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Genomics 71, 200–213 (2001)doi:10.1006/geno.2000.6392, available online at http://www.idealibrary.com on

Cloning and Characterization of Three Novel Genes, ALS2CR1,ALS2CR2, and ALS2CR3, in the Juvenile Amyotrophic LateralSclerosis (ALS2) Critical Region at Chromosome 2q33–q34:

Candidate Genes for ALS2

Shinji Hadano,*,† Yoshiko Yanagisawa,* Jennifer Skaug,‡ Keith Fichter,§ Jamal Nasir,§Duane Martindale,¶ Ben F. Koop,¶ Stephen W. Scherer,‡ Donald W. Nicholson,\

Guy A. Rouleau,** Joh-E Ikeda,*,† and Michael R. Hayden§,1

*NeuroGenes, International Cooperative Research Project, Japan Science and Technology Corporation, Tokai University School ofMedicine, Isehara, Kanagawa 259-1193, Japan; †Department of Molecular Neuroscience, Molecular Medicine Research Center,The Institute of Medical Sciences, Tokai University, Isehara, Kanagawa 259-1193, Japan; ‡Department of Genetics, The Hospitalfor Sick Children, Toronto, Ontario M5G 1X8, Canada; §Department of Medical Genetics, and Centre for Molecular Medicine

and Therapeutics, University of British Columbia, 980 West 28th Avenue, Vancouver, British Columbia V5Z 4H4, Canada;¶Center for Environmental Health, Department of Biology, University of Victoria, Victoria, British Columbia V8W 3N5, Canada;

\Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Point Claire-Dorval,Quebec H9R 4P8, Canada; and **Centre for Research in Neuroscience, McGill University, and Montreal General

Hospital Research Institute, Montreal, Quebec H3G 1A4, Canada

Received July 5, 2000; accepted September 7, 2000

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Amyotrophic lateral sclerosis is a progressive neuro-degenerative disease that manifests as selective upperand lower motor neuron degeneration. The autosomalrecessive form of juvenile amyotrophic lateral sclerosis(ALS2) has previously been mapped to the 1.7-cM inter-val flanked by D2S116 and D2S2237 on human chromo-ome 2q33–q34. We identified three novel full-lengthranscripts encoded by three distinct genes (HGMW-ap-roved symbols ALS2CR1, ALS2CR2, and ALS2CR3)ithin the ALS2 critical region. The intron–exon orga-izations of these genes as well as those of CFLAR,ASP10, and CASP8, which were previously mapped to

his region, were defined. These genes were evaluatedor mutations in ALS2 patients, and no disease-associ-ted sequence alterations in either exons or intron–exonoundaries were observed. Sequence analysis of over-

apping RT-PCR products covering the whole codingequence for each transcript revealed no aberrant mRNAequences. These data strongly indicate that ALS2CR1,LS2CR2, ALS2CR3, CFLAR, CASP10, and CASP8 are not

causative genes for ALS2. © 2001 Academic Press

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a heteroge-neous group of progressive neurodegenerative disor-ders characterized by a selective loss of motor neurons

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AB038949–AB038985.

1 To whom correspondence should be addressed. Telephone: (604)

875-3535. Fax: (604) 875-3819. E-mail: mrh@cmmt.ubc.ca.

2000888-7543/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

in the anterior horn of the spinal cord and brain stem,as well as pyramidal cells of the cerebral cortex (Sid-dique et al., 1996). Although most of the cases reportedare sporadic, 5–10% are familial (FALS) (Siddique etal., 1996). Four types of FALS have been mapped todistinct loci of the human genome. The gene for anautosomal dominant form with late onset (ALS1) mapsto chromosome 21q22.1–q22.2 and is associated withmutations in the copper-zinc superoxide dismutase(SOD-1) gene (Rosen et al., 1993). A dominant form ofjuvenile ALS (ALS4) has recently been mapped to chro-mosome 9q34 (Chance et al., 1998). Two types of auto-somal recessive forms for juvenile ALS, ALS2 andALS5, have also been mapped to chromosome 2q33–q35 (Hentati et al., 1994) and 15q12-q21 (Hentati et al.,998), respectively. However, the genetic causes ofLS2, ALS4, and ALS5 have not been identified. Ped-

grees with adult-onset, autosomal dominant ALS thats neither linked to chromosome 21 nor associated withOD-1 mutations suggest additional loci for FALS,hich is currently referred to as ALS3 (Siddique et al.,991).ALS2, also known as type 3 autosomal recessive ALS

RFALS type3), was originally reported in a large con-anguineous Tunisian kindred (Hamida et al., 1990)

and was characterized by a loss of upper motor neuronsand spasticity of limb and facial muscles accompanyingdistal amyotrophy of hands and feet. Hentati et al.(1994) have previously reported that the ALS2 locuslies within the 8-cM segment flanked by D2S115 and

