Patent Application: US-83090201-A

Abstract:
the invention concerns the identification and characterization of the spg4 gene encoding spastin , and some mutations thereof responsible for the most frequent form of autosomal dominant familial spastic paraplegia , to the cloning and characterization of its cdna and the corresponding polypeptides . the invention also concerns vectors , transformed cells and transgenic animals as well as diagnostic methods and kits , and methods for selecting a chemical or biological compound capable of directly or indirectly interacting with said polypeptide .

Description:
twelve bacs originating from two human genomic libraries , citb — 978_skb ( sold by research genetics ) and rpci - 11 ( osoegawa et al ., 1998 ), and covering the spg4 range , were selected to be sequenced ( hazan et al ., genomics , 60 ( 3 ), 309 - 19 , 1999 ). 40 μg of the dna of each bac were partially digested with the cviji restriction enzyme ( chimerx ) and separated by electrophoresis on 0 . 4 % lmp agarose gel ( fmc ). dna fractions , the sizes of which vary in the region of 3 , 5 and 10 kb , were eluted with β - agarase ( biolabs ) and ligated to a plasmid vector pbam3 , which had been digested with smai and dephosphorylated , beforehand , in a ratio of 1 × insert per 5 × vector . electrocompetent e . coli dh10b bacteria ( gibco - brl ) were transformed with the various ligations , by electroporation . approximately 1 000 to 1 500 subclones per bac ( 8 to 10 equivalent genomes ), consisting of 20 % of clones with inserts at 10 kb , 40 % of clones with inserts at 5 kb and 40 % of clones with inserts at 3 kb , were isolated . the ends of the inserts of these clones were sequenced on a licor 4200 automatic sequencer . for each bac , the sequences were assembled into a backbone consisting of several contigs , using the phred and phrap programs . the holes between each contig were sequenced with labeled dideoxynucleotides on an abi 377 sequencer ( pe - applied biosystems ). the exons contained in these sequence contigs were predicted with the grail ii , genscan , fgeneh and genie computer programs . the sequences were also compared in the embl and genbank nucleic acid and protein databases , with the blastn and blastx programs . the determination of the promoter sequences was carried out using the tssg and tssw computer programs . the results of all these sequence analyses were visualized using the genotator sequence annotation program . the cdna of the spg4 gene was isolated through 5 ′ and 3 ′ race - pcr experiments on polya + rnas of fetal brain , adult brain and adult liver , using the marathon cdna amplification kit ( clontech ) according to the supplier &# 39 ; s instructions . a first pcr followed by an internal pcr were carried out with various pairs of primers , the sequences of which are indicated in table 1 hereinafter : the race - pcr products were cloned with the ta - cloning kit ( invitrogen ) and the corresponding clones were sequenced on an abi 377 ( pe - applied biosystems ). the sequence of the spg4 transcript was varified by sequencing pcr products amplified from a cdna population originating from the lymphoblasts of 6 healthy individuals . the total rnas were extracted from lymphoblast lines of one affected individual per family studied and of 6 control individuals , using the rna plusr kit ( bioprobe system ). the cdna synthesis was carried out on 500 ng to 1 μg of rna , with 100 pmol of random hexameric primers ( pharmacia ) and 200 units of superscript ii reverse transcriptase ( gibco brl ), under standard conditions . four pcr amplifications , generating overlapping fragments which cover all of the spg4 open reading frame , were carried out on the cdnas of the patients and controls . fragment i was amplified with the spa_db / spa_dm primers , and then by internal pcr with the spa_dc / spa_dn primers . fragments ii , iii , and iv were amplified with the spa_ad / spa_am , spa_ba / spa_bm and spa_ca / spa_cm primers ( cf . the sequences of these primers in table 1 ), respectively . each amplification was carried out in a total volume of 50 μl containing 4 μl of cdna (˜ 1 / 7th of the prep . ), 20 pmol of each primer , 200 μm of dntps , 50 mm of kcl , 10 mm of tris , ph 9 , 1 . 5 mm mgcl 2 , 0 . 1 % of triton x - 100 , 0 . 01 % of gelatin and 2 . 