7310.4990.6150.5430.3800.022287 Val-Tyr0.3030.2290.2730.1380.1870.024195 15627 (NLGN4 A289T)G1 16309 (SHANK3 c.1304+48C>T + microdel 18) G2 17702 (NLGN4 S259P)G3 cms13961 (twin)G4 cms13963 (twin)G5 cms17711G6 cms4613G7 12718 (SHANK3 c.1304+48C>T + EGR2 SNPs) G8 16453 (SHANK3 c.1304+48C>T)G9 CMS 6693 (t 14;15)G10 CMS 6276a FMR1O1 CMS 9549 FMR1O2 CMS 15810 FMR1O3 CMS 15811 FMR1O4 CMS 1836 (MECP2 R168X)P1 CMS 3294 (MECP2 M158T)P2 14071 (ZNF711)Z1 25/26 (96.1%) Trp (p
  • Background and Hypothesis Trp->Kynurenine: the balance between kynurenic acid and quinolinic acid (final products of the kynurenine pathway) is critical for neuronal growth, maturation and apoptosis Quinolinic acid: neurotoxic, induces mitochondrial dysfunction and increases free radical generation, is a potent NMDA agonist, maybe the endogenous ligand during early programmed apoptosis Kynurenic acid: neuroprotective, NMDA antagonist, and prevents glutamate-induced neuronal death, particularly in the hippocampus Nitric oxide (NO): influences expression of both molecules, plays a critical role in the interaction between inflammation and neuronal circuits, causes mitochondrial dysfunction, and has been reported elevated in ASD patients
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  • Background and Hypothesis Trp->Serotonin: Trp depletion can cause abnormal behavior, Serotonin (5-HT) has been associated with mood and behavioral regulation. During the first trimester of gestation, 5-HT is produced by the placenta starting from maternal Trp. This endogenous 5-HT is critical in the development of frontal lobes and in the organization of glutamatergic synapses. from McKay, Nature 2011
  • Slide 14
  • ChiSquaredInfoGainGainRatioAttributeSymmetricalUncertOneRAttributeRelieFAttributeSVM Trp-Ala Trp-AspTrp-Ala Trp-Arg Trp-AspTrp-GlyTrp-Arg Trp-Glu Trp-GlyTrp-SerTrp-Asp Trp-Gly Trp-LysTrp-TyrTrp-Ser Trp-Leu Trp-TyrTrp-ValTrp-Trp Frequency of metabolites that are selected by the 10 different feature selection algorithms after testing 37 ASD patients vs. 20 controls CountMetabolitesFrequencyComment 1Trp-Gly10Recommended 2Trp-Lys8Recommended; new on the list 3Trp-Ala7Recommended 4Trp-Arg7Recommended 5Trp-Leu5Recommended 6 Trp-Phe4Considered; new on the list Frequency of metabolites that are selected by 7 different feature selection algorithms after testing 17 ASD patients vs. 18 controls
  • Slide 15
  • A1 -D- Glucose A2 -D- Glucose A3 -D- Glucose A4 -D- Glucose A5 -D- Glucose A6 -D- Glucose A7 -D- Glucose A8 -D- Glucose A9 -D- Glucose A10 -D- Glucose A11 -D- Glucose A12 -D- Glucose B1 Empty B2 Empty B3 Empty B4 Empty B5 Empty B6 Empty B7 Empty B8 Empty B9 Empty B10 Empty B11 Empty B12 Empty C1 L- Tryptophan C2 L- Tryptophan C3 L- Tryptophan C4 L- Tryptophan C5 L- Tryptophan C6 L- Tryptophan C7 L- Tryptophan C8 L- Tryptophan C9 L- Tryptophan C10 L- Tryptophan C11 L- Tryptophan