2008 Barcelona - European Society of Human Genetics
2008 Barcelona - European Society of Human Genetics 2008 Barcelona - European Society of Human Genetics
Molecular and biochemical basis of disease and to detect new sequence variants within SPG4 gene in 51 HSP patients in Estonia . Fifty healthy individuals were used as controls . The majority of patients had pHSP and only five patients had cHSP form of the disease . cHSP was the most frequent in sporadic cases . All 51 samples were screened with DHPLC and abnormal elution profiles were sequenced. Nine changes (G609A, A810G, 1299-1g>c, 1310delA, 1370+215g>c, 1370+202delG, C1503A, 1477-1481del- GAGAA, 1966insA) in SPG4 gene were detected showing no gender predisposition . Seven were new and only found in 11 HSP patients, but two (one new - 1370+215g>c and other previously described - 1370+202delG) were detected both in patients and controls . Only two mutations (1299-1g>c, C1503A) showed familial segregation, in which three and two family members were affected, respectively . Other mutations were found in individuals from different families . In conclusion we associate above-mentioned seven new mutations with 11 AD-HSP cases, since new sequence variants were found only in patients compared to healthy controls . The rest of the patients must be checked for other changes (larger INDELs) in SPG4 and other genes involved in this disease should be considered for further analysis . P05.187 in depth investigation of -1 frameshifting in expanded cAG repeat tracts using time-lapse live cell imaging S. J. Stochmanski 1 , C. Gaspar 1 , D. Rochefort 1 , J. Laganiere 1 , P. Hince 1 , G. Di Cristo 2 , G. A. Rouleau 1 ; 1 Centre of Excellence in Neuromics, CHUM Research Centre, Montréal, QC, Canada, 2 Centre de Recherche, Hôpital Ste-Justine, Montréal, QC, Canada. Spinocerebellar ataxia type 3 (SCA3) results from an expansion of a polyglutamine-encoding CAG tract in the ATXN3 gene . We have previously demonstrated that this expanded CAG tract is subject to -1 ribosomal frameshifting into the alanine frame, which seems to confer an increased toxicity, and that the antibiotic anisomycin reduces both -1 frameshifting and cell toxicity . Currently, dual-tagged ATXN3 reporter constructs were created to express DsRED in the main (glutamine) frame and EGFP in the -1 (alanine) reading frame, and these reporter constructs contained either 14 CAG repeats, 89 CAG repeats, or 92 CAA repeats . Constructs were transfected into COS-1 cells as well as mouse cortical organotypic slice culture preparations, and were monitored for the production of red or green fluorescence signals using time-lapse live-cell two-wave fluorescent microscopy. Employing this technique, we have confirmed the occurrence of -1 frameshifting for the CAG89 construct, whereas the constructs bearing wild-type CAG or expanded CAA repeats did not show significant frameshifting. We also determined that there is a marked time delay between the onset of glutamine-containing protein expression and the production of frameshifted species, as well as a correlation between frameshifitng and cell death. These findings argue in favour of local glutamine codon starvation, followed by a shift in the reading frame to resume translation of the protein in the alanine frame and seem to confirm the implication of -1 ribosomal frameshifting in the pathogenesis of SCA3 . P05.188 Quantitative analysis of SMN gene based on real-time PcR M. Šimášková 1 , H. Poláková 2 , L. Kádaši 1,2 ; 1 Comenius University, Faculty of Natural Sciences, Department of Molecular Biology, Bratislava, Slovakia, 2 Institute of Molecular Physiology and genetics Slovak Academy of Science, Bratislava, Slovakia. Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by the homozygous absence of the survival motor neutron gene (SMN1) in approximately 94% of patients . Since most carriers have only one SMN1 gene copy, several quantitative analyses have been used for the SMA carrier detection . We performed a SMN1quantitative real-time PCR analysis using an allele specific primer for the carrier detection of SMA. We compared the sensitivity, specificity, advantages and disadvantages recently described in the quantitative method, using TaqMan probes and a newly developed Plexor TM technology, which had not previously been used for identifying heterozygotes . Using a comparative threshold cycle (C T ) method and the DNA fragment of human beta globine gene as a reference gene, the gene copy number of SMN1 was quantified. The sensitivity and specificity of the TaqMan and Plexor TM technologies were similar; moreover, the assay efficiency was almost ideal when using the Plexor TM technology . The incidence of SMA (1:6000-10000) and the notable carrier frequency (1:35-50) as well as the severity of disease, and the lack of effective treatment may justify the implementation of such analysis in DNA diagnostic laboratories . P05.189 molecular Genetics and Epidemiology in spanish spinocerebellar Ataxia H. San Nicolás 1 , J. Corral 1 , I. Banchs 1 , L. De Jorge 1 , O. Combarros 2 , J. Berciano 2 , C. Serrano 3 , M. Calopa 4 , A. Matilla 5 , D. Genís 6 , V. Volpini 1 ; 1 Center for Molecular Genetic Diagnosis of Hereditary Diseases (CDGM)- IDI- BELL, Hospitalet de Llobregat, Spain, 2 Dep of Neurology. Hosp Univ Marqués de Valdecilla, Santander, Spain, 3 Dep of Neurology. Hosp Sant Joan de Deu, Martorell, Spain, 4 Dep of Neurology. Hosp Univ De Bellvitge. IDIBELL, Hospitalet de Llobregat, Spain, 5 Health Sciences Research Institute Germans Trias i Pujol, Badalona, Spain, 6 Dep of Neurology. Hosp Univ Josep Trueta, Girona, Spain. Autosomal dominant cerebellar ataxias (ADCA) are a clinically and genetically heterogeneous group of neurodegenerative disorders in which several spinocerebellar ataxia (SCA) genes have been cloned: SCAs1-3, SCAs6-7, SCA12 and SCA17; sharing a CAG repeat expansion mutations which generally encodes a polyglutamine tract . In SCA8 the mutation is an untranslated CTG repeat . We have analyzed 340 unrelated familial and 1,013 sporadic and idiopathic cases of SCA . Over the familial cases 6 .04% were SCA1; 26 .85% SCA2; 32 .89% SCA3; 7 .38% SCA6; 6 .71% SCA7; 14 .77% SCA8; and 1 .34% SCA17 . In 22 familial index cases with SCA8 expansions the allele range goes from 85 to 470 repeats (129 .36% ± 67 .55%; Pearson Coef . =52 .22%) . Maternal transmissions presented elongations of the CTG combined sequence ranging from +2 to +13 repeats (7 .50 ± 5 .5; Pearson Coef . = 73 .33%) . In contrast, paternal transmissions presented contractions ranging from 1 to -17 repeats (-6 .83 ± 7 .51; Pearson Coef = - 109 .93%) . Several giant SCA8 expansions ranges from 401 to 1,126 (N= 9), carried by unaffected adult individuals and being originated from homozygous SCA8 females with alleles of moderate size . In contrast, the homozygous males have transmitted contracted alleles, as in heterozygous cases occurs . We have tested 90 individuals from general population and the distribution of SCA8 alleles could be classified in two groups: 15 to 34 CTGs, with frequency 98% and 77 to 86 CTGs, with frequency 2% . About 60% of familial ADCA cases remained genetically unclassified. No SCA mutations were detected in the 1,013 isolated and idiopathic cases of spinocerebellar ataxia . P05.190 Novel mutations found in ABcA4 in spanish families J. Aguirre-Lamban1,2 , R. Riveiro-Alvarez1,2 , D. Cantalapiedra1,2 , M. Garcia- Hoyos1,2 , E. Vallespin1,2 , A. Avila-Fernandez1,2 , J. Gallego-Merlo1,2 , M. Lopez- Martinez1,2 , M. Trujillo-Tiebas1,2 , C. Ramos1,2 , C. Ayuso1,2 ; 1 2 Fundacion Jimenez Diaz, Madrid, Spain, Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain. Introduction: ABCA4 mutations have been associated with Stargardt disease (STGD) . A few cases with autosomic recessive cone-rod dystrophy (arCRD) and autosomic recessive retinitis pigmentosa (arRP) have also been found to have ABCA4 mutations . Comparative genetic analyses of ABCA4 variation and diagnostics have been complicated by substantial allelic heterogeneity . Subjects and Methods . 