Patent Application: US-201013391572-A

Abstract:
method for in vitro diagnosis of susceptiblility of developing venous malformations i the extracranial segment of the cerebrospinal veins in a patient comprising the detection of copy number variations in chromosome 6p21 in a sample of genomic dna of the patient , wherein the venous malformations are associated with the development of multiple sclerosis .

Description:
in the following description , numerous specific details are given to provide a thorough understanding of embodiments . the embodiments can be practiced without one or more of the specific details , or with other methods , components , materials , etc . in other instances , well - known structures , materials , or operations are not shown or described in detail to avoid obscuring aspects of the embodiments . reference throughout this specification to “ one embodiment ” or “ an embodiment ” means that a particular feature , structure , or characteristic described in connection with the embodiment is included in at least one embodiment . thus , the appearances of the phrases “ in one embodiment ” or “ in an embodiment ” in various places throughout this specification are not necessarily all referring to the same embodiment . furthermore , the particular features , structures , or characteristics may be combined in any suitable manner in one or more embodiments . the headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments . the present inventors to investigate the occurrence of copy number variations ( cnvs ) underlining genome unbalances in the major locus ( hla , chromosome 6p21 ) associated with multiple sclerosis ( ms ) recur to a comparative genomic hybridisation ( cgh ) array in order to overcome the inevitable errors associated with the snps - based arrays . it is now well recognised that copy number variations ( cnvs ) typically ranging from 1 kb to several mb do contribute to genetic variations and disease susceptibility . cnvs account for more nucleotide variations between individuals ; in addition , the functional meaning of cnvs might be more immediate , especially for those located within genes , regulatory regions or known imprinted regions , since the possible consequence of the genome unbalance ( s ) may be interpretable in a more straight forward way . the advantage to search for cnvs by the cgh technique is the possibility to directly correlate the known , validated cnvs with a possible function , both related to the specific gene unbalanced and to other genomic regions with copy variations . in fact , cgh approach allows to finely mapping the identified cnvs in non - genic regions , possibly correlated to genes regulatory function , epigenetic changes or other non - coding functions . the development of robust high throughput platforms based on comparative genomic hybridisation ( cgh ) capable of identifying thousand genomic variations has greatly improved the research in this direction , as recently demonstrated in a major neurodegenerative disorder , the amiotrophic lateral sclerosis , in which non - polymorphic sub - microscopic duplications and deletions seem to be frequent in sporadic cases . the present inventors designed a locus - specific cgh array in order to explore the occurrence of cnvs in the hla - dra locus region ( 6 , 899 , 999 bp ; chromosome 6p21 : 29 , 900 , 001 - 36 , 800 , 000 bp ) in 15 patients with the peculiar association of chronic cerebrospinal venous insufficiency ( ccsvi ) and ms phenotype . the present inventors already described the peculiar association of chronic cerebrospinal venous insufficiency ( ccsvi ) in patients with ms in the international patent application no . pct / it2008 / 000129 . in such an application the inventors demonstrated that ccsvi is due to stenosing venous malformations ( vm ) affecting the azygous and the jugular veins , leading to significant anomalies in cerebral venous outflow haemodynamics . the hampered cerebral venous return in consequence of the extracranial venous malformations is peculiar to ms , and was not found in a miscellaneous of patients affected by other neurodegenerative disorders , such as parkinson &# 39 ; s , alzheimer &# 39 ; s , amyotrophic lateral sclerosis . in total patients showed 322 cnvs of which 225 extragenic and 97 intragenic . the present inventors identified 234 known polymorphic cnvs in the 15 patients having such a “ plus ” phenotype ( i . e . vm - ccsvi and ms phenotype ). looking at the distribution of the polymorphic cnvs identified in these patients , the present inventors observed a peak of cnvs number within the hla region . outside this specific region however , the number of cnvs per patients remains high , though with variable distribution . interesting , the overall number of cnvs showed a correlation with the number of stenosing vm as demonstrated by venography in the extracranial segments of the cerebrospinal veins , with a trend toward significance . the contribution to the correlation is certainly due to the extragenic cnvs , being significantly correlated to the number of stenosing vm ( r = 0 . 53 , r = 0 . 28 , p & lt ; 0 . 