5 units of taq polymerase ( cetus - pe ). the pcr reactions were carried out according to the “ hot start ” process : the taq polymerase is added at 92 ° c ., after a first denaturation step of 5 min at 94 ° c . the samples are subsequently subjected to 35 cycles of denaturation ( 94 ° c . for 40 sec ), of hybridization ( 55 ° c . for 50 sec , with the exception of fragment i ; 58 ° c . for 50 sec ) and of elongation ( 72 ° c . for 1 min ), followed by a final elongation step ( 5 min at 72 ° c .). the pcr products are sequenced on an abi 377 automatic sequencer ( pe - applied biosystems ), with the spa_dc / spa_dn , spa_ac / spa_an , spa_bb / spa_bn and spa_cb / spa_cm primers for fragments i , ii , iii and iv , respectively . the mutations were also sought or confirmed by sequencing the 17 predicted exons of the spg4 gene in the patients and controls . each exon was amplified with the corresponding “ a + m ” pair of primers ( cf . table 2 hereinafter ), with the exception of exon 1 ( gspaex1c / gspaex1m ), and exons 10 , 11 and 12 which were co - amplified with the gspaex10a / gspaex12m and gspaex11a / gspaex12m pairs of primers . other than for exon 1 , which is amplified using the advantage gc genomic pcr kit ( clontech ) according to the supplier &# 39 ; s instructions , four slightly different pcr programs ( 1 , 2 , 3 and 4 ) were used to amplify the spg4 exons ( see table 2 ). the amplifications were all carried out in a volume of 50 μl containing 100 ng of genomic dna , 50 pmol of each primer , 250 μm pf dntps , 1 × takara buffer and 1 unit of takara la taq taq polymerase ( shuzo co .). the pcr reactions were carried out according to the “ hot start ” process : the taq polymerase is added at 94 ° c ., after a first denaturation step of 5 min at 96 ° c . the samples are subsequently subjected to 30 cycles of denaturation ( 94 ° c . for 40 sec ), of hybridization ( prog . 1 : 60 ° c . for 50 sec ; prog . 2 : 58 ° c . for 50 sec , prog . 3 and 4 : 55 ° c . for 50 sec ) and of elongation ( prog . 1 and 4 : 72 ° c . for 1 min , prog . 2 and 3 : 72 ° c . for 40 sec ), followed by a final elongation step ( 10 min at 72 ° c .). the sequencing of these pcr products was carried out on an abi 377 sequencer ( pe - applied biosystems ), using either the pcr primers or the internal primers termed “ b ” and “ n ” ( see table 2 ). the cdna clones 977312 ( est aa560327 ) and 568234 ( est aa107866 ) derived from the mouse blastocyst and e8 embryo cdna libraries , which both correspond to the murine ortholog of spg4 , were isolated using the image consortium and sequenced in the laboratory on an abi 377 sequencer ( pe - applied biosystems ). in order to analyze the expression profile of spg4 and of its murine ortholog spg4 , the collections of cdna from various fetal and adult human tissues , and also from mouse tissues ( mtc panels , clontech ), were tested by pcr according to the supplier &# 39 ; s protocol , with the spa_ca / spa_cm pair of primers for the human cdnas and the spa_ca / spam ( spam : 5 ′- accgaagtcaagagcctatc - 3 ′) pair for the mouse cdnas . the pcr conditions are those used for amplifying spg4 from lymphoblast line cdna ( cf . § detection of mutations ), except that these samples were subjected to 32 cycles for the cdnas derived from adult human tissues and from mouse tissues , and to 28 cycles for the cdnas derived from fetal tissues . the amplification products migrated by electrophoresis on 2 % agarose gels . the histological and histo - enzymatic analyses were carried out on a muscle biopsy from a patient derived from an spg4 locus - linked family according to the standard techniques described in casari et al ., 1998 . the spg4 ( or spast ) cdna and the deduced protein sequence , genbank / embl aj246001 ; the incomplete spg4 cdna clone , genbank / embl aj246002 ; the spg4 ( or spast ) gene , genbank / embl aj246003 . the analysis of the recombination events made it possible to reduce the spg4 candidate region to a genetic range of 0 cm between the d2s352 and d2s2347 markers ( 19 , 20 ). a presequencing map of the spg4 range composed of 37 bacs was constructed ( hazan et al ., in press in genomics ); the candidate region covers a physical distance of approximately of 1 . 5 mb . twelve overlapping bacs , stretching over the spg4 region , with the exception of a single 4 kb hole between clones a and e , were selected to be sequenced ( fig1 a ). seven of these bacs ( a , b , c , d , e , f and g ), covering approximately 70 % of the region of interest , have already been sequenced . the sequences of these 7 bacs were compared with those of the nucleic acid and protein databases , and analyzed with four exon prediction programs . these preliminary sequence - analyses made it possible to reveal 14 potential transcription units , including three corresponding to the genes encoding xanthine dehydrogenase , steroid 5α - reduclase 2 and a tgfβ - binding protein . of the 14 genes detected by the sequence analysis , 9 had been previously identified in the est ( for “ expressed sequence tag ”) databases and located - in the spg4 range ( hazan et al ., in press in genomics ); the 5 remaining genes could only be identified by sequencing the candidate region . one of these 5 novel genes showed homology in 3 ′ of its coding region , with the genes encoding the aaa protein family ( confalonieri et al ., 1995 ). more thorough sequence analyses showed that this gene , named spg4 ( or spast ), was composed of 17 exons and extended over a region of approximately 90 kb , covered by two adjacent