31 unrelated families, were previously studied with the ABCR400 genotyping microarray . In patients with either none or only one mutant allele were analysed by dHPLC, sequencing and multiplex ligation-dependent probe amplification (MLPA). Haplotype analysis was also performed . Results . 27 ABCA4 mutations were found in 31 Spanish patients with the ABCR400 microarray. We confirmed that the p.Arg1129Leu mutation is the most frequent in Spanish patients. dHPLC allowed us to find eleven novel mutations and were not found in the 100 chromosomes . Using both tools, the mutation detection rate obtained was incremented in a 24 .2% with respect to the use of the microarray alone . We detected 1 .6 % of false positives and 1 .6 % of false negatives . In 17/31 patients (54 .8%) in which the second or neither mutation was found by these methodologies, they were studied with MLPA; however no deletion or duplication was found in these samples . No mutation was found in 4/31 patients (12 .9%) . Conclusions . The ABCR400 microarray is a comprehensive screening tool for genetic variation in patients with ABCR-associated retinal pa-
Molecular and biochemical basis of disease thology . We consider that the combination of microarray, dHPLC and MLPA is the optimal screening strategy for mutation analysis in this huge gene in Spanish patients . P05.191 Genomic structural Variation in patients with multiple sclerosis E. Docampo 1,2 , R. Rabionet 1,2 , M. García 1,2 , J. E. Martínez 3 , E. Munteis 3 , J. Roquer 3 , L. Armengol 1,2 , X. Estivill 1,2 ; 1 Center for Genomic Regulation, Barcelona, Spain, 2 Public Health and Epidemiology Network Biomedical Research Center (CIBERESP), Barcelona, Spain, 3 Neurology Service, Hospital del Mar-IMAS, Barcelona, Spain. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease that affects the central nervous system . Several studies provide evidence that MS is a complex disorder involving genetic and environmental factors . Several different approaches have been undertaken to elucidate the genetic causes of MS, including linkage and association studies . These have consistently shown association to the major histocompatibility complex (MHC) region, specifically the DR15 haplotype. Other MS genes have been more elusive, and only ILR7 has shown up as a clear risk factor . The aim of this study was to evaluate a possible contribution of genomic structural variation to MS susceptibility . Forty relapsing-remitting MS samples were divided into two pools and comparative genomic hybridization (CGH) against a pool of 50 control samples was performed using Agilent 244K arrays . With the initial selection criteria (three consecutive probes with a log2 ratio above 0 .29), only the chromosome 6 MHC region, spanning four to six probes (depending on the pool), showed differential hybridization between cases and controls . Relaxing the criteria to only two consecutive probes, five additional regions showed a difference in cases versus controls and were selected for follow-up by RTqPCR experiments . In addition, based on the association shown by the MHC region, a SNPlex experiment with 48 SNPs from HLA class II region was designed, including SNPs from potential CNV regions and SNPs not analyzed by other platforms, and three of these SNPs were shown to be significantly associated to MS . P05.192 Analysis of raw data from mLPA-based assays: development of a new web-based software ,,emLPA“ J. Camajova, M. Krhounek, M. Hancarova, J. Vejvalka, M. Macek, M. Macek Jr; Department of Biology and Medical Genetics, Charles Univ.