C12 L- Tryptophan D1 Trp-Gly D2 Trp-Gly D3 Trp-Gly D4 Trp-Gly D5 Trp-Gly D6 Trp-Gly D7 Trp-Gly D8 Trp-Gly D9 Trp-Gly D10 Trp-Gly D11 Trp-Gly D12 Trp-Gly E1 Trp-Lys E2 Trp-Lys E3 Trp-Lys E4 Trp-Lys E5 Trp-Lys E6 Trp-Lys E7 Trp-Lys E8 Trp-Lys E9 Trp-Lys E10 Trp-Lys E11 Trp-Lys E12 Trp-Lys F1 Trp-Ala F2 Trp-Ala F3 Trp-Ala F4 Trp-Ala F5 Trp-Ala F6 Trp-Ala F7 Trp-Ala F8 Trp-Ala F9 Trp-Ala F10 Trp-Ala F11 Trp-Ala F12 Trp-Ala G1 Trp-Arg G2 Trp-Arg G3 Trp-Arg G4 Trp-Arg G5 Trp-Arg G6 Trp-Arg G7 Trp-Arg G8 Trp-Arg G9 Trp-Arg G10 Trp-Arg G11 Trp-Arg G12 Trp-Arg H1 Trp-Leu H2 Trp-Leu H3 Trp-Leu H4 Trp-Leu H5 Trp-Leu H6 Trp-Leu H7 Trp-Leu H8 Trp-Leu H9 Trp-Leu H10 Trp-Leu H11 Trp-Leu H12 Trp-Leu Customized Biolog Tryptophan plate
  • Slide 16
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  • Substrate Aut 15057 Aut 13669 Aut 18454 Aut 15301 Aut 17160 Aut CMS 14869 Control CMS 11237 Control CMS 11959 Control CMS 11210 Control CMS 12225 Control CMS 12223 Control CMS 12808 P value (t test) a-D-Glucose1.7551.2592.1821.8151.1241.4361.5961.8171.7430.9161.5491.2470.6205 Empty0.1290.1090.1080.1410.2470.1160.3030.0980.120.1110.1470.1380.8271 L-Trptophan0.3840.2590.370.4430.5550.5130.670.5640.6510.6960.9490.6270.0041 Trp-Gly0.2970.20.2960.3660.4350.3390.6640.4480.5190.5240.6940.4370.0028 Trp-Lys0.3230.2210.2370.3560.4210.3340.4330.3890.4540.4860.7060.4660.0068 Trp-Ala0.3590.2340.2890.3980.4340.3610.4410.4570.5430.5420.7160.560.0032 Trp-Arg0.3080.2040.2690.3830.380.2740.4390.4360.4940.480.6830.430.0024 Trp-Leu0.7270.3150.8630.6350.5710.4680.7630.8210.7080.6110.9130.5950.1687 New customized Biolog tryptophan plate 6 ASDs vs. 6 Controls Note: the t test was performed on the log-transformed data.
  • Slide 18
  • New customized Biolog tryptophan plate 6 ASDs vs. 6 Controls
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  • Substrate Schizo CMS 20844 Schizo CMS 20845 Schizo CMS 20846 Schizo CMS 20847 Schizo CMS 20848 Schizo CMS 20849 Schizo CMS 20850 Schizo CMS 20851 Schizo CMS 20852 Schizo CMS 20853 Control CMS 12225 p value (Mann- Whitney test) Control CMS 12223 p value (Mann- Whitney test) a-D-Glucose2.1421.1041.7461.5191.0850.9661.5862.3422.0911.420.9090.001951.2680.10547 Empty0.1790.1840.1270.1450.1080.1120.120.2290.1420.1580.1690.105470.2260.00391 L-Trptophan0.660.5290.5510.5280.5090.2910.360.8050.4880.5150.6250.064450.7550.00391 Trp-Gly0.5730.4490.4640.4320.440.3030.3110.5860.3570.4010.420.769530.5550.00977 Trp-Lys0.5520.4370.4870.3930.3750.2680.2830.6430.3550.3770.410.530.01953 Trp-Ala0.5360.4480.4980.4020.4390.2590.3080.6160.3780.4250.4520.3750.570.00586 Trp-Arg0.5490.3990.4260.3620.3290.210.2490.6880.370.4240.4050.556640.5260.01953 Trp-Leu0.6240.5120.590.4970.4980.4210.4510.7961.0340.5040.5530.921880.7420.01953 New customized Biolog tryptophan plate 10 Schizophrenia patients vs. 2 Controls
  • Slide 21