bac clones , d and g ( cf . fig1 b ). the first three predicted exons of this gene were identified in bac d , by two of the four exon prediction programs used , grail ii and genscan ; they show strong homology with a mouse blastocyst est , aa560327 . the last 14 exons are found in bac g . the protein sequence deduced from exons 7 to 17 is significantly homologous to a subclass of the aaa family , which includes the yta6p ( schnall et al ., 1994 ), tbp6 ( schnall et al ., 1994 ) and end 13 yeast proteins , and also the skd1 mouse protein ( perier et al ., 1994 ). of the four exon prediction programs fgeneh appears to be the most reliable and the most powerful , enabling detection of most of the genes of this chromosomal region at 2p21 - p22 . this observation also applies to the spg4 gene , for which 15 exons could be demonstrated using this program , while only 4 , 9 or 11 exons could be located using the genie , grail ii and genscan programs , respectively . the genomic organization of this gene ( fig1 b ) could subsequently be confirmed by determining the sequence of the spg4 cdna . the intron / exon junctions are represented on table 3 hereinafter : the exon size ranges from 41 bp ( exon 16 ) to 1 . 410 kb ( exon 17 ), that of the introns ranging from 140 bp ( intron 11 ) to 23 . 247 kb ( intron 1 ). several successive amplifications by 5 ′ and 3 ′ race - pcr were carried out on collections of adult liver and brain and fetal brain cdna , in order to characterize the spg4 transcript . all the 5 ′ race - pcrs gave amplification products terminating at nt position 263 of the spg4 cdna ( fig2 ), which was probably due to the rich gc content of the 5 ′ region of the transcript ( 90 % of gc in the 60 bp preceding nt position 263 ). four overlapping pcr products , covering all of the coding region , were amplified from the cdnas derived from the lymphoblasts of six control individuals , and entirely sequenced with the aim of verifying the sequence of the spg4 transcript . aligning the sequences of all the pcr and race - pcr products made it possible to reconstitute a 3263 bp sequence comprising a 1848 bp open reading frame preceded by a 125 bp untranslated 5 ′ region ( 5 ′ utr for “ 5 ′ untranslated region ”) and followed by 1290 bp 3 ′ utr region including a polyadenylation site between nt positions 3227 - 3232 , ˜ 35 bp upstream of the polya tail ( fig2 ). comparing the sequence of the spg4 cdna with the est databanks made it possible to detect significant homology with 6 human ests , including est n47973 which contains a more extended 3 ′ noncoding region (+ 180 bp ) comprising a second polyadenylation site . the translation initiation site was identified by the presence of a kosak consensus sequence ( ctgtgaatga ) defined as a “ suitable context ” for translation initiation given that a purine is located 3 nt upstream of the initiator atg , itself preceded by a stop codon . the 3263 bp cdna sequence is identical to the transcribed sequence deduced from the 17 exons of the spg4 gene . the analysis of the sequence of the 5 ′ region using the tssg and tssw computer programs suggests the presence of a promoter sequence of the tata box type located 43 bp upstream of nt position 1 of exon 1 . heterozygous mutations were sought in the spg4 cdna originating from lymphoblasts of 14 patients derived from spg4 locus - linked families ( 1 affected individual per family ). four overlapping pcr fragments , i , ii , iii and iv , covering the open reading frame of the spg4 cdna , were amplified and sequenced in the 14 patients , and also in 6 healthy control individuals . the agarose gel electrophoresis of pcr fragment iv showed three bands of equal intensity in 3 patients from families 2992 , 5226 and 5330 originating from the same region of switzerland , which would suggest a microdeletion or a mutation of a splice site ; the two additional bands were not present in 2 healthy individuals derived from families 2992 and 5330 ( fig3 a ). the genomic sequence of exon 16 revealed a heterozygous a → g mutation of the splice acceptor site ( ag ) of intron 15 in the affected individuals of these three families ( fig3 b ); this mutation engenders the loss of exon 16 , followed by a reading frame shift in the abnormal transcript . none of the healthy members , including husbands and wives , carry this mutation of the splice site . the identification of the same mutation in all the affected members of these three swiss families demonstrates the existence of a common ancestor , which had probably been suggested by the study of the haplotypes . three point mutations , 1210c → g , 1468g → a and 1620c → t , which introduced amino acid substitutions into the protein sequence ( s362c , c448y and r499c ), were respectively revealed by sequencing pcr fragments iii and iv in the affected individuals of families 624 , 4014 and 618 . these three substitutions all involve a cysteine residue , inducing the loss or