-2nd Med. School and Univ. Hospital Motol, Prague, Czech Republic. MLPA allows detection of large deletions/duplications that would otherwise remain undetected by standard PCR-based techniques . MLPA is based on relative quantification of PCR-multiplexed specific sequences, and for interpretation, computerized analysis of data is necessary . Available commercial, proprietary or freely distributed software packages differ in user friendliness, diagnostic utility, sensitivity and specificity. Usually, these software applications also do not allow optimization of utilized algorithms by long-term “feedback learning” from stored data . Our “eMLPA” software aims to provide a universal computational interface for all MLPA-based assays . While other software packages use defined algorithms for data analysis, eMLPA allows selection of variant methods for e .g . probe normalization . As none of the individual steps (peak discrimination, e .g .) is a trivial task, instead of rigid computations our approach suggests solutions to be verified, corrected or rejected in interaction with users . Data transformation via independent procedures allows for flexibility and comparison of results at various analytical steps . Data manipulation (management of users, probes, samples and results) is implemented in a web-based system, with no other requirements than a browser . To support the logistics of teamwork properly, eMLPA allows users to create, manage and collaborate on defined projects. eMLPA also offers long-term storage of anonymized intermediary data for evaluation of different numerical methods, for data quality control or for assessment of variance of results of specific probes within a given MLPA mix . Supported by VZFNM 000064203 and Medigrid 1ET202090537 . P05.193 Gene expression signature of adrenaline biosynthesis and inflammatory pathways in women with left ventricular apical ballooning syndrome N. Marziliano1 , M. Grasso1 , A. Repetto1 , E. Porcu1 , A. Pilotto1 , M. Diegoli2 , M. Tagliani1 , E. Disabella1 , A. Eloisa1 ; 1 2 Fondazione IRCCS Policlinico San Matteo, PAVIA, Italy, University of Pavia, PAVIA, Italy. Marked elevation of plasma catecholamine levels has been reported in transient left ventricular apical ballooning syndrome (LVABS) . Using quantitative PCR (Q-PCR), we investigated the expression profiles of inflammatory and adrenergic pathways in the RNA isolated from peripheral leukocytes, and from lymphocyte and monocyte subsets of 16 female patients with LAVBS, and compared with 7 age-matched women with acute myocardial infarction (AMI) and 17 presenting with chest pain and angiographically normal coronary arteries . Three of the 351 genes belonging to the adrenaline biosynthesis pathway [DAT (Neurotransmitter transporter, dopamine), AT (Sodium-dependant amino acid transporter) and NET (Norepinephrine transporter)] were over-expressed in LVABS than in AMI, whereas COMT (Catechol-Omethyltransferase) and PMNT (Phenylethanolamine N-methyltransferase) were under-expressed. Similarly 3 of the 343 genes of the inflammatory pathway [IL1β (Interleukin 1β), TNFα (Tumor necrosis factoralpha), IL10 (Interleukin-10)] were overexpressed in AMI while TBX21 (T-cell-specific T-box transcription factor) in LVABS; although higher in LVABS, MAD-homolog was underexpressed in both AMI and LVABS than in controls; CCR5 (Chemochine receptor 5) was underexpressd in AMI than in LVABS and controls . In addition 3 of 178 genes of the angiogenesis pathway [AT1R (Angiotensin receptor 1), AT2R (Angiotensin receptor 2) and HIF2A (Endothelial-pas domain protein 1)] were overexpressed in the AMI than in LVABS and controls, while iNOS was more expressed in controls than LVABS and AMI . The lymphocytes did not express DAT, NET, AT, DOPA and COMT . Our gene expression results suggest an overactivity of adrenergic and inflammatory pathways in LVABS and replicate the biochemical profile documented previously in LVABS . P05.194 Research Report: Analysis of the tcR transciptome by tcLandscape: a tool for t cell immune response characterization and patient follow-up C. Ruiz1 , P. Miqueu1 , L. Xu2 , S. Jankowski2 ; 1 2 TcLand Expression, Nantes, France, Applied Biosystems, Foster City, CA, United States. The understanding of T cells during the immune response follow-up is a crucial step in the development and improvement