Dra. Carmen Espinós - 'Neuropatías periféricas hereditarias'
•
2 recomendaciones•1,512 vistas
Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
Recomendados
Recomendados
Más contenido relacionado
La actualidad más candente
La actualidad más candente (20)
Similar a Dra. Carmen Espinós - 'Neuropatías periféricas hereditarias'
Similar a Dra. Carmen Espinós - 'Neuropatías periféricas hereditarias' (20)
Más de Fundación Ramón Areces
Más de Fundación Ramón Areces (20)
Último
Último (20)
Dra. Carmen Espinós - 'Neuropatías periféricas hereditarias'
- 1. Charcot-Marie-Tooth disease GENETIC DIAGNOSIS Carmen Espinós Miguel Servet Researcher -CIBERER Genetics and Genomics of Neuromuscular Disorders - CIPF
- 2. Charcot-Marie-Tooth Disease: Genetic Heterogeneity DEMYELINATING CMT CMT Type Gene CMT Type Gene CMT1A PMP22 CMT4A GDAP1 CMT1B MPZ CMT4B1 MTMR2 CMT1C LITAF CMT4B2 MTMR13 CMT1D EGR2 CMT4C SH3TC2 CMT1F NEFL CMT4D NDRG1 CMTX1 GJB1 CMT4F PRX CMTX5 PRPS1 CMT4G HK1 SURF1 SOX10 CMT4H FGD4 CMT4J FIG4 AXONAL CMT CMT Type Gene CMT Type Gene CMT2A MFN2 CMT2K GDAP1 CMT2B RAB7 AR-CMT2K CMT2C TRPV4 CMT2L HSP22 CMT2D GARS CMT2M DNM2 CMT2E NEFL CMT2N AARS CMT2F HSP27 DHTKD1 VCP CMT2I/J MPZ CMT4C1 LMNA COX6A1 CMT4C3 MED25 CMTX1 GJB1 KARS LRSAM1 INTERMEDIATE CMT CMT Type Gene DI-CMTB DNM2 DI-CMTC YARS DI-CMTD MPZ RI-CMTA GDAP1 RI-CMTB KARS - Mutations in a gene can cause several forms of CMT - Different patterns of inheritance for the same gene
- 3. Saporta & Shy Neuron Clin 2013 Algorithm for the genetic diagnosis of CMT patients
- 4. Saporta & Shy Neuron Clin 2013 Genetic diagnosis: - 91.2% of CMT1 - 43% of CMT2 Algorithm for the genetic diagnosis of CMT patients
- 5. Test for a specific gene More frequent genes Many possible causative genes Interesting phenotype Interesting family Gene-to-gene Gene Panel Whole Exome Sequencing (WES) Whole Genome Sequencing (WGS) Genetic diagnosis
- 6. Test for a specific gene More frequent genes Many possible causative genes Interesting phenotype Interesting family Gene-to-gene Gene Panel Whole Exome Sequencing (WES) Whole Genome Sequencing (WGS) Genetic diagnosis
- 7. Fridman et al. JNNP 2014 The most common genes: PMP22 duplication (CMT1A) Cx32 (CMTX1) MFN2 (CMT2A) MPZ (CMT1B) Inherited Neuropathies Consortium 17 sites: 10 in USA 1 in the UK 1 in Italy 1 in Australia
- 8. 1009 patients evaluated 571 Excluded (not CMT) 275 Demyelinating 163 Axonal 34 Gypsy241 Caucasian • 42 GDAP1 - 18 AR - 24 AD (CMT2K) • 31 GJB1 (CMTX1) • 10 MPZ (CMT2I/J) • 7 HSPB1 (CMT2F) • 4 MFN2 (CMT2A) • 3 NEFL (CMT2E) • 3 HSPB8 (CMT2L) • 1 GARS (CMT2D) • 1 KARS CMT1 •184 PMP22a (CMT1A) •25 GJB1 (CMTX1) •9 MPZ (CMT1B) •2 PMP22b (CMT1A) •1 NEFL (CMT1F) CMT4 •4 PRX (CMT4F) •2 SH3TC2 (CMT4C) •2 FGD4 (CMT4H) 12 Uncharacterized 61 Uncharacterized • 26 SH3TC2 (CMT4C) • 6 HK1 (CMT4G) • 2 NDRG1 (CMT4D) 438 CMT 0 Uncharacterized Sivera et al. Neurology 2013