insertion of a cysteine in the protein sequence . a 1 bp deletion , 1520delt , which creates the appearance of a stop codon inducing a truncated protein composed of 465 amino acids ( aa ), was detected in the affected individuals of family a . none of the five mutations summarized in table 4 hereinafter was found in the control individuals tested , whether they belong to the healthy siblings or to the spouses of the seven families analyzed herein . these five mutations significantly affect the protein sequence in a very conserved domain , or aaa cassette ( beyer , 1997 ), which is composed of several protein motifs presumed to be responsible for the atpase activity in all the members of the aaa family . in addition to these five mutations described above , searches for heterozygous mutations , carried out on patients suffering from ad - hsp derived from 36 other families , made it possible to reveal 34 other mutations which modified or were likely to modify the product of expression of the spg4 gene . the characteristics of these 34 other mutations are summarized in table 5 hereinafter , into which the first five mutations mentioned above have also been inserted . the open reading frame of spg4 encodes a 616 aa protein which we have named spastin and the molecular weight of which is approximately 67 . 2 kdaltons ( kd ). the comparison of this amino acid sequence in the protein databases , using the blast programs , made it possible to reveal a region of strong homology with several members of the aaa family , at the c - terminal end of spastin . the “ typical ” motifs of the aaa family , encompassed in the aaa cassette , are located between aa positions 342 and 599 ( see fig2 ) according to the sequence comparisons in the prodom and prosite protein domain databases . the three conserved typical domains , including the walker a and b motifs and also the minimum consensus motif of the aaa proteins are located in the aaa cassette at aa positions 382 - 389 , 437 - 442 and 480 - 498 , respectively , ( fig2 ). the walker a motif , “ gppgngkt ”, also called p - loop , which corresponds to the atp - binding domain , and the b motif , “ iifide ”, are very conserved among all the members of the aaa family , including spastin . the comparison of the aaa cassettes present in 150 proteins of this atpase family , derived from organisms which are very far apart in evolution made it possible to classify this set of proteins into several subgroups , as a function of the number of aaa cassettes identified ( 1 or 2 ) and of the sequence homologies between these various cassettes ( beyer , 1997 ). among all the proteins of the aaa family , spastin shows stronger homology with a particular subclass of the aaas , and more specifically with the following proteins , most of which were identified through the complete sequencing of the genome of the organism in question : two proteins of caenorhabditis elegans , o16299 and q18128 ; two subunits of the 26s proteasome of saccharomyces cerevisiae , yta6p ( q02845 ) and tbp6 ( p40328 ) ( schnall et al ., 1994 ); a subunit of the proteasome of schizosaccharomyces pombe ( o43078 ); the sap1 ( p39955 ) and end13 ( p52917 ) proteins of s . cerevisiae and the murine skd1 protein ( p46467 ) ( perier et al ., 1994 ). the multiple alignment of these 8 proteins with spastin is represented in fig4 a . of the 257 amino acids encompassing the aaa cassette ( aa positions 342 - 599 ), spastin shows 52 %, 51 % and 50 % sequence identity with the yta6p ( q02845 ) yeast protein , the o16299 nematode protein and the tbp6 ( p40328 ) yeast protein , respectively . similar results were obtained by analyzing the protein sequence of spastin in the prodom database , which showed the existence of three domains of homology ( named 92 , 179 and 6226 , and corresponding to aa positions 342 - 409 , 411 - 509 and 512 - 599 ) found in the putative subunits of the 26s proteasome of yeast . in addition , the members of this aaa subgroup most commonly contain motifs of the leucine - zipper type , two of which could be detected in the protein sequence of spastin at aa positions 50 - 78 and 508 - 529 , by analyzing the sequence in the prosite database ( see fig2 ). this analysis was also able to predict the presence of a dimerization motif of the helix - loop - helix type , located between aa positions 478 and 486 . the comparison of the protein sequence of spastin with those of the mitochondrial metalloproteases , such as the afg3 , rca1 and yme1 − yeast proteins , and also paraplegin , which is implicated in a rare form of ar - hsp , shows that the homology between these five members of the aaa family is limited to the 257aa region encompassing the aaa cassette ( fig4 b ). in this region , the sequence identity between spastin and paraplegin is only 29 %, whereas paraplegin and the afg3 yeast protein are 57 % identical over this same portion of the protein sequence . this sequence comparison