D2S155 on chromosome 2q33–q35, and they further

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201ALS2 CANDIDATE GENES

narrowed the candidate region to a 1.7-cM segment bylinkage and haplotype analyses (Hosler et al., 1998).Yeast artificial chromosome (YAC)-based physicalcloning and mapping revealed that this intervalspanned approximately 3 Mb of genomic DNA andallowed generation of a transcript map of the ALS2critical region (Hosler et al., 1998; Hadano et al., 1999).

ithin this interval, nine known genes, usurpinCFLAR) (Rasper et al., 1998; Shu et al., 1997),aspase-10 (CASP10) (Fernandes-Alnemri et al., 1996;incenz and Dixit, 1997), caspase-8 (CALS8) (Boldin etl., 1996), human origin recognition complex proteinORC2L) (Takahara et al., 1996), proteosome compo-ent C3 (PSMA2) (Tamura et al., 1991), Abl interactor(AIP-1) (Dai and Pendergast, 1995), T-cell membranelycoprotein (CD28) (Buonavista et al., 1992), cytotoxic-lymphocyte-associated protein (CTLA4) (Buonavistat al., 1992; Dariavach et al., 1988), and sentrin (UBL1)Okura et al., 1996; Desterro et al., 1998), and morehan 30 expressed sequence tag (EST) sequencesHiller et al., 1996) representing possible novel genesave been assigned (Hadano et al., 1999).To understand the molecular mechanism of the

athogenesis of ALS2 and other forms of ALS, identi-cation of the gene and mutation that links to ALS2 isssential. A recent study showed that no sequence al-erations associated with ALS2 have been found in fourxons encoding CD28 and three exons encoding

CTLA4, suggesting that CD28 and CTLA4 are notcausative genes for ALS2 (Hosler et al., 1998). Despitesubstantial efforts to identify genes in this region, asystematic approach for the identification of ALS2-associated sequence alterations in the candidate geneshas yet to be attempted. To accomplish this, we haveemployed a three-step approach that consists of: (1)isolation of full-length transcription units from theALS2 critical region, (2) determination of intron–exonorganization, and (3) scanning for mutations by DNAsequencing.

We hereby succeeded in the cloning and charac-terization of three novel full-length transcripts(HGMW-approved symbols: ALS2CR1, ALS2CR2, andALS2CR3) that mapped within the ALS2 critical re-gion. We defined the intron–exon organizations ofthese novel genes as well as those of CFLAR, CASP10,and CASP8, which were previously mapped to thisregion. We also show the results of mutational analy-ses for these six genes in ALS2 patients.

MATERIALS AND METHODS

Genomic DNA and lymphoid cell lines of the ALS2 family. Sam-ples of the genomic DNA and lymphocyte of the affected and unaf-fected individuals from the Tunisian ALS2 family (Hamida et al.,1990; Hentati et al., 1994; Hosler et al., 1998) were used in thismutational study.

Generation of bacterial artificial chromosome (BAC)/P1 artificialchromosome (PAC) contigs. To generate a physical map of the ALS2

candidate region, screening of the human BAC (Shizuya et al., 1992) c

and PAC (Ioannou et al., 1994) genomic DNA libraries was per-formed using DNA markers and cDNA clones that mapped withinthe candidate region (Hosler et al., 1998; Hadano et al., 1999) asprobes. The position of each BAC/PAC clone was confirmed by PCR-based STS/EST content mapping as previously described (Hadano etal., 1999).

Identification of candidate transcripts. To identify the tran-scribed DNA sequences mapped within the candidate region forALS2, we searched the public databases in the Genome Data Base(GDB) (http://gdbwww.gdb.org), Gene Map of the Human Genome(Deloukas et al., 1998), and UniGene at The National Center forBiotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov).Furthermore, genomic DNA sequences derived from the high-throughput sequencing of two PAC clones (251L7 and 562F5)(Hadano et al., 1999) and derived from a public database (HumanChromosome 2 Sequencing Project) were used as queries for theBLAST search (Altschul et al., 1997). DNA sequences of ESTs/cDNAswere aligned using BLAST and Sequencher Version 3.0 (Gene CodesCorp.) programs, and putative nonoverlapping transcriptional unitswere established. Of these nonoverlapping transcriptional units,novel transcribed sequences were named as “CALS” standing for“candidate for ALS,” because those units represent the candidategenes for ALS2.

Mapping of candidate transcripts. Representative PCR primersets for each nonoverlapping transcribed sequence were retrievedfrom the dbEST database (NCBI) and used for mapping onto YACand BAC/PAC contigs by determining the presence or absence ofeach EST in individual YAC, BAC, or PAC clones by PCR. For allPCR amplification, a standard procedure was employed as describedpreviously (Hadano et al., 1999).

Reverse transcription-polymerase chain reaction (RT-PCR) analy-sis of nonoverlapping transcriptional units. To amplify the tran-scribed DNA sequences, total RNA extracted from the human brain,spinal cord, and lymphocytes was subjected to RT-PCR using theSuperScript preamplification system (Gibco BRL) according to thesupplier’s recommendations.