- 9. Mutational analysis gene-to-gene - More frequent genes: PMP22, MPZ, Cx32, MFN2, GDAP1 - Candidate Genes according to clinical phenotype Associated symptom CMT type Gene Optic atrophy CMT2A CMTX5 MFN2 PRPS1 Hearing loss CMT4D/HSMN-Lom NDRG1 Diaphragm paralysis CMT4A GDAP1 Scoliosis CMT4C CMT4H SH3TC2 FGD4 Renal failure ID-CMT type E IFN2 Glaucoma CMT4B2 SBF2/MTMR13 Cataracts ID-CMT type B/CMT2M DNM2 Predominant hand wasting CMT2D GARS
- 10. Mutational analysis gene-to-gene Frederik Sanger Sanger method - Time-consuming - Tedious Hospital No. DNAs No. Analysis Identified mutation H. La Paz (Madrid) 78 118 25 H. La Fe (Valencia) 167 119 47 H. V. del Rocío (Seville) 48 75 28 H. Bellvitge (Barcelona) 26 15 7 289 327 107 More Frequent Genes - Candidate Genes - Over 90% of CMT1 and 50% of CMT2 can be diagnosed - In most of the hospitals, this is the only approach to be applied Department of Genomics and Translational Genetics - CIPF Biobank CIBERER prelimary results
- 11. Test for a specific gene More frequent genes Many possible causative genes Interesting phenotype Interesting family Gene-to-gene Gene Panel Whole Exome Sequencing (WES) Whole Genome Sequencing (WGS) Genetic diagnosis
- 12. Gene panels •All known CMT genes •Cost can be reduced •Provide a comprehensive view of the mutational load in all CMT genes Much better characterization of genotype-phenotype correlations A technical drawback for this approach is the less than 100% coverage of a sequence of interest - Technology will outperform Sanger sequencing in sensivity and specifity - Next Generation Sequencing platforms become more and more accessible
- 13. Gene panels 81 CMT families Genetic analysis: CMT1A duplication PMP22 GJB1 MPZ LITAF MFN2 EGR2 Mutation is identified in 22 families Panel with 51 genes 59 families studied by NGS 22 CMT1 29 CMT2 1 I-CMT 7 unknown CMT 20%
- 14. DHTKD1 DNM2 EGR2 FGD4 FIG4 GAN GDAP1 GJB1* GNB4 HARS HINT1 HK1** KARS KIF1B KIF5A LITAF LMNA LRSAM1 MARS MED25** MFN2 MICAL1 MPZ MTMR2 NDRG1 NEFL PDK3 PMP22 PRSP1 PRX RAB7A SBF1 SBF2 SH3TC2 SLC12A6 TDP1 TFG TRIM2 TUBA8 YARS ATP7A BICD2 BSCL2 DCTN1 HSPB3 PLEKHG5 SETX ATP7A BICD2 BSCL2 DCTN1 HSPB3 PLEKHG5 SETX [9 genes] AARS DNAJB2 DYNC1H1 FBLN5 GARS HSPB1 HSPB8 IGHMBP2 TRPV4 [9 genes] AARS DNAJB2 DYNC1H1 FBLN5 GARS HSPB1 HSPB8 IGHMBP2 TRPV4 CMT Genes [40 genes] dHMN genes [7 genes] *Promotor region included **Only founder mutation • 56 genes • 862 targets • 186.34 Kb • 99.98% coverage Gene panel_CMT&dHMN: based on Haploplex technology