suggests that spastin does not belong to the same aaa subgroup as paraplegin and other mitochondrial metalloproteases . in addition , the computer analysis of the spastin sequence using the psort ii program , which makes it possible to predict the subcellular location of the proteins , appears to indicate that spastin is a nuclear protein . a possible nuclear localization signal ( nls ), rgkkk , was revealed between aa positions 7 and 11 , whereas no signal peptide characteristic of importation into mitochondria could be detected , unlike what had been observed for paraplegin . the comparison of the nucleic acid sequence of spg4 in the est databanks made it possible to detect several human , murine and rat ests showing strong homology with spg4 . the mouse blastocyst and e8 embryo cdna clones corresponding to two of the murine ests , aa560327 and aa107866 , were obtained from the image consortium and entirely sequenced . the assembly of the sequences of these cdna clones made it possible to reconstitute a 1689 bp consensus sequence including a 1514 bp incomplete open reading frame . the comparison between the human spg4 cdna and this mouse cdna showed that the murine transcript lacks approximately 460 bp at the 5 ′ end , including the translation initiation codon . the mouse open reading frame is followed by a 175 bp 3 ′ noncoding region ( 3 ′ utr ) containing a polyadenylation site located ˜ 20 bp upstream of the polya tail ( fig5 ). the nucleic acid sequence of spg4 and the protein sequence of human spastin show 89 % ( between nt positions 460 and 1982 ) and 96 % ( between aa positions 113 and 616 ) identity , respectively , with the mouse cdna and deduced protein sequences . this considerable degree of homology makes it possible to affirm that this mouse transcript corresponds to the murine ortholog of spg4 , which was therefore named spg4 . the hybridization of northern blots comprising the mrnas of various human and murine tissues ( clontech ) with the spg4 and spg4 cdna clones did not give any convincing results , except a very weak band corresponding to a 2 . 5 kb transcript in the mouse testicle after exposure for 10 days . because of the low level of expression of this gene , the expression profiles for spg4 and spg4 were determined by pcr experiments on normalized collections of cdna originating from various adult and fetal tissues ( see fig6 a to 6 c ). the murine spg4 gene is expressed ubiquitously in the adult tissues of mice , and also from the e7 stage to the e17 stage of mouse embryos ( fig6 a ). higher expression of spg4 was detected in the liver , skeletal muscle and testicles , and also at the e15 stage of embryos . the early expression of spg4 during embryonic development was confirmed by the presence of ests originating from blastocyst , e8 embryo and embryonic carcinoma cdna libraries in the public est databanks . the human spg4 gene is , itself , also expressed ubiquitously in adult ( fig6 b ) and fetal ( fig6 c ) tissues , with perhaps more marked expression in fetal brain . in order to determine whether spastin mutations induced an oxidative phosphorylation ( oxphos ) impairment in mitochondria , in the same way as had been observed for paraplegin , a muscle biopsy was performed on a patient from one of the spg4 locus - linked ad - hsp families . the morphological and histo - enzymatic analyses of this muscle biopsy did not reveal any muscle fibers of the rrf ( for “ ragged red fiber ”) type , characteristic of oxphos impairments in mitochondria . the fact that all the muscle fibers appear to be normal , and also the prediction of a nuclear localization for spastin , seem to indicate that spg4 locus - linked ad - hsp is not a mitochondrial disease of the oxphos type , unlike spg7 locus - linked ar - hsp . using a positional cloning approach based on sequencing a 1 . 5 mb region , we have identified the spg4 ( or spast ) gene responsible for the most common form of ad - hsp , previously located on chromosomal bands 2p21 - p22 . thirty nine mutations which modify or are likely to modify the gene product , named spastin , could be detected in the affected individuals from forty one families with ad - hsp showing a link to the spg4 locus . spastin is a novel member of the aaa protein family , which appears to have a nuclear localization and which shows strong homology with the subunits of the 26s proteasome of yeast . despite great homology restricted to a domain of 230 to 250 aa , termed aaa cassette , the many members of this protein family can participate in very varied cellular mechanisms , such as the transport of proteins in vesicles , cell cycle regulation , organelle biogenesis , i . e . control of transcription , etc . however , all these cellular mechanism involve the assembly , the functioning or the degradation of protein complexes , which suggest that the members of the aaa family are so - 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