59-Rapid amplification of cDNA ends (RACE). To achieve thefull-length cloning of the ALS2CR3 transcript, we used a RACEtechnique. Human fetal brain Marathon-Ready cDNA (Clontech)and human brain whole Marathon-Ready cDNA (Clontech) wereused to amplify the transcript ends. One hundred nanograms ofMarathon-Ready cDNA was subjected to the amplification usingadaptor primer 1 (AP1), 59-CCATCCTAATACGACTCACTAT-AGGGC-39, and gene-specific primer, CC-T3-2RR, 59-TAACTCGTG-CAGCTCCATTG-39. After 3 min of denaturation at 94°C, 30 cycles ofPCR (10 s at 95°C, 30 s at 62°C, 10 min at 68°C), followed by 7 minat 68°C, were performed using a Hot-Start PCR technique and Ex-pand Long Template PCR system (Boehringer Mannheim) in 20 ml ofeaction mixture according to the manufacturer’s protocol. The pri-ary PCR product was subjected to secondary PCR using the nested

rimers AP2, 59-ACTCACTATAGGGCTCGAGCGGC-39, andALSC1w1R, 59-ACACAGTCGCTGACAAGCTG-39. PCR productsere directly ligated into pCR2.1 vector using the TA cloning system

Invitrogen).

DNA sequencing and analysis of DNA sequence data. Represen-ative EST clones were obtained from Research Genetics, Inc.lasmid DNA was extracted and purified using the QIAprep minind maxi columns (Qiagen) and subjected to DNA sequencing toetermine the entire insert of the clones. Double-stranded DNAas sequenced by dideoxy sequencing using the BigDyeTermina-

or Cycle Sequencing kit (ABI) and the ABI 373A or ABI 377 DNAequencer. All primers used in DNA sequencing were designedsing Primer 3.0 (http://www-genome.wi.mit.edu). CompositeNA sequence was constructed by aligning the entire DNA se-uence of the selected cDNA clones using Sequencher Version 3.0Gene Codes Corp.).

The DNA sequence and the translated amino acid sequences were

ompared with the nonredundant nucleotide and protein sequence

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202 HADANO ET AL.

databases using BLASTN and BLASTP, respectively (Altschul et al.,1997). Identification of protein motifs and domains was performed bythe MOTIF server at GenomeNet Japan (http://www.genome.ad.jp)and the BCM search launcher (http://www.hgsc.bcm.tmc.edu/SearchLauncher).

Northern blot analysis. Multiple human adult tissue Northern(MTN) blots (Clontech) were hybridized with [a-32P] dCTP-labeledALS2CR1, ALS2CR2, and ALS2CR3 or human actin cDNA in Ex-pressHyb hybridization solution (Clontech) at 68°C. Membraneswere washed with 23 SSC containing 0.1% SDS at 68°C and exposedto X-ray film (Bio-MAX, Kodak).

Intron–exon organization. To define the intron–exon organiza-tion of the ALS2CR1 and ALS2CR2 genes, the cDNA sequence wascompared with the genomic DNA sequence data published in theGenBank database using BLAST and Sequencher Version 3.0 (GeneCodes Corp.) programs. The accession numbers for the genomic DNAsequence data for ALS2CR1 and ALS2CR2 were AC005037 andAC007282, respectively.

To define the intron–exon organization of the ALS2CR3 CFLAR,CASP10, and CASP8 genes, two PAC clones (251L7 and 562F5) weresubjected to high-throughput sequencing according to a method pre-viously described (Rowen and Koop, 1994). DNA sequences forAS2CR3 CFLAR, CASP10, and CASP8 cDNA were compared withthose of the PAC clones, and the contigs that contained the part ofexonic sequences for ALS2CR3 CFLAR, CASP10, and CASP8 weredetected. Further, direct BAC/PAC DNA sequencing was also carriedout using primers derived from exonic regions. The size of eachintron was then determined by PCR amplification of PAC clones orhuman genomic DNA using the Expand Long Template PCR system(Boehringer Mannheim) according to the supplier’s protocol.

Mutational analysis of genomic DNA. To detect gross deletionsin the ALS2 candidate region, STS/EST content mapping usingPCR analysis to determine the presence or absence of each DNAmarker and EST that mapped within the ALS2 candidate regionwas performed on the genomic DNA of affected individuals. Fur-thermore, amplification of the region containing both exons andintron– exon boundaries, i.e., exon-PCR, in ALS2CR1, ALS2CR2,ALS2CR3, CFLAR, CASP10, and CASP8 genes was carried out.Genomic DNAs from three family members affected with ALS2(Hamida et al., 1990; Hentati et al., 1994; Hosler et al., 1998) andthree normal individuals from a non-ALS2 family were investi-gated. Oligonucleotide primers for exon-PCR were designed usingPrimer 3.0 (http://www-genome.wi.mit.edu). The sequences ofprimers are listed in Table 1. Approximately 50 ng of genomicDNA was subjected to PCR amplification using ExTaq polymerase(TaKaRa) with parameters consisting of 95°C for 15 s, 58°C for30 s, and 72°C for 30 s for 40 cycles.