- 15. ID no. Clinical form Inheritance Transmission Gene Nucleotide change Amino acid change SGT/036 CMT2 AD Heterozygosis MFN2 c.310C>T p.R104W DNA_121 CMT1 AD Heterozygosis MPZ c.21_26dupTGCCCC p.P9_A10dup DNA_837 CMT2 X-linked Heterozygosis GJB1 c.44_45delinsTT p.R15L DNA_872 CMT2 X-linked Heterozygosis GJB1 c.-540G>C No aa change DNA_554 CMT1 AR Heterozygosis PRX c.642insC p.R215QfsX8 c.589G>T p.E197XHeterozygosis DNA_571 CMT1 AR Homozygosis FGD4 c.1886delGAAA p.K630NfsX5 SGT/047 CMT1 AR Homozygosis HK1 g.9712G>C No aa change DNA_708 CMT2 AR Heterozygosis GDAP1 c.172_173delCTinsTTA p.P59AfsX4 Heterozygosis c.311-1G>A No aa change SGT/047 CMT1 AR Homozygosis HK1 g.9712G>C No aa change Heterozygosis SH3TC2 c.3325C>T p.R1109X SGT/044 CMT1 AR Heterozygosis SH3TC2 c.3325C>T p.R1109X Heterozygosis c.2211_2213delCCC p.C737_P738delinsX DNA_621 CMT1 AD Heterozygosis HSPB1 c.418C>G p.Arg140Gly Gene panel_CMT&dHMN: control group
- 16. Depth > 250x
- 17. Previous genetic analysis: •CMT1A duplication •Frequent genes - PMP22 - MPZ - GJB1 - GDAP1 - MFN2 Gene panel_CMT&dHMN: patients
- 18. fCMT-391 fCMT-416 fCMT-245 Mutational screeningGene Panel dHMN Gene panel_CMT&dHMN 2 cases → DNAJB2 c.352+1G>A Blumen et al. Ann Neurol 2012 Exome Sequencing fCMT-83
- 19. ID no. Clinical form Gene dbSNP/1000G Nucleotide Change Amino acid change Pathogenic effect DNA_1138 I-CMT HARS rs78741041/0.0055 c.14C>A p.Ala5Glu possibly damaging SGT/018 dHMN PLEKHG5 rs140202670/0.0023 c.1225C>T p.Arg409Trp deleterious SBF1 rs201776298/0.0045 c.868G>A p.Ala290Thr possibly damaging SGT/031 CMT2 MICAL1 rs201447051/0.0014 c.374T>C p.Leu125Pro deleterious SGT/029 I-CMT PLEKHG5 Novel c.800G>A p.Arg267His deleterious SETX rs151117904/0.0027 c.7727T>C p.Ile2576Thr possibly non-damaging SETX rs148568105 c.6013G>A p.Val2005Met deleterious SGT/030 CMT2 KIF1B rs121908162/0.0009 c.2480C>T p.Thr827Ile possibly non-damaging SETX rs61742937/0.0096 c.2975A>G p.Lys992Arg possibly non-damaging SETX rs79740039/0.0064 c.59G>A p.Arg20His possibly non-damaging SGT/068 CMT2 PRX Novel c.4077_4079delGGA p.Glu1360del possibly damaging SLC12A6 Novel c.1421A>G p.His474Arg possibly non-damaging SGT/072 CMT2 IGHMBP2 Novel c.1582G>A p.Ala528Thr possibly damaging SGT/081 CMT2 DNM2 Novel c.2201A>G p.Asn734Ser possibly non-damaging SGT/139 CMT2 HARS Novel c.989A>G p.Tyr323Cys deleterious HARS Novel c.679T>G p.Ser227Ala possibly damaging SGT/142 CMT2 MFN2 rs140234726 c.749G>A p.Arg250Gln possibly non-damaging LRSAM1 Novel c.2136_2143delCATCGCCCinsC p.Ile713SerfsTer20 possibly damaging SGT/106 CMT2 LRSAM1 Novel c.2083_2095delTGCTGCCAGCAGTinsT p.Cys696_Cys699del possibly damaging SGT/109 CMT2 PLEKHG5 Novel c.718G>A p.Asp240Asn possibly non-damaging SETX rs61742937/0.0096 c.2975A>G p.Lys992Arg possibly non-damaging SGT/114 I-CMT SETX Novel c.4289C>T p.Ser1430Phe possibly damaging SGT/170 CMT2 AARS rs138081804/0.0009 c.2185C>T p.Arg178Trp deleterious KARS Novel c.1603C>T p.Arg535Trp possibly damaging Gene panel_CMT&dHMN: results 14 cases → novel changes and/or variants with frequency ≤ 1%