05 ). this result suggests that the contribution of extragenic cnvs in the development of the associated vm is related to their regulatory action in the process of angiogenesis . the region studied contains 211 known genes . using the functional bioinformatics tool the present inventors identify many genes interacting in both neurodegenerative and angiogenesis circuits . notably , hspa1l , hsp1a and hsp1b and the hladq2 genes network on both pathways . heat - shock proteins ( hsps ) represent a group of regulatory proteins involved in a variety of processes , including immunity and angiogenesis . in particular , hspa1l expression is modulated by ets1 transcription factor and by sp100 a nuclear autoimmune antigen . interestingly , genes negatively regulated by ets1 and up - regulated by sp100 , as hspa1l , have anti - migratory or anti - angiogenic properties . ms has a very well known major heritable component since its susceptibility is associated with the mhc class ii region , especially hla - drb5 * 0101 - hla - drb1 * 1501 - hla - dqa1 * 0102 - hladqb1 * 0602 haplotypes , which dominate genetic contribution to ms risk [ 20 ]. interestingly hladqa2 is known to be involved with pro - inflammatory cd4 (+) t - cell - mediated autoimmune diseases , such as ms and type 1 diabetes [ 21 ]. cd4 is also a very well known inhibitor of tumor angiogenesis [ 22 ], so supporting a link between the two pathways . the interpretation of the pathway interaction is obviously complex , but it suggests biological and functional links among these genes as well as , intriguingly , between angiogenesis and immunity . consistently with the general meaning of extragenic cnvs , putatively involved in gene expression regulation , this finding is interesting , since it supports the possibility that the number of structural variations laying within regulatory regions may represent a genomic “ perturbation ” increasing susceptibility for vm phenotype associated with ms the present inventors described . regarding specific candidate genes which expression could be potentially disturbed by genomic unbalances or “ perturbation ”, the pathways analysis suggests that genes involved in angiogenesis and immunity are the more interesting proteins . fifteen patients affected by the relapsing - remitting clinical course of ms diagnosed according to the revised mc donald criteria as disclosed in [ 11 ] entered the study . the present inventors determined the expanded disability disease score ( edss ) as disclosed in [ 12 ], as well as the multiple sclerosis severity score ( ms - ss ) as disclosed in [ 13 , 14 ]. the kurtzke expanded disability status scale ( edss ) is a method of quantifying disability in multiple sclerosis . the edss categorizes a person &# 39 ; s level of disability . edss scores range from 0 - 10 , with higher scores indicating more severe disability . the ms - ss is a relationship between the edss and the disease duration , being the ms clinical course more aggressive in accordance with the time in which a certain score has been reached . in the present population ms was associated with ccsvi venous malformation documented by a sequential colour - doppler / selective venography protocol as disclosed in the international patent application no . pct / it2008 / 000129 as well as in [ 9 ]. the clinical and demographic characteristics of the selected patients are given in the table 1 . patients signed an informed consent . detailed information about each of the 15 patients is provided in table 2 . dna from the 15 patients was extracted by protocol recommended by agilent using the qiagen dneasy blood & amp ; tissue kit . dna from the 15 patients was extracted by protocol recommended by agilent . highly concentrated dna was checked with a nanodrop for quality ( 260 / 280 ratio − 1 . 8 and 260 / 230 ratio = 2 . 0 ). dna integrity was evaluated on agarose gel at 1 % in tbe 1 ×. clinical data are given as median and interquartile range . genotype - phenotype correlations were analyzed by the means of linear regression analysis , including evaluation of slope , x and y intercept , followed by run test . p values & lt ; 0 . 05 were considered to be significant . ms - cgh microarray design was carried out using the web based agilent earray database version 5 . 4 ( agilent technologies , santa clara , calif .) [ 16 ]. the high density acgh search function within earray was used to turn the genomic region chr6 : 29 , 900 , 001 - 36 , 800 , 000 ( march 2006 human reference sequence , ncbi build 36 . 1 , hg18 ;) into a probe set by selecting the maximum number of exonic , intronic and intragenic 60mer oligonucleotide cgh probes available in the database . this probe set included 43102 probes that were used to reach the array format of 4 × 44 k , creating four identical 44 k arrays on a single slide for simultaneous analysis of four different samples . this platform called ms - cgh is a high - density microarray with a resolution of one probe every 160 bp which allow the rapid determination of the molecular profile identifying the presence of copy number variations ( cnvs ) in heterozygosity or in homozygosity in the genomic region studied . labelling and hybridisation were performed following the protocols provided by agilent ( agilent oligonucleotide array - based cgh for genomic dna analysis protocol v5 . 0 ) as described in [ 17 ]. briefly , genomic dna from a control and a patient in the same quantity were digested by restriction enzymes alui and rsai in separate tubes . the obtained fragments of dna from each samples were amplified in presence of cyanine 3 - dutp ( control sample ) and cyanine 5 - dutp ( patient sample ). after a step of purification and quantitation of the incorporation of the cyanines samples were combined and hybridized onto the microarray for 17 hours at 65 ° c . the array was analysed with the agilent scanner and the feature extraction software ( v9 . 1 ). a graphical overview and analysis of the data were obtained using the dna analytics software ( v4 . 0 . 36 ). for identifying duplications and deletions the present inventors used the standard set - up of the adm - 2 statistical analysis provided by dna analytics software ( agilent genomic workbench 5 . 0 ) freely available at agilent technologies website . according to this set - up and in the case of autosomal genes , heterozygous deletions are visualised with values of minus 1 and homozygous deletions as minus infinite (− 4 in cgh analytics ). the corresponding values for heterozygous and homozygous duplications are plus 0 . 5 and plus 1 respectively . at least 4 consecutive non - overlapping probes reaching these values were needed for a positive call , together with absence of known snps in the region covered by the significant probes . all the 15 patients were done in duplicate on the array in order to provide robustness and validity to the results . pathway analysis and literature mining was performed using pathway studio software from ariadne genomics inc . pathway studio database contains millions of regulatory and interaction events from all pubmed abstracts and more than 350 , 000 full - text articles extracted by medscan natural language processing technology . the present inventors have used graph navigation tool in pathway studio called “ build pathway ” to find literature evidence supporting functional association of measured genes with angiogenesis and other processes linked to blood vessel formation as well as immunity and neurodegeneration . the present inventors identified 234 known polymorphic cnvs in the 15 patients by comparing with the cnvs database [ 18 ]. this finding confirmed that the hla locus is highly polymorphic in terms of genomic unbalances as expected considering the known high density of snps . the distribution of the cnvs among patients both in terms of number and density of them is showed in fig1 a and 1b . table 3 reports all the cnvs identified in patients . cnvs in the last column of the table are identified with same catalog number of the database of genomic variants and can be searched either in ucsc genome browser and in the database of genomic variants itself . fig8 reports on the cgh profile identified in patient pf , as example . the present inventors correlated the number of the cnvs , with the clinical parameters of the patients ( fig2 , top panel , table 1 ). for the oversized number of cnvs significantly affecting x and y intercept , slope and run test the present inventors excluded from linear regression analysis the patient coded as mc ( 52 cnvs vs median ( ir ) 23 ( 13 ) of our patients population , table 1 ). linear regression analysis demonstrated that the overall number of cnvs is correlated with the number of extracranial vm demonstrated by venography in the extracranial segments of the cerebrospinal veins , with a trend toward significance ( r = 0 . 52 , r = 0 . 27 , p = 0 . 0545 ) ( fig3 and fig6 panel a ). by splitting the analysis correlating either the extragenic cnvs ( fig5 and fig6 panel c ) or the intragenic cnvs ( fig4 and fig6 panel b ) with the extracranial vm the present inventors found a robust and significant correlation with the former ( r = 0 . 53 , r = 0 . 28 , p & lt ; 0 . 05 ). 211 genes are contained within the region the present inventors covered by the present cgh array . since the phenotype in focus is characterised by multiple sclerosis and venous malformation , the present inventors applied a bioinformatics tool to select genes known to be involved in angiogenesis and venous development as well as linked to multiple sclerosis , immunity and neurodegeneration . interestingly some genes are linked to these processes . hspa1l and hspa1a are linked to ms and diabetes and other immunity disorders , and regulatory functions as chromatin remodelling , neuroprotection , protein folding , and regenerative - degenerative tools as neurodegeneration , neuron toxicity , cell survival , neuroprotection , synaptic transmission or even aging factors ( senescence and telomere maintenance ). also the gene grm4 seems to interact with many proteins linked to ms ( fig7 a ) focusing on specific functional pathways , as angiogenesis , obviously considering the plus phenotype of the present patients ( venous malformation ) the present inventors obtained a more selective puzzle of interactions ( fig7 b ). grb2 and hspa1a and b genes directly act on angiogenesis , taf11 is known to be involved in artery passage and e2f1 transcription factor is known to be an angiogenesis positive inducer in hepatitis and cancer . interesting hla - dqa2 may also be implicated in angiogenesis by interacting with cd4 . the correlation found between genotype ( cnvs ) and phenotype ( vm / ccsvi ) are further supported by the above reported regulatory functions in the angiogenic process . both extragenic and intragenic cnvs with known interaction in human angiogenesis support the hypothesis that the identified genetic variants may determine disregulation in the embryological process of venous angiogenesis , possibly leading to the development of vm . vm in turn create overtime a status known as ccsvi which is strongly associated to ms . naturally , while the principle of the invention remains the same , the details of construction and the embodiments may widely vary with respect to what has been described and illustrated purely by way of example , without departing from the scope of the present invention . 2 . ramagopalan s v , et al . bmc med genet 2009 ; 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