To detect small deletions or base substitutions in the exons orintron–exon boundaries, DNA sequencing of the exon-PCR productswas performed. Exon-PCR products were purified by ultrafiltrationusing C3TK (Millipore) and subjected to DNA sequencing analysisusing the same oligonucleotides as primers. The DNA sequences ofPCR products were analyzed using Sequencher Version 3.0 (GeneCodes Corp.).

Amplification of the whole coding region by RT-PCR. Total cellu-lar RNA was extracted from lymphoblastoid cell lines establishedfrom lymphocytes derived from family members of the ALS2 pedi-gree by using the Total RNA extraction kit (Qiagen) according to themanufacturer’s instructions.

To amplify the whole coding regions of ALS2CR1, ALS2CR2,ALS2CR3, CFLAR, CASP10, and CASP8 transcripts, several sets ofPCR primers that allowed amplification of overlapping fragmentscovering the whole coding region of each transcript were designed(Table 2). RT-PCR was performed on human total RNA extractedfrom brain and lymphocytes. RT-PCR products were subjected to

DNA sequencing analysis.

RESULTS

A Transcript Map of the ALS2 Candidate Region

Fifty-four putative nonoverlapping transcriptionalunits were assigned to this interval (Fig. 1). Thoseunits contain 42 unknown transcriptional units andfour known genes, KIAA0005 (Nomura et al., 1994),CDC-like kinase (CLK1) (Johnson and Smith, 1991),bone morphogenic protein type II receptor (BMPR2)(Rosenzweig et al., 1995), and NADH dehydrogenase(ubiquinone) 1 b-subcomplex 3 (NDUFB3) (Ton et al.,997), in addition to eight genes that previouslyapped in this interval: ORC2L, CFLAR, CASP10,ASP8, UBL1, AIP-1, CD28, and CTLA4. Proteasome

omponent HC3 (PSMA2) was previously assigned tohis interval (Hosler et al., 1998; Hadano et al., 1999).owever, DNA sequencing and mapping of the PSMA2

ene revealed that the intron-less sequence which wasighly similar to that of PSMA2, was localized withinhe ALS2 region on chromosome 2q33–q34, suggestinghat the PSMA2-like sequence at the ALS2 regionight represent a pseudogene. According to the GDB

atabase, PSMA2 was recently mapped to chromosomeq27 (http://gdbwww.gdb.org).

xpression of Candidate Transcripts in the Brain

We analyzed the expression of transcribed sequencesy RT-PCR on total RNA extracted from normal hu-an brain, spinal cord, and lymphocytes. Thus far, 33

f 54 units including 8 known genes have been ana-yzed for expression, and 32 of 33 units were expressedt least in the brain or spinal cord (Table 3). Of 32nits, 3 units, ALS2CR1, ALS2CR2, and ALS2CR3,ontained large open reading frames (ORFs) encodingutative novel proteins. Their consensus DNA se-uences were obtained by extensive sequence align-ent (see below). These three units were subjected to

urther characterization as primary candidates forLS2.

LS2CR1

Initially, approximately 1.6 kb of the consensus se-uence for the ALS2CR1 transcript was generated byligning data from 83 ESTs compiled in the UniGeneerver under the ID number of Hs.21943. One cDNAlone (IMAGE Clone 32584) was found to cover thentire region of this consensus sequence and thereforeas subjected to full-length DNA sequencing as a rep-

esentative clone for ALS2CR1. The ALS2CR1 se-uence encompassed 1606 nucleotides with a single053-bp (286–1338 nt.) ORF, and it was predicted toncode a 39.0-kDa protein consisting of 350 amino ac-ds (Fig. 2a). The sequence of the putative translationtart site (tccttcATGG) was similar to the Kozak con-

sensus sequence (Kozak, 1987). Both the polyadenyla-tion signal (AATAAA; 1566–1571 nt) and the poly(A)

tail have been identified at the 39 end of the ALS2CR1

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203ALS2 CANDIDATE GENES

cDNA. The nucleotide sequence of ALS2CR1 has beendeposited with the DDBJ, EMBL, and GenBank DataLibraries under Accession No. AB038949.

