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Genetica clinica diaria_sin_videos

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Genetica clinica diaria_sin_videos

  1. 1. Genética en la Clínica Diaria Carlos E. Prada, MD Clinical & Biochemical Geneticist Division of Human Genetics Cincinnati Children’s Hospital Medical Center Director, Centro de Medicina Genómica y Metabolismo Fundación Cardiovascular de Colombia
  2. 2. • Genetic testing uses laboratory methods to look at your genes. • Identify increased risks of health problems • Choose treatments • Response to therapies What Can I learn from genetic testing?
  3. 3. Types of Genetic Testing • Diagnostic testing • Predictive and pre-symptomatic genetic tests • Carrier testing • Prenatal testing • Newborn screening • Pharmacogenomic testing • Research genetic testing
  4. 4. Benefits and Drawbacks • Rule in or rule out a disease • Eliminating uncertainty • Treatment recommendations • Decision making process - Screening • Emotional burden (Guilt, angry, depression) • Financial difficulties • Discrimination – GINA law 2008 • Limitations of techonology
  5. 5. How do I decide about tests? • Doctors recommendations based on personal and family history • Testing is voluntary • Talk about genetic test benefits and limitations • Emotional support
  6. 6. What is Whole Exome Sequencing? • The genome is the entirety of an individual’s DNA • The exome is the coding region of the genome (1.5%) – Location of majority of mutations responsible for disease
  7. 7. Whole Exome Sequencing (WES) • Characteristics: – Examines the coding regions of most genes at one time. – One of the most comprehensive genetic tests available • Mutations found in ~25% • Results may be used to: – Guide a patient’s treatment or management – Genetic counseling – End diagnostic odyssey for some cases
  8. 8. History of Next Generation Sequencing
  9. 9. Differences between NGS and traditional sequencing
  10. 10. Next generation sequencing: • is a short read shotgun approach • has no precise control over the sequence initiation point in the template • is dependent on the existence of a reference sequence and requires bioinformatic processing for alignment Differences between NGS and traditional sequencing
  11. 11. Next Generation Sequencing
  12. 12. Aligned reads Data Processing Image analysis Sequencing by synthesis Cluster generation Library preparation Template NGS Process Illumina HiSeq2000
  13. 13. – Break down template • 200-500bp fragments • Random breakpoints • Sonication, nebulization, enzymatic cleavage – Ligate Y-adapter at ends • Anchor point for sequencing primer • Produce asymmetric ends when denatured Aligned reads Data Processing Image analysis Sequencing by synthesis Cluster generation Library preparation Template A AT T NGS Process
  14. 14. • Cluster generation – Isolate individual library molecules (ssDNA) on the sequencing slide • Equivalent to plating step – Replicate the molecule • Boost signal Aligned reads Data Processing Image analysis Sequencing by synthesis Cluster generation Library preparation Template NGS Process
  15. 15. Cluster Generation
  16. 16. Aligned reads Data Processing Image analysis Sequencing by synthesis Cluster generation Library preparation Template 1. Bind sequencing primer 2. Elongate with fluorescent nucleotides – 3’ blocked (single incorporation) – 4 color system, one dye per base 3. Record the color incorporated – Laser excitation of dyes 4. Remove 3’ block – Reversible terminator chemistry 5. Repeat cycle NGS Process
  17. 17. NGS Process • Image analysis – Transform image at each cycle into strings of base calls • Data processing – Align reads to a reference genome/transcriptome Aligned reads Data Processing Image analysis Sequencing by synthesis Cluster generation Library preparation Template NGS Process
  18. 18. mRNA- seq Exome Genomic Enrich. ChIP-seqmiR-seq Genome Meta genomics NGS Applications