- 20. ID no. Clinical form Gene dbSNP/1000G Nucleotide Change Amino acid change Pathogenic effect DNA_1138 I-CMT HARS rs78741041/0.0055 c.14C>A p.Ala5Glu possibly damaging SGT/018 dHMN PLEKHG5 rs140202670/0.0023 c.1225C>T p.Arg409Trp deleterious SBF1 rs201776298/0.0045 c.868G>A p.Ala290Thr possibly damaging SGT/031 CMT2 MICAL1 rs201447051/0.0014 c.374T>C p.Leu125Pro deleterious SGT/029 I-CMT PLEKHG5 Novel c.800G>A p.Arg267His deleterious SETX rs151117904/0.0027 c.7727T>C p.Ile2576Thr possibly non-damaging SETX rs148568105 c.6013G>A p.Val2005Met deleterious SGT/030 CMT2 KIF1B rs121908162/0.0009 c.2480C>T p.Thr827Ile possibly non-damaging SETX rs61742937/0.0096 c.2975A>G p.Lys992Arg possibly non-damaging SETX rs79740039/0.0064 c.59G>A p.Arg20His possibly non-damaging SGT/068 CMT2 PRX Novel c.4077_4079delGGA p.Glu1360del possibly damaging SLC12A6 Novel c.1421A>G p.His474Arg possibly non-damaging SGT/072 CMT2 IGHMBP2 Novel c.1582G>A p.Ala528Thr possibly damaging SGT/081 CMT2 DNM2 Novel c.2201A>G p.Asn734Ser possibly non-damaging SGT/139 CMT2 HARS Novel c.989A>G p.Tyr323Cys deleterious HARS Novel c.679T>G p.Ser227Ala possibly damaging SGT/142 CMT2 MFN2 rs140234726 c.749G>A p.Arg250Gln possibly non-damaging LRSAM1 Novel c.2136_2143delCATCGCCCinsC p.Ile713SerfsTer20 possibly damaging SGT/106 CMT2 LRSAM1 Novel c.2083_2095delTGCTGCCAGCAGTinsT p.Cys696_Cys699del possibly damaging SGT/109 CMT2 PLEKHG5 Novel c.718G>A p.Asp240Asn possibly non-damaging SETX rs61742937/0.0096 c.2975A>G p.Lys992Arg possibly non-damaging SGT/114 I-CMT SETX Novel c.4289C>T p.Ser1430Phe possibly damaging SGT/170 CMT2 AARS rs138081804/0.0009 c.2185C>T p.Arg178Trp deleterious KARS Novel c.1603C>T p.Arg535Trp possibly damaging Gene panel_CMT&dHMN: results 12 NOVEL VARIANTS 6 DELETERIOUS 9 POSSIBLY DAMAGING 9 POSSIBLY NON-DAMAGING 14 cases → novel changes and/or variants with frequency ≤ 1%
- 21. • Panel with 56 genes involved in CMT/dHMN • Depth > 250x DIAGNOSIS TOOL • Wide genetic heterogeneity of this group of neuropathies • Difficulties in order to establish the phenotypical consequences of the novel variants Relevance of segregation analysis Databases: www.treat-cmt.es GENETIC TESTING Gene panel_CMT&dHMN: conclusions
- 22. Test for a specific gene More frequent genes Many possible causative genes Interesting phenotype Interesting family Gene-to-gene Gene Panel Whole Exome Sequencing (WES) Whole Genome Sequencing (WGS) Genetic diagnosis
- 23. WES-WGS Majewski J et al. J Med Genet 2011 Metodology Year In how much time Cost Sanger 2001, Human Genome Project 13 years $3 billion NGS 2008 5 months $1 million NGS 2014 Some days $1.000 How much does it cost to sequence a genome?