The amino acid sequence predicted from the

TAB

Primer Sequences for the Amplification of Ex

Gene Exon No. Product (bp) Forward primer

ALS2CR1 1 378 59-CAGAGAAGTTTCCGGACTG-392 495 59-TTCTGTATGTTGTCATCTTGCG-393 286 59-CAAAACTCCATTTTCCCAC-394 216 59-AATGGAGTGATTTTTCCCCC-395 251 59-AGGACTAACAATGCACACCG-396 257 59-TGGTGATGAGACCAGGAGTG-397 293 59-AATTTGCTCTTCCCTTTTTGG-39

LS2CR2 1 251 59-ACTGGTGCAGAGTTCCAAGC-392 250 59-GGATGGGTTTAAGGCAACTG-393 312 59-CCACCCCTGCTTATCCTGTA-394 239 59-CCTAATGAGCCACCTCTGTGTTTG-395 345 59-GGCCTGTTTAAATCTTCTGCC-396 280 59-AGTATTAGCTGCCACGACCG-397 257 59-GCTGGAAGCAAAATCATTTC-398 258 59-TTCTTCATTGGCCAGTTTCC-399 269 59-TCTTGATTTGTCGCTTATGGG-39

10 349 59-TTGAAGGAAAGTTCTGTTTCTTTT-3911 226 59-GCTAAGAATTGGGCTTCTGAC-3912 350 59-GATGACCAGAATCAAAGGCAG-39

LS2CR3 1 284 59-AGTGGCTCTCCTTTGGCTTC-392 627 59-TTGTCCTGCCCCTTAG-393 364 59-TGGATTCATTTACATAGACTCTCCTCT-394 235 59-AAGGCTGGGGTTTTTGTTG-395 270 59-CTGAACTGCCATTTAACCTTG-396 261 59-TGAAAATCAAGCATTTTATATGGAC-397 302 59-GGATGAGCCAGGTAGAGATG-398 376 59-CACCACAGTCCAGTGCTTGT-399 232 59-GGCTTTAAGATGATACCCAG-39

10 257 59-TGTTTTGGGAAGGCAATAAAG-3911 278 59-TCCCCTGAATGTTAGCCTTG-3912 349 59-GGGTGGCTTTATTTCTGATGG-3913 385 59-ATCAGTGAACGCGATTTTCC-3914 412 59-TGGGAAATGTAAAACATGCC-3915 333 59-TTCAATCTTGTATTTGCTCCC-3916 367 59-CTTGGAGCTTGCTTTTTATTC-39

480 59-TTCCACACCACCAAATTCAC-39FLAR 1 461 59-TAACGCGTGGAACTTGTGTC-39

2 566 59-CTCCTAGTAGGTGGTGGGGG-393 273 59-CCTACTGAGGCCATTAAACCC-394 599 59-TAAGTCAGGGAGACCACCCA-395 300 59-TGCTGTTTTATGAGGGAGGG-396 338 59-TCCCAAAGTGCTGGGATTAC-397 313 59-GCGCCCTATACTCCATTCTTC-398 184 59-TCCTAAAGGCAGCTGTTGTG-399 349 59-CAGAATTTTCCCTGTTCTTTGG-39

10 274 59-ACTCAGCCAGTGTAGGGGTG-3911 250 59-TGCCAAAGGAAATCCAAAAG-3912 ;11000 59-AGAACCAGTGAAGAAATCCATTCAGGAA

13 621 59-CCAGTCCTCTTGAGCTCTCC-3914a 220 59-TGTTGCACAGTGTTTGCTTTC-3914b 619 59-TTGCTAAGCAGCTTGTGGTG-39

ASP10 1 324 59-TTAACCCTGGAAAAGGGGAG-392 470 59-ACTCTCTAACTCCCTGCCCC-393 349 59-TCAGAAGGCCTGCTTCTACC-394 348 59-CCCCTACATTCCTGAGCAAC-395 208 59-GGTTCAAGCAATTCTCCTGC-396 230 59-TCCTATGTTTCATGCCCTCC-397 299 59-TGAATTTGTCTCAGGAGGGC-398 262 59-TGGGGAGATACATCCTGCTC-399 679 59-CGGCCTTGTTTCAGTTCTTC-39

10 315 59-TCACTTGAATCCAGGAGGTG-3911 264 59-CCACTCAGTCAAAGTTCCCC-39

ASP8 1 271 59-GTTGGAGCAAAGGTTTGGAG-392 271 59-ACATGGGCTTCTACTCACCG-393 349 59-ACCCTGCAGTTCCTTCTGTG-394 258 59-GAAGTAGAGCTGGCTGGCTG-395 442 59-GATGAACAAGCCAGCAAATG-396 247 59-GTCCTAAAAACCCACAGCCC-397 259 59-ATCAAGCCCACTGTGACTCC-398 276 59-TCCTTGGTTGGAGAAATTGG-399 204 59-TGAAACTGACAAATCGCTCC-39