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  • * ** ** Substrate Mann-Whitney P value Bonferroni correction P value a-D-Glucose0.0013440.010756 Empty0.0231020.184815 L-Trptophan0.0000190.000148 Trp-Gly0.0000030.000024 Trp-Lys0.0000070.000059 Trp-Ala0.0000620.000495 Trp-Arg0.0000200.000163 Trp-Leu0.0000040.000030 * P value
  • SLC3A2 variants (107 ASD patients) PatientVariant Effect/ Bioinformatics Parents/controlsSNP (1000 genomes/NHLBI) 17550 (BM)c.6 G>A (Exon 1) p.Glu2Glu; 4 new ESEs; no qPCR changes Mother: negative; 2/274 (0.7%) controls vs 1/107 patients (0.9%) rs114958414 G: 2183 (99.96%); A: 1 (0.04%) NHLBI G: 12994 (99.96%); A: 6 (0.04%) 12382 (WM) 1 c.826 G>A (Exon 5) p.Asp276AsnBenign Father; 0/281 controls rs80190679 (G: 99.9%; A: 0.1%) NHLBI G: 12981 (99.9%); A: 17 (0.1%) c.902-24 T>A (Intron 5) None Negative (de novo) No 18454 (BM) 2 c.1027 G>T (Exon 7) p.Asp343Tyr Deleterious (6/8 websites)-Splice changes No parents available; 0/542 controls vs. 1/107 patients (0.9%) No 17410 (WM) 3 c.1352 G>A (Exon 10) p.Arg451Gln Deleterious (5/8 websites) Father (affected brother: Negative) rs148443520 G: 10755 (99.97%); A: 3 (0.03%) NHLBI G: 12995 (99.98%); A: 3 (0.02%) 17843 (WM) c.1316 G>T (Exon 10) p.Arg439Met - Borderline (4/8 websites) Father (mother not available) rs116829615 (G: 99.7%; T: 0.3%) NHLBI G: 12979 (99.9%); T: 19 (0.1%) 15294 (BF)c.594 T>G (Exon 4)p.Ala198AlaNot testedNo 17226 4 - 9754 - 18454 2 (BM) c.1606 C>T (Exon 12)p.Leu536Leu 17226: mother and unaffected twin sister= Negative rs76688902 (C: 98.1%; T: 1.9%) NHLBIG (C: 97.5%; T: 2.5%) 17817-14037- 12970 -12053 (BM) c.902-11 C>T (Intron 5) Higher Signal Quantile (69->78) at Acceptor Splice Site 12970: both parents are heterozygous rs59762129 (C: 95.5%; T: 4.5%) 1 del (20)(p11.22;p11.23), 2 EGR2 SNPs, 3 MET pol C/C, 4 FRAXE 0 repeats
  • Slide 33
  • SLC7A5 variants (107 ASD patients) PatientVariantEffect/BioinformaticsParents/controlsSNP (1000 genomes/NHLBI) 10471 (HM) 1 c.1123 C>A (Exon 7) p.Pro375Thr Deleterious Mother and relatives: negative; 0/542 controls vs. 1/57 patients (1.75%), p value: 0.0952 No c.1347 C>G (Exon 9) p.Val449Val rs11541881 C: 2166 (99.2%); G: 18 (0.8%) NHLBI C: 12969 (99.98%); G: 3 (0.02%) CMS 14985 (WM- PDD) c.1141-98 T>A (Intron 7)Acceptor splice site changesNo parents availableNo 15301 (BM)c.1290+44 G>A (Intron 8)Lost 1 ESEMother: negativeNo CMS 15811 (WM) 2 c.816-18 C>T (Intron 4) Higher Signal Quantile (57 -> 59) at Acceptor Splice Site No parents availableNo 14988 (BM)c.1141-93 C>T (Intron 7)NoneNot testedNo 4 patients (1 Hom) c.690 C>G (Exon 3) p.Asn230Lys Lost 2 ESEs Father (12970); 10/282 controls (3.5%) rs1060250 (C: 98.7%, G: 1.3%) NHLBI C: 12768 (98.3%); G: 228 (1.7%) 12970 (BM) 3 c.387 C>T (Exon 1)p.Ala129AlaFather rs33913122 (C: 99.8%, T: 0.2%) NHLBI (12966) C: 99.97%, T: 0.03% 12 patients, 11.2%c.843 C>T (Exon 5)p.Ser281SerNot tested rs33992288 (C: 99.8%; T: 