of immunotherapy treatments. TcLand has developed a new workflow and tool: the Tc- Landscape . This method provides a global and detailed analysis of the T Cell Receptor (TCR) β-chain transcriptome. The TcLandscape consists of a quantitative analysis of TCR β-chain mRNA expression by real-time PCR, followed by a qualitative diversity study and T cell selection based on the collection of the TCR Complementarity Determining Region 3 (CDR3) length distribution (CDR3-LD) with a capillary electrophoresis sequencer . An integrated analysis is then performed to assess the contribution of each Vβ chain length with the total T cell repertoire . The result is displayed in a 4-dimensional visualization graph. Novel dedicated statistical analysis methods, including specific signal treatment algorithms and multidimensional data reduction methods, allow the identification of significant differences between the different groups of patients . The ability to analyze whole T cell populations, as well as specific T cells, such as CD4+/CD8+ or Treg cells, provides a powerful tool to highlight sub-populations of interest in order to understand auto-immune diseases, transplantation, vaccination and other immune responses. In this presentation, we will outline the workflow to conduct a TcLandscape study . P05.195 characterization of a spontaneous, recessive, missense mutation arising in the Tecta gene M. Moreno-Pelayo 1,2 , R. Goodyear 3 , A. Mencía 1,2 , S. Modamio-Høybjør 1,2 , K. Legan 3 , L. Olavarrieta Scappini 1,2 , F. Moreno 1,2 , G. Richardson 3 ; 1 Unidad de Genética Molecular-Hospital Ramón y Cajal, Madrid, Spain, 2 Centre
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Molecular and biochemical basis <strong>of</strong> disease<br />
thology . We consider that the combination <strong>of</strong> microarray, dHPLC and<br />
MLPA is the optimal screening strategy for mutation analysis in this<br />
huge gene in Spanish patients .<br />
P05.191<br />
Genomic structural Variation in patients with multiple sclerosis<br />
E. Docampo 1,2 , R. Rabionet 1,2 , M. García 1,2 , J. E. Martínez 3 , E. Munteis 3 , J.<br />
Roquer 3 , L. Armengol 1,2 , X. Estivill 1,2 ;<br />
1 Center for Genomic Regulation, <strong>Barcelona</strong>, Spain, 2 Public Health and Epidemiology<br />
Network Biomedical Research Center (CIBERESP), <strong>Barcelona</strong>, Spain,<br />
3 Neurology Service, Hospital del Mar-IMAS, <strong>Barcelona</strong>, Spain.<br />
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease<br />
that affects the central nervous system . Several studies provide<br />
evidence that MS is a complex disorder involving genetic and environmental<br />
factors . Several different approaches have been undertaken to<br />
elucidate the genetic causes <strong>of</strong> MS, including linkage and association<br />
studies . These have consistently shown association to the major histocompatibility<br />
complex (MHC) region, specifically the DR15 haplotype.<br />
Other MS genes have been more elusive, and only ILR7 has shown<br />
up as a clear risk factor . The aim <strong>of</strong> this study was to evaluate a possible<br />
contribution <strong>of</strong> genomic structural variation to MS susceptibility .<br />
Forty relapsing-remitting MS samples were divided into two pools and<br />
comparative genomic hybridization (CGH) against a pool <strong>of</strong> 50 control<br />
samples was performed using Agilent 244K arrays . With the initial selection<br />
criteria (three consecutive probes with a log2 ratio above 0 .29),<br />
only the chromosome 6 MHC region, spanning four to six probes (depending<br />
on the pool), showed differential hybridization between cases<br />
and controls . Relaxing the criteria to only two consecutive probes,<br />
five additional regions showed a difference in cases versus controls<br />
and were selected for follow-up by RTqPCR experiments . In addition,<br />
based on the association shown by the MHC region, a SNPlex experiment<br />