  19. 19. Technical Limitations of WES • Capturing the exome – 85-99% examined • Certain types of mutations not detected – Large del/dups, rearrangements, trinucleotide repeat expansions, mtDNA • Bioinformatics pipeline – Assumptions made based on clinical info – Accurate medical and family histories are crucial • Current knowledge of the exome
  20. 20. Future Developments in NGS Life Technologies Ion Proton sequencer, chip Nanopore Technologies GridION and MinION sequencing nodes $1,000 genome in a day Single molecule sequencing Native DNA
  21. 21. Newborn Screen by NGS
  22. 22. Methods for Exome Sequencing • Singleton • Only proband • Trio • Affected + two parents • Trio + unaffected sibling • Affected + two parents + unaffected sibling
  23. 23. Exome Data • Variant Filtering – Quality – Read depth – False positives removed – Common variants removed – Variant calls: Non-pathogenic, non-exon removed – Inheritance pattern: AR (homozygous, compound heterozygous), AD, XL, De novo
  24. 24. Data Analysis • Heuristic filtering to identify novel genes for Mendelian disorders Stitziel et al, Genome Biol 2011
  25. 25. Pipelines for Filtering Data Inheritance models to trio-D, AR, XLD, XLR, digenic Prioritize phenotype filter, ACMG – 57 genes, common mutation (>1% in HGMD) Annotate all model files Visual inspect variants to identify and remove false-positives Apply in-house control script , keep freq <5% Gene function, functional domain, pseudogene OMIM/HGMD genes AD AR XLD XLR Digenic Variant MAF< 1% , dbSNP, 1000K G, ESP5400, OMIM, HGMD SIFT, Conservation phyloP, Gratham score Organize by AD, AR, XLD, XLR, digenic Mutation type – nonsense, frameshift, splicing, missense, in-frame, reported synonomous segregation Pathway analysis Gene expression Gene phenotype association – human, animal models Literature support Candidate gene ranking NextGENe Golden Helix
  26. 26. Dominant De novo Germline mosaicism XL Male Sex-limited Recessive Homozygous Compound Het Filtering by Inheritance Model
  27. 27. Compound heterozygotes Rare / Common De novo / De novo Rare / Rare Rare / De novo Filtering by Inheritance Model
  28. 28. Glaucoma with and without aniridia FOXC1 (c.313_314insA;p.Tyr105*)
  29. 29. Indications for Clinical WES • The patient’s symptoms or family history suggest a genetic condition but – there is an atypical clinical presentation – negative previous genetic testing • The suspected condition is genetically heterogeneous and multi-gene panels are unavailable/impractical • Requires a relation with managing physician
  30. 30. – Probable disease-causing mutation(s) related to the patient’s phenotype with supporting evidence – Additional gene variants of unknown clinical significance related to patient’s phenotype What Is Included in the Report?
  31. 31. Genetic Counseling Considerations in WES • Patient perception of WES as definitive test • Looking for diagnosis • Genetic discrimination questions • Decision making regarding incidental findings • Coping with uncertainty • Parental guilt
  32. 32. Patient 1 9 month old male with: • Immunodeficiency: T-,B+, NK+ SCID with dermatitis and hair loss • Congenital anomalies: cervical and lumbar kyphosis, basilar skull anomaly, short stature • Dysmorphic features: bilateral microtia, malar prominence, narrow alae nasi, cupid bow lip, retrognathia
  33. 33. Cervical vertebral body hypoplasia, --“wedged”, “beaked” Basilar skull anomaly—narrow foramen magnum Lumbar vertebral anomalies—”wedged”, “beaked” Skeletal changes not consistent with storage disorders
  34. 34. Patient 1 • Previous genetic testing: – SCID panel – DOCK8 – VCFS FISH – CHD7 – FOXP3 • Microarray revealed pericentric region on chromosome 20 with LOH: – 20p11.23p11.1(18,236,237-26,293,985)x2 hmz, – 20q11.21q12(29,522,520-40,987,446)x2 hmz • Exome Sequencing Revealed: – Homozygous c.463_465del (p.Asn155del) mutation in PAX1: Genetic diagnosis of Otofaciocervical syndrome • SCID phenotype not explained – Mouse model have showed T cell developmental defects.
  35. 35. Patient 2. Familial Dominant Parkinson 45 yo Onset of symptoms at age 30 years Improves with alcohol per patient report (DYT15?) No cognitive decline Normal Brain MRI Dystonia and SCA panel negative
  36. 36. Patient 3. Familial Dominant Parkinson 38 yo – Sister Onset of symptoms at age 28 years No cognitive decline Dystonia on exam
  37. 37. Patient 4. Familial Dominant Parkinson 34 yo Onset of symptoms at age 25 years No cognitive decline
  38. 38. Patient 5. Familial Dominant Parkinson 8 year old Difficulty writing and tremors Normal development
  39. 39. Curr Genomics. Dec 2013; 14(8): 560–567. Genetic Causes
  40. 40. 126,752 103,431 28,875 14,312 2,943 1,801 769 14 heterozygous variants No known disease genes – 4 with brain expression Quality Control Exome Focus on parts that make protein Focus on important protein changes Healthy Population 1 Dominant analysis Healthy Population 2 Healthy Population 3 Filtered out, do not review
  41. 41. Candidate Genes Gene Symbol Alignment Chromosome AKAP5 Real 14 ATXN2 Real 12 SH2D2A Real 1 ADORA3 Real 1 LENG8 Real 19 ERAP2 Real 5 CHRM2 Real 7
  42. 42. AKAP5 Important in depolarization of neurons.
  43. 43. UCSC Genome Browser - conservation
  44. 44. Patient 6. Polyneuropathy and Parkinsonism 38 yo previously healthy Tremor and difficulty walking. Chronic pain. Normal brain MRI. No cognitive changes. Neurophysiological studies: polyneuropathy
  45. 45. Patient 6. Polyneuropathy and Parkinsonism
  46. 46. Pathway analysis
  47. 47. PLP1 network
  48. 48. Patient 7. Grandson of patient 6. Spasticity Nystagmus Unable to walk Brain MRI - hypomyelination
  49. 49. Patient 8 • 17 year old female with leukoencephalopathy, global developmental delay, hypotonia, cryptogenic partial complex epilepsy, and dysphagia. • Seizures and developmental delay began in infancy and progressively worsened with age • Epilepsy Panel NGS: – de novo c.1217G>A(p.H406R) in STXBP1
  50. 50. • STXBP1 (MUNC18-1) encodes syntaxin binding protein 1 • An evolutionarily conserved protein expressed in the brains of humans and rodents • Involved in release of neurotransmitters through regulation of syntaxin
  51. 51. Previously reported de novo STXBP1 mutations in patients with EIEE
  52. 52. Patient 9 6 yo with multiple joint subluxations and dislocations (larsen-like phenotype). Epileptic encephalopathy. Previous tessting: FLNB sequencing negative. Normal chromosomes. Development: holds head, smiles, tracks lights and noises. Not ambulatory. No language development.
  53. 53. • Family history: Parents are first cousins. Healthy brother. No other affected members. • Exam: hypotonia, sterotypies, hypermobility, rotoscoliosis, clinodactyly, and brachydactily. • Brain MRI: frontotemporal atrophy. No leucodystrophy. • No prenatal complications. • Epilepsy panel study detected a homozygous mutation in PGAP1. Patient 9
  54. 54. Patient 10 • 3 y.o. male • Immunological phenotype: Hypogamma- globulinemia, recurrent infections, fevers • Other features: fine motor and speech delay, feeding problems/FTT, gait abnormality, hypotonia, macrocephaly, deep set eyes, prominent forehead, thin upper lip, long fingers and toes, persistent fetal fingerpads.
  55. 55. Previous testing • Normal microarray • Normal 22q deletion testing • MRI: “prominence of subarachnoid space over both cerebral convexities”
  56. 56. De novo mutation in FBN1 - Marfan syndrome • Missense mutation: c.5873G>A(p.1958C>Y) affecting cysteine residue • Previously reported as pathogenic in literature, (Ogawa et al.) • De novo mutation • Sanger confirmed
  57. 57. PREGUNTAS Contacto: carlosprada@fcv.org
  58. 58. OFTALMOGENETICA Carlos E. Prada, MD Clinical & Biochemical Geneticist Division of Human Genetics Cincinnati Children’s Hospital Medical Center Director, Centro de Medicina Genómica y Metabolismo Fundación Cardiovascular de Colombia
  59. 59. Visual perception and loss
  60. 60. WUSTL Hofer et al, 2005 Color vision in the vertebrate retina
  61. 61. • Retinitis Pigmentosa • Leber Congenital Amaurosis • Congenital Stationary Night Blindness • Cone-Rod Dystrophy • Cone Dystrophy • Achromatopsia (complete vs incomplete) • Enhanced S-cone syndrome • Stargardt Disease • Syndromic Retinopathies (PNPLA6) Retinal Dystrophies and Degeneration
  62. 62. Retinitis Pigmentosa • Affects 1:2,500-3,000 • Rods initially affected, later cones • Peripheral – Central progression • Vision loss late 1st-2nd decades • >60 associated genes or loci • AD, AR, and XL forms Rpfightingblindness.uk Molecular Vision
  63. 63. Leber Congenital Amaurosis • Affects 1:80,000-100,000 • Cone and Rod involvement • Early vision loss (often <1yo) • Myopia, nystagmus • Progressive, ~Severe RP • AD and AR (XL reported) • Foveal hypoplasia • Associated finding in ciliopathies (Joubert, Meckel-Gruber, Senor- Loken, Bardet-Biedl) • 15+ associated genes or loci
  64. 64. Allelic and Locus Heterogeneity in LCA
  65. 65. LCA and Foveal Hypoplasia A B
  66. 66. Incomplete Achromatopsia • = Blue cone monochromacy (BCM) • Affects 1:100,000 • Cone function defect, no loss? • Early, static vision loss • Incomplete (Red+Green) – XL • (OPN1MW + OPN1LW) • Typically normal fovea exam, can have mild foveal hypoplasia • Good quality of life, usually cannot drive
  67. 67. Stargardt Disease • Affects 1:8,000-10,000 • Photoreceptor-RPE disease • Early macular degeneration (2nd-3rd decade) • RPE atrophy  PR death • Areolar choroidal dystrophy • Progressive, similar to AMD • ABCA4, ELOVL4
  68. 68. OFTALMOGENETICA Profound neuropathy target esterase impairment results in Oliver- McFarlane Syndrome
  69. 69. Oliver-McFarlane Syndrome • Hypopituitarism-chorioretinopathy-trichomegaly • 14 patients reported since 1965 (2 sib pairs) • Congenital anterior hypopituitarism (short stature, ID) • Progressive chorioretinal degeneration in childhood • Ataxia, neuropathy, spastic paraplegia (2nd-4th decade) • Our patients: 3rd sibship, 7yo+9yo • No family history • Normal SNP microarray • No genetic cause
  70. 70. A D E F
  71. 71. 99,710 Quality >20, Depth >10 Whole exome analysis Recessive inheritance 74,187 Remove if synonomous, noncoding, predicted likely benign/tolerated 11,363 108 Homozygous AR Compound Heterozygous 59 Remove if frequency >1% Visual inspection Remove false positives 0 >1% 3 1PNPLA6 p.Arg1099Gln;p.Gly1176Ser