0.2%) NHLBI (12994) C: 99.95%, T: 0.5% 9754 (BM)c.939+20 C>T (Intron 5)NoneNot testedrs33993343 (C: 99.8%; T: 0.2%) 9754-14988 (BM)c.1008 C>T (Exon 6)p.Phe336PheNot tested rs1060251 (C: 99.2%; T: 0.8%) NHLBI (12996) C: 98.9%, T: 1.1% 12388 (BF)-17226 (BM) 4 c.1043+6 C>T (Int 6) Higher Splice Score (10.1 -> 12.2) at Donor Splice Site Not testedrs33966404 (C: 98%; T: 2%) 1 ZNF711 mut; 2 FMR1 full mutation; 3 47,XY +del(15)(q14); 4 FRAXE 0 repeats
  • Slide 34
  • SLC7A8 variants (107 ASD patients) PatientVariantEffect/BioinformaticsParents/controlsSNP (1000 genomes/NHLBI) 12388 (BF) c.47 C>G (Exon 1) p.Pro16Arg Borderline (4/8 websites) Mother and affected brother rs147920363 C: 2180 (99.8%); G: 4 (0.2%) NHLBI C: 12964 (99.7%); G: 42 (0.3%) 6320 (WM) 1 17673 (BM) c.52 G>T (Exon 1) p.Gly18Trp Deleterious Decreased Signal Quantile (19->8) at Donor Splice Site Negative (6320-de novo; 17673-Mother); 2/542 controls vs 1/107 patients (0.36% vs 0.9%,) rs144958980 G: 2175 (99.6%); T: 9 (0.4%) NHLBI G: 12930 (99.4%); T: 76 (0.6%) 4947 (WM) c.86 C>T (Exon 1) p.Ser29Phe Deleterious (5/9 websites) No parents available; 2/542 controls vs 1/57 patients (0.36% vs 0.9) rs149980964 C: 2181 (99.9%); T: 3 (0.1%) NHLBI C: 12970 (99.8%); T: 36 (0.2%) 15318 (WM) 1 c.1016-49 T>C (Intron 7)NoneNegative (de novo)No CMS 6276 (WM) 2 c.1125 C>T (Exon 9)p.Thr375ThrNo parents available rs141169165 (C: 99.9%; T: 0.1%) NHLBI (13006) C: 99.92%, T: 0.08% CMS 1836 (WF) 3 c.1113+19 G>A (Intron 8)NoneNo parents availableNo 23 patients, 21.5% (2 Hom, 1.9%) c.120 C>T (Exon 1) p.Ile40Ile, 2 new ESEs Not tested rs1884545 (C: 94%; T: 6%) NHLBI (13006) C: 89.6%, T: 10.4% 12713 (WM) c.634+10 G>T (Intron 4) 1 new ESE Father c.634+10 G>A is SNP rs35329117 (no frequency data, no ESE change) 20 patients, 18.7% (5 Hom, 4.6%) c.635-83 T>A (Int 4) 4 1 new ESENot tested rs10151002 (T: 93%; A: 7%). Genotypes (1092): T/T: 87.8%, T/A: 10.4%, A/A: 1.7% NHLBI (13006 alleles) T: 99.992%, A: 0.008% (1) 66 patients (16 Hom) c.913-13 G>T (Intron 6) Increased Signal Quantile (42 ->63) at Acceptor Splice Site Not testedrs1015089 (G: 65.1%; T: 34.9%) 89 patients, 83.1% (31 Hom, 29%) c.1170 T>C (Exon 9)p.Tyr390Tyr, 1 new ESENot tested rs7157021 (T: 33.7%; C: 66.3%) NHLBI (13006): T: 41.3%, C: 58.7% 1 ZBTB20 mut ; 2 FMR1 full mutation ; 3 MECP2 (c.502 C>T; p.R168X); 4 T>A genomic, A>T cDNA
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  • Fragile X syndrome Moderate to severe ID, macroorchidism, and distinct facial features, including long face, large ears, and prominent jaw. About 1/3 of the patients show autistic traits, other behavioral issues (i.e.: ADD/ADHD, anxiety/OCD) are also common. First monogenic cause of ID (1/4,000 males). Unstable expansion of a CGG repeat in the FMR1 promoter and abnormal methylation suppression of FMR1 transcription and decreased protein levels in the brain.