- 24. WES-WGS: how to know which variants have phenotypic effect? Need of databases with variants associated with phenotypeNeed of databases with variants associated with phenotype
- 25. WES-WGS: NGS as a diagnosis tool Whole Genome Sequencing in a CMT patient 9.069 single nucleotide variants (SNVs) 159 missense SNVs associated with an inherited trait 21 associated with a Mendelian disease 16 detected in heterozygosis in the 5 changes were erroneously associated with disease SH3TC2 p.R954X/p.Y169H Whole Exome Sequencing in a CMT patient Some genes involved in CMT and related neuropathies identified by WES TUBB3 Tischfield et al. Cell 2010 SLC5A7 Barwick et al. AJHG 2012 MARS Gonzales et al. JNNP 2013 PDK3 Oates et al. AJHG 2013 SCN11A Leipold et al. Nat Genet 2013
- 26. CMT fCMT-248 I:1 I:2 II:2 1088 II:1 III:1 712 III:2 IV:1 III:4 IV:2 III:3 IV:3 ? II:4II:3 II:6II:5 III:5 ? III:7III:6 IV:4 IV:5 III:8 III:9 III IV:6 EGR2/Krox20 p.R409QEGR2/Krox20 p.R409Q WES-WGS: new genes, new mutations dHMN fCMT-83 DNAJB2 c.352+1G>ADNAJB2 c.352+1G>A
- 27. 1. Prioritize non synonymous SNVs 2. Changes segregating in heterozygosis 3. dbSNP variants excluded fCMT-248 118263 variants 920 non-synonymous SNVs 895 SNPs 25 non- synonymous SNVs
- 28. 1. Prioritize non synonymous SNVs 2. Changes segregating in heterozygosis 3. dbSNP variants excluded fCMT-248 118263 variants 920 non-synonymous SNVs 895 SNPs 25 non- synonymous SNVs Novel nucleotide change in EGR2/Krox20 chr10:64573172 c.1226G>A p.R409Q EGR2/Krox20
- 29. fCMT-248 CMT-248 healthy individuals CMT-248 affected individuals A CONFIRMATION – COSEGREGATION chr10:64573172 c.1226G>A p.R409Q EGR2/Krox20
- 30. fCMT-248 CMT-248 healthy individuals CMT-248 affected individuals A CONFIRMATION – COSEGREGATION chr10:64573172 c.1226G>A p.R409Q EGR2/Krox20 c.1226 G>A has not been found in any of 214 healthy individuals of Spanish population by dHPLC analysis.
- 31. fCMT-248 Polyphen 2 Arg409 conservation SIFT: Substitution at pos 409 from R to Q is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00 chr10:64573172 c.1226G>A p.R409Q EGR2/Krox20
- 32. fCMT-248 chr10:64573172 c.1226G>A p.R409Q EGR2/Krox20 R409Q mutation behaves similarly to DBD mutations Luc * * p value <0.005 Cx32-Promoter LIGHT EGR2 WT or Mutants Luciferase assay * *
- 33. Axonal CMT fCMT-248 I:1 I:2 II:2 1088 II:1 III:1 712 III:2 IV:1 III:4 IV:2 III:3 IV:3 ? II:4II:3 II:6II:5 III:5 ? III:7III:6 IV:4 IV:5 III:8 III:9 III IV:6 EGR2/Krox20 p.R409QEGR2/Krox20 p.R409Q I:1 I:2 II:2 1051 II:1 1050 III:2 676 III:1 1052 IV:1 1053 IV:2 1054 III:3 II:3 II:4 III:4 1089 III:5 Axonal CMT fCMT-237 I:1 I:2 II:2II:1 III:1 II:4II:3 III:2 III:3 III:4 II:6II:5 III:5 II:7 III:6 Hereditary Recurrent Neuropathy fCMT-266 Axonal CMT fCMT-197 Novel GeneNovel Gene Novel GeneNovel Gene WES-WGS: new genes, new mutations The beginning of a new story… Has the identified variant phenotypic effects? Why does the novel gene lead to a neuropathy? The beginning of a new story… Has the identified variant phenotypic effects? Why does the novel gene lead to a neuropathy? dHMN fCMT-83 DNAJB2 c.352+1G>ADNAJB2 c.352+1G>A
- 34. ControlRNAi 3-5d5weeks3-5d Sciatic nerve Brain 9 months Spinal cord Skeletal muscle Heart Liver Spleen Kidney Lung WES-WGS: new genes, new mutations “You are completely free to carry out whatever research you want, so long as you come to these conclusions” ControlHeat Shock The beginning of a new story… Has the identified variant phenotypic effects? Why does the novel gene lead to a neuropathy? The beginning of a new story… Has the identified variant phenotypic effects? Why does the novel gene lead to a neuropathy? WGS-WES • Next Generation Sequencing platforms become more and more accesible • Whole Exome Sequencing probably more suitable than Whole Genome Sequencing • Difficulties in order to establish the phenotypical consequences of the novel variants • Interesting phenotype/Interesting family • Research project
- 35. Thanks to… And to you for your attention to all the colleagues who participate in this research Funds: to the probands and their relatives for their kind colaboration
Notas del editor
- By exome sequencing we have achieved the genetic diagnosis in four families. In two of them, the gene and even the mutation had been previously related to CMT. We have also detected the same mutation in two unrelated families, this is a novel gene and we are currently investigating the function of this gene,. Something similar happens with the family 266. Thus, when a new gene is identified, a new story begins: