Patent Application: US-201414221460-A

Abstract:
a nucleotide sequence signal amplification composition that includes an isolated , synthetic nucleotide sequence of greater than 7 nucleotides . the sequence is a fragment of seq id no : 3 and further comprising a t nucleotide at position 1438 of seq id no : 3 and one or more primers that bind to the synthetic nucleotide sequence , a thermostable dna polymerase , a restriction enzyme , or a combination thereof .

Description:
a truncating mutation in the trappc9 gene on chromosome 8q24 has been identified that associates with the developmental disabilities : non - syndromic autosomal recessive mental retardation ( ns - armr ) and autism . trappc9 is a 23 exon gene that encodes a 1246 amino acid protein , nik and ikkβ binding protein ( nibp ). this protein is expressed at high levels in the muscle and kidney , and to a lesser extent in the brain , heart and placenta ( hu et al , 2005 ). only isoform 1 of the gene is present in the brain ( hu et al , 2005 ). nibp is involved in the nf - kappa - β signaling pathway , and directly interacts with ikkβ and map3k14 ( hu et al , 2005 ). it is likely involved in both classical and alternative activation of the nf - kappa - β signalling pathway ( hu et al , 2005 ). it potentially plays a role in neuronal differentiation , but this requires further investigation ( hu et al , 2005 ). it is expressed in the cell bodies and processes of neurons ( hu et al , 2005 ). nibp contains one known conserved region originally identified in saccharomyces cerevisiae called trs120 ( pfam08626 ; sacher et al , 2000 ). it is known to function in er to golgi traffic ( sacher et al , 2000 ). one mutation that causes the truncation of the nibp protein resides at position 1438 of the nucleic acid sequence shown in seq id no . 1 ( fig1 ). the single nucleotide polymorphism at this position results in a thymidine ( t ), as shown in seq id no . 3 ( fig3 ) instead of the wild - type cytosine ( c ) (“ the c allele ”). for the purposes of present invention , this polymorphism will be referred to as the c1438t polymorphism . however , it is to be noted that the polymorphism is also termed c1423t in mir et al ., 2009 ( the american journal of human genetics 85 , 1 - 7 , december 11 which is hereby incorporated by reference ) due to differences in numbering conventions when counting from the coding sequence of genbank accession number nm — 031466 . the presence of t at position 1438 of seq id no . 3 (“ the t allele ”), results in a termination codon , instead of the wild - type arginine in the amino acid sequence shown by seq id no . 2 ( fig2 ). the resulting truncated protein comprises 475 amino acids compared to the full - length protein , which is made up of 1246 amino acids . it has been found that the t allele is inherited in an autosomal recessive manner . as a result , individuals heterozygous for the allele may be carriers of the allele without having the phenotype of the disorder . identifying and counselling these individuals may limit or prevent the possible transmission of the recessive genotype onto offspring . the sample obtained from a subject may comprise any biological sample from which genomic dna may be isolated , for example , but not to be limited to a tissue sample , a sample of saliva , a cheek swab sample , blood , or other biological fluids that contain genomic dna . in a preferred embodiment , which is not meant to be limiting in any manner , the sample is a blood sample . in another embodiment , rna or mrna is isolated from the subject . the method of obtaining and analyzing dna or rna is not critical to the present invention and any method or methods may be used ( e . g . ausubel , et al . ( eds ), 1989 , current protocols in molecular biology , green publishing associates , inc ., and john wiley & amp ; sons , inc ., new york , at p . 2 . 10 . 3 , or maniatis et al ., in molecular cloning ( a laboratory manual ), cold spring harbor laboratory , 1982 , p . 387 - 389 ). for example , which is not to be considered limiting in any manner , dna may be extracted using a non - enzymatic high - salt procedure ( lahiri and nurnberger 1991 ). alternatively , the dna may be analyzed in situ . rna can isolated , for example , by phenol chloroform extraction and analyzed using rt - pcr . genotyping of the c1438t marker ( or any of the other markers as described herein ) may be performed by any method known in the art , for example pcr , sequencing , ligation chain reaction ( lcr ) or any other standard method known in the art that may be used to determine snps ( single nucleic acid polymorphisms ). in an embodiment , which is not meant to be limiting in any manner , amplifying the nucleic acid sequence containing the c1438t marker and genotyping the same is performed by pcr analysis using appropriate primers , probes and pcr conditions . in one embodiment , the step of amplifying the sequence containing the c1438t marker involves subjecting the nucleic acid sample to pcr , wherein the program for denaturing , annealing , amplifying is stored on a computer readable medium for execution by a microprocessor . the program causes a machine containing the samples to cycle through various temperatures for set periods of time . a similar or different machine comprising one or more programs may be employed to convert physical information , for example , but not limited to binding of nucleic acids or probes to target sequences , amplification or the like to a different state , such as electronic or otherwise , for example a signal that can be printed , displayed pictorially or digitized . in a further embodiment , the restriction enzyme taq i is used to detect the presence of the t allele at c1438t marker . taq i recognizes the consensus sequence , t c g a , which corresponds to the wild - type sequence of the trappc9 gene in the area of the polymorphism . the t allele will disrupt this consensus sequence and taq i will not be able to cut the sequence . as such taq i ( or an isoschizomer of taqi , or other restriction enzyme recognizing this or the complementary wild - type or mutated sequence ) can be used to easily determine the genotype of the subject . other methods also may be used . an apparatus , such as microarray or dna chip , can be used to detect the presence or absence of the c1438t marker or any other nucleic acid which results in a truncated nibp protein or mutated nibp protein as described herein . in this case , but without wishing to be limiting in any manner , an oligonucleotide may be bound to a substrate , which is suitable for this type of application . in an embodiment the oligonucleotide preferably comprises a contiguous nucleic acid , for example , the sequence from seq id no . 3 ( fig3 ) containing position 1438 of the sequence or a sequence substantially identical thereto . another oligonucleotide can also be bound to the substrate . for example , but not wishing to be limiting , a nucleotide sequence comprising a complement of the nucleic acid sequence from seq id no : 3 containing position 1438 of the sequence may be employed . in a further embodiment , the oligonucleotide comprises a contiguous nucleic acid sequence from seq id no . 1 containing position 1438 of the sequence , or a complement thereof or a sequence substantially identical thereto . in one embodiment the oligonucleotides are 7 , 10 , 12 , 15 , 16 , 17 , 19 , 21 , 23 , 25 or more nucleotides in length . in another embodiment , the oligonucleotides are 60 nucleotides in length or more . alternatively , the oligonucleotides may be defined by a range of any two of the values noted above or any two values therein between . a person skilled in the art will recognize that the length of the oligonucleotides can be altered based on the parameters of the assay . it is envisaged that the apparatus can contain other oligonucleotide sequences to confirm the subject &# 39 ; s susceptibility to the developmental disability or to test for the susceptibility of additional diseases or disorders , comorbid or otherwise . as noted above , the c1438t marker in trappc9 gene , results in a truncation of the nibp protein . in a separate study , sequence analysis also identified a 4 base pair deletion resulting in a frameshift and premature truncation : pleu772trpfsx7 in exon 14 . this observation provides a unique opportunity to use the difference in protein length , between the wildtype and the truncation proteins , to predicted the susceptibility of a subject to a developmental disability . as such , the present invention also contemplates screening methods which identify and / or characterize the proteins as defined above within biological samples from subjects . such samples may or may not comprise dna or rna . for example , such screening or testing methods may employ immunological methods , for example , but not limited to antibody binding assays such as elisas or the like , protein sequencing , electrophoretic separations to identify the proteins as described above in a sample . as will be evident to a person of skill in the art , the screening methods allow for the differentiation of the proteins as defined herein from wild type proteins known in the art . also contemplated by the present invention is a nucleic acid comprising or consisting of a sequence selected from the group consisting of : a ) a nucleic acid sequence comprising seq id no . 4 ( fig4 ); b ) a complement of a nucleic acid sequence comprising seq id no . 4 ; c ) a fragment of either a ) or b ); d ) a nucleic acid sequence capable of hybridizing to any one of a ), b ) or c ); and e ) a nucleic acid sequence that exhibits greater than about 70 % sequence identity with the nucleic acid defined in a ), b ) or c ). a nucleic acid sequence exhibiting at least 70 % identity thereto is understood to include sequences that exhibit 70 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99 . 9 % or 100 % identity , or an value therein between to seq id no . 4 . further , the nucleic acid may be defined as comprising a range of sequence identity as defined by any two of the values listed or any values therein between . any method known in the art may be used for determining the degree of identity between nucleic acid sequences . for example , but without wishing to be limiting , a sequence search method such as blast ( basic local alignment search tool : ( altschul s f , gish w , miller w , myers e w , lipman d j ( 1990 ) j mol biol 215 : 403 - 410 ) can be used according to default parameters as described by tatiana et al ., fems microbiol lett . 174 : 247 - 250 ( 1990 ), or on the national center for biotechnology information web page at ncbi . nlm . gov / blast /, for searching closely related sequences . blast is widely used in routine sequence alignment ; modified blast algorithms such as gapped blast , which allows gaps ( either insertions or deletions ) to be introduced into alignments , psi - blast , a sensitive search for sequence homologs ( altschul et al ., ( 1997 ) nucleic acid res . 25 : 3389 - 3402 ); or fasta , which is available on the world wide web at expasy ( embl - european bioinformatics institute ). similar methods known in the art may be employed to compare dna or rna sequences to determine the degree of sequence identity . stringent hybridization conditions may be , for example but not limited to hybridization overnight ( from about 16 - 20 hours ) hybridization in 4 × ssc at 65 ° c ., followed by washing in 0 . 1 × ssc at 65 ° c . for an hour , or 2 washes in 0 . 1 × ssc at 65 ° c . each for 20 or 30 minutes . alternatively , an exemplary stringent hybridization condition could be overnight ( 16 - 20 hours ) in 50 % formamide , 4 × ssc at 42 ° c ., followed by washing in 0 . 1 × ssc at 65 ° c . for an hour , or 2 washes in 0 . 1 × ssc at 65 ° c . each for 20 or 30 minutes , or overnight ( 16 - 20 hours ); or hybridization in church aqueous phosphate buffer ( 7 % sds ; 0 . 5m napo 4 buffer ph 7 . 2 ; 10 mm edta ) at 65 ° c ., with 2 washes either at 50 ° c . in 0 . 1 × ssc , 0 . 1 % sds for 20 or 30 minutes each , or 2 washes at 65 ° c . in 2 × ssc , 0 . 1 % sds for 20 or 30 minutes each for unique sequence regions . also contemplated by the present invention is a method of predicting susceptibility to a developmental disability in a human subject , comprising the steps of : isolating rna from the subject ; hybridizing an oligonucleotide comprising a contiguous nucleic acid of seq id no . 1 to the rna ; wherein the absence of rna complementary to the oligonucleotide is predictive of the developmental disability . the presence of the t allele at position 1438 of seq id no . 4 is believed to lead to nonsense - mediated rna decay , and hence reduction or total loss of the mrna . this presents a unique opportunity to detect the presence of trappc9 mrna in a subject for the purposes of predicting the susceptibility of the subject to a developmental disorder . the present invention will be further illustrated in the following examples . however it is to be understood that these examples are for illustrative purposes only , and should not be used to limit the scope of the present invention in any manner . the family in which the c1438t mutation was identified , mr - 2019 , is a large family with multiple incidences of consanguinity between first cousins . we obtained the dna of 8 affected and 12 unaffected family members . affymetrix 5 . 0 snp microarray analysis was conducted to identify genetic differences between the affected and unaffected family members . the results were analyzed with dchip , using homozygosity mapping as a basis for identifying regions of susceptibility . a 3 . 2 mb region of autozygosity was identified in the family at locus 8q24 , from 139 , 465 , 102 - 142 , 726 , 810 ( ucsc march 2006 build ), consisting of a run of 606 consecutive homozygous snps . this region overlaps with a 6 . 8 mb locus identified in an iranian ns - armr pedigree ( najmabadi et al , 2007 ). the 3 . 2 mb region contains 12 genes , only one of which has been previously implicated in mr . this gene , kcnk9 ( mim 605874 ), has been shown to be causal in recently identified birk - barel syndrome ( barel et al , 2008 ) ( mim 612292 ). kcnk9 was sequenced and found to be normal in the family used in this example . additional genes in the region were sequenced that appeared to be good candidates for mr based on functional and expression data obtained from the ucsc database . an expression - based algorithm was used to identify genes in our region that co - expressed with known causal mr genes ( genome . ucla . edu / projects / uget ). a mutation in trappc9 was identified . the mutation , at c1438t causes the gene to be truncated at the end of its 7th exon . each of the 8 affected individuals were homozygous for the t allele , whereas all of the 12 unaffected individuals were either homozygous for the c allele or carriers of the t allele . the present invention has been described with regard to one or more embodiments . however , it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims . abrahams b s , geschwind d h . 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