with 48 SNPs from HLA class II region was designed, including<br />
SNPs from potential CNV regions and SNPs not analyzed by other<br />
platforms, and three <strong>of</strong> these SNPs were shown to be significantly associated<br />
to MS .<br />
P05.192<br />
Analysis <strong>of</strong> raw data from mLPA-based assays: development <strong>of</strong><br />
a new web-based s<strong>of</strong>tware ,,emLPA“<br />
J. Camajova, M. Krhounek, M. Hancarova, J. Vejvalka, M. Macek, M. Macek<br />
Jr;<br />
Department <strong>of</strong> Biology and Medical <strong>Genetics</strong>, Charles Univ.-2nd Med. School<br />
and Univ. Hospital Motol, Prague, Czech Republic.<br />
MLPA allows detection <strong>of</strong> large deletions/duplications that would otherwise<br />
remain undetected by standard PCR-based techniques . MLPA<br />
is based on relative quantification <strong>of</strong> PCR-multiplexed specific sequences,<br />
and for interpretation, computerized analysis <strong>of</strong> data is necessary<br />
. Available commercial, proprietary or freely distributed s<strong>of</strong>tware<br />
packages differ in user friendliness, diagnostic utility, sensitivity and<br />
specificity. Usually, these s<strong>of</strong>tware applications also do not allow optimization<br />
<strong>of</strong> utilized algorithms by long-term “feedback learning” from<br />
stored data .<br />
Our “eMLPA” s<strong>of</strong>tware aims to provide a universal computational interface<br />
for all MLPA-based assays . While other s<strong>of</strong>tware packages use<br />
defined algorithms for data analysis, eMLPA allows selection <strong>of</strong> variant<br />
methods for e .g . probe normalization . As none <strong>of</strong> the individual steps<br />
(peak discrimination, e .g .) is a trivial task, instead <strong>of</strong> rigid computations<br />
our approach suggests solutions to be verified, corrected or rejected<br />
in interaction with users . Data transformation via independent procedures<br />
allows for flexibility and comparison <strong>of</strong> results at various analytical<br />
steps . Data manipulation (management <strong>of</strong> users, probes, samples<br />
and results) is implemented in a web-based system, with no other<br />
requirements than a browser . To support the logistics <strong>of</strong> teamwork<br />
properly, eMLPA allows users to create, manage and collaborate on<br />
defined projects. eMLPA also <strong>of</strong>fers long-term storage <strong>of</strong> anonymized<br />
intermediary data for evaluation <strong>of</strong> different numerical methods, for<br />
data quality control or for assessment <strong>of</strong> variance <strong>of</strong> results <strong>of</strong> specific<br />
probes within a given MLPA mix .<br />
Supported by VZFNM 000064203 and Medigrid 1ET202090537 .<br />
P05.193<br />
Gene expression signature <strong>of</strong> adrenaline biosynthesis and<br />
inflammatory pathways in women with left ventricular apical<br />
ballooning syndrome<br />
N. Marziliano1 , M. Grasso1 , A. Repetto1 , E. Porcu1 , A. Pilotto1 , M. Diegoli2 , M.<br />
Tagliani1 , E. Disabella1 , A. Eloisa1 ;<br />
1 2 Fondazione IRCCS Policlinico San Matteo, PAVIA, Italy, University <strong>of</strong> Pavia,<br />
PAVIA, Italy.<br />
Marked elevation <strong>of</strong> plasma catecholamine levels has been reported in<br />
transient left ventricular apical ballooning syndrome (LVABS) .<br />
Using quantitative PCR (Q-PCR), we investigated the expression pr<strong>of</strong>iles<br />
<strong>of</strong> inflammatory and adrenergic pathways in the RNA isolated from<br />
peripheral leukocytes, and from lymphocyte and monocyte subsets <strong>of</strong><br />
16 female patients with LAVBS, and compared with 7 age-matched<br />
women with acute myocardial infarction (AMI) and 17 presenting with<br />
chest pain and angiographically normal coronary arteries . Three <strong>of</strong><br />