10 232 59-ACCAGTGTACCTTTCCTGCC-3911 350 59-GCAATGGAAAGCAAGTCCTC-3912 343 59-AATGTTATGCCCACTGTGCTC-39

323 59-TTCACTGGTTTGAAGTGCCC-3913 236 59-CCCCACAGACAGTCACAATG-39

ALS2CR1 ORF was subjected to a sequence homology

search using the BLAST server. We have identifiedsignificant homology (P 5 6 3 10222) betweenALS2CR1 and ngg1-interacting factor 3, a yeast pro-tein whose function has not been defined. No obvious

1

s (Exon-PCR) for the ALS2 Candidate Genes

Reverse primer Remarks

59-AGCTGGAAACAACTCCCGTC-3959-AAAGTTTCAAAGTATTTGCAGGG-3959-TTTCAAGCCTAAATACCAGGG-3959-TCAATTATACTGTTAGAAAATGTGTGC-3959-CCAAGAGTTTCGATCTTTGTCC-3959-TTTGCCATTTTTGATTTTGC-3959-CACACCAGTCCACTGACTC-3959-CAAGGCTCACTCCCTCACAC-39 3% formamide/15% glycerol59-ATGGCTTGGCATTAACATCC-3959-GGCAAAGTTAGAGCATTCATTG-3959-TGAAGGGTCAATTAGAAATTAGG-3959-TGTATCATTTTCTTCATATTGTTACC-3959-GGCAAATCATAAATCTAAAATCCC-3959-TGCTCCTTCCTAAAACCTCC-3959-GACACAGACAACCCAATCCA-3959-GAAAGGCAAGTAGCTCAGGG-3959-TGGAAGGGCAATGTTACAAA-3959-AAAACATCGAGACAACGGATG-3959-AAAAAGCAGCAAGGAGAAAGTG-3959-AGTTGTCAAGGGCAAGTTCG-3959-GAGGCATTTGCATTTTCTCC-3959-TCCCGTGAAATCAGTTCAAA-3959-CCAAATGATCCTCAAGTTTTCC-3959-TATGCCATCATTTCAGGGAC-3959-AAATGGCAATTAGGTTGAACG-3959-ACTTCCGAGGTGGAGATACC-3959-CACGACAGTCCAGTGCTTGT-3959-AGTCACTCTTTCCCCTCCTG-3959AACCATCAAACTTTTAGGACAAAC-3959-CCACTTTGGATTGTGGCTTC-3959-CGGCAATGTGAAAAGAAATG-3959-CCAGGGAAAGGAACTTCCAG-3959-CTCCAGTGAGCAGAGGTAGC-3959-TACTCACTTGCCCTGCCTAC-3959-CAAAGGGTAAAGGAGAAGGG-39 Part of exon59-TGCATATCTATCCTTCATGTGC-39 Part of exon59-CTGCAATGCTGACTCAGGG-39 3% formamide/15% glycerol59-CCAAAGCGAGGACTCAATC-3959-TATGGGCTCTTTCCTTCTGG-3959-CCTCGTTTCTGCCATTCAGT-3959-TCCCACTTCTGATAGGCTCC-3959-CATCCAGGTTCCATTCCATC-3959-ACGGTTTCATGCTGGGATTC-39GAATGATTAAGTAGAGGCAGTTCC-3959-GGAAATGACCAACTCTAAAAATGC-3959-TTCAGGGTTCCCTTGTATGG-3959-TGAATACAGCTTGCCTGTGC-39

9 59-TGAGATGCAAGAATTTCTGGACTTCATAGC-39 Expand-PCR59-GCATGGTGGCACATG-39 Sequence primer59-GAAAATAAGCAGGTGGTCCG-3959-GGAGAATTCAGGGACACAGC-3959-TTGAGTAAGATAGTTCCTGTC-3959-GCCATGAAGCAAAGGAAAAG-3959-CTTCCTTTCAAAGCCCAATG-3959-GCTCACACAGACCCATTCTG-3959-GCATGGCCAATTGGTTTAAG-3959-TAAGCATTGACCAGCACACC-3959-CATGCATTCAATGTCTTGCC-3959-GAGCCCCTAAAGAAACCGTC-3959-CCCCAATTTGCATTTGATAAAC-3959-TCACCCTTACCAAAGGTGTTG-3959-ACCCAATGATTCGTTTGAGG-3959-GCTGTAGGGCGTTGTCAATC-3959-TGCTTGGTAAGGTTGCTGTG-3959-AATAACCAAAGAGAGGGCGG-3959-CTGTTGAAGAGGCCACCTTG-3959-CCCATCCACACTCACATACG-3959-CATTCAAAACCACCTCCC-3959-GCAGTAAGTCAGGATTGCCC-3959-CTTTGGTCTCAGAAATGCCC-3959-AGTAGGTTTTGCCGATTCCC-3959-ATGTGGTAGAAGGCTGTGGG-3959-AAGTTTCTGCCCCTTGTCAG-3959-ATTTTCTTGAAACATGGCCC-3959-AACACTTTGGGTTTTCCAGC-39 Part of exon59-TGTAACAGTGAGGAGGGCTG-39 Part of exon59-GCGACAGAGCGAGATTCTG-39

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204 HADANO ET AL.

Northern blot analysis revealed the presence of twotranscripts of approximately 4.3 and 1.6 kb of theALS2CR1 gene in various tissues (Fig. 3). Both tran-scripts showed a similar pattern of expression, with ahigher level of expression in the heart, skeletal muscle,and testis. The small 1.6-kb transcript, which mightrepresent the full-length sequence reported in thisstudy, was expressed at a higher level than the largertranscript.

ALS2CR2

Approximately 2.1 kb of the consensus sequence forthe ALS2CR2 transcript was generated by aligningdata from 44 ESTs compiled in the UniGene serverunder the ID number of Hs.5771. One cDNA clone(IMAGE Clone 35296) was subjected to the full-lengthDNA sequencing as a representative clone forALS2CR2, and it encompassed 2043 nucleotides withan additional 110 nucleotides from the 59 end of theconsensus sequence, but lacking 125 nucleotides at the39 end of the consensus sequence. The composite DNAsequence of ALS2CR2 encompassed 2168 nucleotideswith a single ORF that was 1257 nucleotides long(242–1498 nt) and predicted to encode a 47.0-kDa pro-tein consisting of 418 amino acids (Fig. 2b). The se-quence of the putative translation start site did notmatch the Kozak consensus sequence. Two putativepolyadenylation signals (ATTAAA, 2020–2025 nt;AATAAA, 2147–2152 nt) and a poly(A) tail have beenidentified at the 39 end of the ALS2CR2 compositesequence. The nucleotide sequence of ALS2CR2 hasbeen deposited with the DDBJ, EMBL and GenBankData Libraries under Accession No. AB038950.

The amino acid sequence predicted from theALS2CR2 ORF was subjected to a sequence homologysearch using the BLAST server. ALS2CR2 was signif-icantly homologous to more than 30 protein sequences

TAB

Primer Sequences for RT-PCR o

Gene Fragment Product (bp) Forward primer

LS2CR1 A 696 59-TCCGGTTTGTAGATTCCCTG-39B 756 59-TACAGAGGGAAACCACCGAG-39

LS2CR2 A 652 59-CCTGAAGAGAGTCGCTGGC-39B 728 59-ATACTCCCACGGGAACACTG-39C 539 59-CCCACAGTTCAGCACATCAG-39D 582 59-GAGAAAAGGCCATCAGCAAG-39

LS2CR3 A 920 59-GCTCGTTGCTACGGATGTC-39B 358 59-GGAAATGCTGCAAGAAAAGC-39C 1611 59-CCTGCAAGCTTCCAAAGATG-39D 536 59-CTTGCCCTTCTCCTTTACCC-39

FLAR A 507 59-GAAAGGATTCTGAAAGAAATGAAGTCAGCB 407 59-GCAGAGATTGGTGAGGATTTG-39C 324 59-GAGCAGGGACAAGTTACAGGAATGTTCTCD 709 59-AGAACCAGTGAAGAAATCCATTCAGGAATE 541 59-GTCTCACAGCTCACCATCCC-39

ASP10 A 1029 (900) 59-ACCTTGGAGCACACAGAGGATTCTACTTTCB 683 59-ATGCAAGGAGCATCTGCTAACACTCTAAACC 378 59-GAAGCAGATGCTCTGAACCC-39

ASP8 A 482 59-GCTGAATTTGCTAGTCAACTCAACAGGAAGB 344 59-AAAGAGAATGTTGGAGGAAAGCAATCTGTC 473/428 59-CAGAAGAAGTGAGCAGATCAGAATTGAGGD 399 59-GGACAAAGTTTACCAAATGAAAAGCAAACE 485 59-TTCACTGGTTTGAAGTGCCCTTCCC-39

encoding serine/threonine protein kinases. We queried

the PROSITE database with the ALS2CR2 amino acidsequence, and it was revealed that ALS2CR2 con-tained a typical protein kinase domain at 58–369 res-idues in its translated sequence. Therefore, ALS2CR2might encode a novel serine/threonine protein kinase.

A single 2.4-kb transcript was detected by the North-ern blot analysis using the ALS2CR2 probe (Fig. 3).This transcript was expressed in a similar manner asALS2CR1 with a higher level of expression in theheart, skeletal muscle, testis, and colon. The transcriptdetected in the testis was slightly larger (approxi-mately 2.6 kb). These data suggest that the small por-tion of the ALS2CR2 transcript at either the 59 or the39 end could still be missing in the cDNA clones ana-lyzed in this study.