  72. 72. Pt3. c.1390-27_2287+1200[2];p.Val1215Ala Pt4. c.1973+2T>G;p.Val1215Ala
  73. 73. Pt5. Patton et al. Am J Ophthalmol (1986). p.Arg1031Glnfs*38;p.Gly1129Arg Pt6. p.Arg1031Glnfs*38;p.Gly1129Arg
  74. 74. Pt5. p.Arg1031Glnfs*38;p.Gly1129Arg H&E SMI31 GFAP IBA1 Human Cerebellar Degeneration
  75. 75. Oliver and McFarlane, 1965 Pt2 Pt2 Pt1 Pt4 1965 Pt6
  76. 76. Phenotype Oliver-McFarlane 1965 Laurence-Moon 1866 Bardet-Biedl 1920-1922 Trichomegaly, Alopecia + – – Intellectual disability + + + Retinal degeneration + + + Choroidal atrophy + + – Anterior hypopituitarism + + – Short stature + + – Hypogonadism + + +/– Ataxia, SP, PN + +++ – Obesity – – + Polydactyly – – + Oliver-McFarlane and Laurence-Moon Syndromes
  77. 77. Laurence-Moon Syndrome Poster 2929S: H. Dollfus, M. Prasad, C. Stoetzel Pt7-10. Chalvon-Demersay et al. Archives de Pédiatrie (1993). p.Gly726Arg;p.Arg1031Glnfs*38
  78. 78. PNPLA6 (Neuropathy Target Esterase) Poster 2976T: G. Arno, S. Hull, V. Plagnol, T. Moore Hou et al, 2009
  79. 79. Spastic Paraplegia 39 (SPG39) Adult onset Ataxia, +/- Cerebellar atrophy Spastic Paraplegia Peripheral Neuropathy Gordon-Holmes Syndrome Boucher-Neuhauser Syndrome Late childhood/Adolescent onset Ataxia, SP, PN, Cerebellar atrophy Hypogonadotropic Hypogonadism Chorioretinal Degeneration (BNHS) Laurence-Moon Syndrome Oliver-McFarlane Syndrome Congenital/Childhood onset Ataxia, SP, PN, Cerebellar atrophy Anterior Hypopituitarism, atrophy Chorioretinal Atrophy Hair Anomalies (OMS) Synofzik et al, 2013 Topaloglu et al, 2014 Rainier et al, 2008 PNPLA6 spectrum of disorders
  80. 80. PNPLA6 spectrum of disorders Hypothesis: spectrum of congenital  adult PNPLA6 diseases corresponds to the severity of NTE loss-of-function: SPG39↓ OMS↓↓↓ 1) Validate OMS/LMS mutation pathogenicity in vivo 2) Examine PNPLA6 expression during embryogenesis 3) Compare NTE enzymatic activity across disease states
  81. 81. Normal Mild Intermediate Severe Zebrafish Morpholino – Rescue OMSSPG39WT Morpholino: Song et al, 2013 vs Wt RNA * p<0.05 ** p<0.01 ***p<0.001
  82. 82. Human Embryonic PNPLA6 Expression CS23 (GA 8 weeks)
  83. 83. Human NTE Activity Assay Skin biopsy Fibroblast culture Phenol Valerate Phenol NTE NTE Cellular Assay Wild type – 2 controls SPG39 – carrier SPG39 – homozygote OMS – parent carriers OMS – affected patientsParaoxon Assay: Hein et al, 2010
  84. 84. OMSSPG39WT Human NTE Activity vs Wt/Wt * p<0.05 ** p<0.01 ***p<0.001
  85. 85. PNPLA6-opathy Disease Model Healthy NTE Activity Phenotype SPG39 Laurence-Moon Oliver-McFarlane GHS BNHS
  86. 86. Summary 1) Oliver-McFarlane and Laurence-Moon syndromes are caused by PNPLA6 mutations and NTE loss-of-function 2) Human expression and pathology studies support a spectrum of tricho-oculo-neurologic PNPLA6-opathies 3) Phenotype is dose-dependent – patients with OMS have three-fold loss of NTE activity compared to SPG39
  87. 87. Acknowledgements The families Robert.Hufnagel@cchmc.org Carlos.Prada@cchmc.org carlosprada@fcv.org

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