the 351 genes belonging to the adrenaline biosynthesis pathway [DAT<br />
(Neurotransmitter transporter, dopamine), AT (Sodium-dependant<br />
amino acid transporter) and NET (Norepinephrine transporter)] were<br />
over-expressed in LVABS than in AMI, whereas COMT (Catechol-Omethyltransferase)<br />
and PMNT (Phenylethanolamine N-methyltransferase)<br />
were under-expressed. Similarly 3 <strong>of</strong> the 343 genes <strong>of</strong> the inflammatory<br />
pathway [IL1β (Interleukin 1β), TNFα (Tumor necrosis factoralpha),<br />
IL10 (Interleukin-10)] were overexpressed in AMI while TBX21<br />
(T-cell-specific T-box transcription factor) in LVABS; although higher in<br />
LVABS, MAD-homolog was underexpressed in both AMI and LVABS<br />
than in controls; CCR5 (Chemochine receptor 5) was underexpressd<br />
in AMI than in LVABS and controls . In addition 3 <strong>of</strong> 178 genes <strong>of</strong> the<br />
angiogenesis pathway [AT1R (Angiotensin receptor 1), AT2R (Angiotensin<br />
receptor 2) and HIF2A (Endothelial-pas domain protein 1)] were<br />
overexpressed in the AMI than in LVABS and controls, while iNOS was<br />
more expressed in controls than LVABS and AMI . The lymphocytes did<br />
not express DAT, NET, AT, DOPA and COMT .<br />
Our gene expression results suggest an overactivity <strong>of</strong> adrenergic and<br />
inflammatory pathways in LVABS and replicate the biochemical pr<strong>of</strong>ile<br />
documented previously in LVABS .<br />
P05.194<br />
Research Report: Analysis <strong>of</strong> the tcR transciptome by<br />
tcLandscape: a tool for t cell immune response characterization<br />
and patient follow-up<br />
C. Ruiz1 , P. Miqueu1 , L. Xu2 , S. Jankowski2 ;<br />
1 2 TcLand Expression, Nantes, France, Applied Biosystems, Foster City, CA,<br />
United States.<br />
The understanding <strong>of</strong> T cells during the immune response follow-up is<br />
a crucial step in the development and improvement <strong>of</strong> immunotherapy<br />
treatments. TcLand has developed a new workflow and tool: the Tc-<br />
Landscape . This method provides a global and detailed analysis <strong>of</strong><br />
the T Cell Receptor (TCR) β-chain transcriptome. The TcLandscape<br />
consists <strong>of</strong> a quantitative analysis <strong>of</strong> TCR β-chain mRNA expression<br />
by real-time PCR, followed by a qualitative diversity study and T cell<br />
selection based on the collection <strong>of</strong> the TCR Complementarity Determining<br />
Region 3 (CDR3) length distribution (CDR3-LD) with a capillary<br />
electrophoresis sequencer . An integrated analysis is then performed<br />
to assess the contribution <strong>of</strong> each Vβ chain length with the total T cell<br />
repertoire . The result is displayed in a 4-dimensional visualization<br />
graph. Novel dedicated statistical analysis methods, including specific<br />
signal treatment algorithms and multidimensional data reduction methods,<br />
allow the identification <strong>of</strong> significant differences between the different<br />
groups <strong>of</strong> patients .<br />
The ability to analyze whole T cell populations, as well as specific T<br />
cells, such as CD4+/CD8+ or Treg cells, provides a powerful tool to<br />
highlight sub-populations <strong>of</strong> interest in order to understand auto-immune<br />
diseases, transplantation, vaccination and other immune responses.<br />
In this presentation, we will outline the workflow to conduct<br />
a TcLandscape study .<br />
P05.195<br />
characterization <strong>of</strong> a spontaneous, recessive, missense<br />
mutation arising in the Tecta gene<br />
M. Moreno-Pelayo 1,2 , R. Goodyear 3 , A. Mencía 1,2 , S. Modamio-Høybjør 1,2 , K.<br />
Legan 3 , L. Olavarrieta Scappini 1,2 , F. Moreno 1,2 , G. Richardson 3 ;<br />
1 Unidad de Genética Molecular-Hospital Ramón y Cajal, Madrid, Spain, 2 Centre
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