  • Slide 36
  • Preliminary data Fragile X syndrome 12 cases (8 with full mutation, 4 female carriers) versus 12 controls Considering just the Fragile X diagnosis, neither the patient nor the carrier cohort showed significant metabolic differences as compared to controls Two subgroups with mental disorders: 3 cases with ADD/ADHD (and Anxiety/OCD) 6 cases with Anxiety/OCD (including the 3 with ADD/ADHD) In both subgroups we noticed a statistically significant increase of utilization of glucose-related sugars, Krebs cycle intermediates, and ketone bodies, as compared to controls.
  • Slide 37
  • Preliminary data - Fragile X patients with ADD/ADHD and Anxiety/OCD (3) Significantly increased utilization of: glucose-related sugars (14/27, 51.8%), particularly glucose* n-acetyl-neuraminic acid: predominant sialic acid in gangliosides (crucial component of neuronal membranes) pyrimidines (2/2): thymidine and uridine* Krebs cycle intermediates (7/9, 77.7%): particularly lactate*, succinate*, and malate* ketone bodies and other carboxylic acids (9/13, 69.2%): particularly acetoacetate*, b-hydroxy-butyrate*, propionate*, and acetate * most amino acids: some (Asn*, His*, Met*, and Val*) were significant for all the wells in the arrays. * Significant in 3/3 cases
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  • Preliminary data Fragile X patients with Anxiety/OCD (6) Significantly increased utilization of: glucose-related sugars (9/27, 33.3%), particularly glucose* n-acetyl-neuraminic acid pyrimidines (2/2): thymidine and uridine* Krebs cycle intermediates (6/9, 66.7%): particularly lactate* and pyruvate* ketone bodies and other carboxylic acids (6/13, 43.1%): particularly acetoacetate*, b-hydroxy-butyrate*, and acetate* Only Asn* was significant for 80% of the wells in the arrays. * Significant in at least 4/6 cases
  • Slide 39
  • Background (ID and ADD/ADHD) Neuronal cells have one of the highest metabolic rates in the human body and use sugars and ketone bodies as primary energy sources. Abnormal metabolism of sugars and ketone bodies may affect neuronal development, signal transmission and synaptic function. Several mental disorders have been associated with increased metabolism within specific brain areas as detected by PET studies.
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  • Hypothesis (ID and ADD/ADHD) Some mental disorders have a late onset (ADD/ADHD, OCD, Tourette syndrome) and can occur after an event causing an increase in metabolic rate (high fever, infections, brain damages, puberty). When compared to controls, cells from patients with ID and developmental mental disorders like ADD/ADHD appear to have increased need of energy sources under situations promoting metabolic stress and the consequences can be more severe in those tissues with higher metabolic rates (i.e. neuronal cells).
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  • Summary Biolog phenotype microarray plates are able to identify significant abnormalities in the metabolic profile of cells from patients with neurodevelopmental disorders, like ASDs, ADD/ADHD, and anxiety/OCD. The metabolic findings reveal new molecular pathways playing an important role in the pathogenesis of those disorders and can provide new tools for therapeutic approaches. Follow-up studies involving kinetics and gene expression analysis are ongoing to better characterize these findings.
  • Slide 42
  • Acknowledgments Dr. Charles Schwartz Dr. Chin-Fu Chen Cindy Skinner Kelly Jones Lauren Cascio Ayla Pittman Mackenzie DeGraff Heather McCartney