ALS2CR3

Initially, consensus sequences for the ALS2CR3transcript were generated by aligning 171 ESTs thatwere compiled in the UniGene server under the IDnumber of Hs.154248 and a partial mRNA sequence(KIAA0549, Accession No. AB011121) encoding a partof the ALS2CR3 transcript. Based on data of the con-sensus sequences obtained, we have chosen threecDNA clones (IMAGE Clones 531437, 28264, and40750) that are likely to cover the maximum portion ofthe ALS2CR3 transcript. To obtain the DNA sequenceof the gapped portion between two cDNA clones (28264and 40750), RT-PCR was carried out using primers F,59-CTGTTTTCAGGCCTTTGAGC-39, and R, 59-CT-TCTGGCTTAACCCCTTCC-39, and the DNA sequenceof its amplified product (294 bp) was determined. Fur-thermore, to clone the 59 end of the ALS2CR3 tran-script, we performed 59-RACE analysis. Six indepen-dent 59-RACE clones were obtained. DNA sequenceanalysis of these 59-RACE clones, RT-PCR products,and three cDNA clones allowed the generation of a

2

he ALS2 Candidate Transcripts

Reverse primer Remarks

59-GCAAAGGCTTCTCCAGTGAC-3959-CCCTCGAAGACACTCTCTGG-3959-TAGTTCAACCCTCTCACGGC-3959-TCCACTGGAGACAAGCACAC-3959-TCCAAGGTAACACTGGAGGC-3959-AGGCCAGAGAATCTGGGAG-3959-ACACAGTCGCTGACAAGCTG-3959-TAACTCGTGCAGCTCCATTG-3959-TTGACCACTCTGGCTATCCC-3959-ATTTCCATTCCTCCATTCCC-3959-TCCTTGCTTATCTTGCCTC-3959-GCTTCTGATTCCTGAATGGATTTCTTCAC-3959-TGAGATGCAAGAATTTCTGGACTTCATAGC-3959-AACTCTGCTGTTCCAATCATACATGTAGCC-3959-TTGAGTAAGATAGTTCCTGTC-3959-ATCTTTATGGGTTCCTTGTCTGTCCTTCAG-39 Minor variant (900-bp product)59-ACAGCAGTGAGGATGGATAAGATGTCTTC-3959-ACCCAATGATTCGTTTGAGG-3959-GTTCCCTTTCCATCTCCTCCTTTCTAGTGT-3959-CACAGACTCTTTTCAGGATGTCCAACTTTC-3959-ATCCAAGTGTGTTCCATTCCTGTCCCTAAT-39 Two variants59-AACACTTTGGGTTTTCCAGC-3959-GCGACAGAGCGAGATTCTG-39

LE

f t

C-39

-39C-39

-39-39

-39C-39T-39

C-39

complete mRNA sequence for the ALS2CR3 transcript.

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205ALS2 CANDIDATE GENES

206 HADANO ET AL.

It encompassed 6470 nucleotides with a single ORFthat was 2745 nucleotides long (382–3126 nt) and was

TAB

Candidate Transcriptional Units for A

cDNA/ESTRepresentative EST/mRNA

(GenBank/RefSeq No.)

1 KIAA0005 NM_0146702 CLK1 NM_0040713 CALS-109 AI7607704 ALS2CR1 (CALS-7) R437055 ORC2L NM_0061906 CALS-144 AA1475687 NDUFB3 NM_0024918 CFLAR NM_0038799 CALS-46 AI126061

10 CASP10 NM_00123011 CASP8 NM_00122812 CALS-B AA46028013 ALS2CR3 (CALS-C) AB01112114 ALS2CR2 (CALS-21) R4524115 CALS-116 AI19058416 CALS-23 AA40454317 CALS-69 H3883118 CALS-117 AA01284019 CALS-71 AA01593020 CALS-24 AA62139021 CALS-122 N3980522 CALS-111 H6893523 CALS-124 AA37573824 CALS112 AA43605425 CALS-70 Z3980226 UBL1 NM_00335227 CALS-72 N7239428 BMPR2 NM_00120429 CALS-26 T9663130 CALS-130 AI52385731 CALS-139 AA81249632 CALS-140 AI67237933 CALS-135 AA27865034 CALS-132 AA89753135 CALS-25 N6633636 CALS-74 AA01905437 CALS-75 AI79920638 CALS-77 AI018304

39 CALS-78 AA45005040 CALS-79 AI65159041 CALS-80 AA19524542 CALS-81 AI09754443 CALS-27 R7090344 AIP-1 NM_005759

45 CALS-29 N5430346 CALS-83 T8161847 CALS-31 R9779948 CD28 NM_00613949 CTLA4 NM_00521450 CALS-145 AI26792851 CALS-131 AA68324152 CALS-125 AI74240053 CALS-32 H6984654 CALS-33 R08362

a This study (see Table 2).b Hadano et al. (1999).

predicted to encode a 101.4-kDa protein consisting of

914 amino acids (Fig. 2c). Two putative polyadenyla-tion signals (AATAAA, 3801–3806 and 6435–6440 nt)

3

2 and Summarized Results of RT-PCR

Marker

RT-PCR

Brain Spinal cord Lymphocyte

stSG2762 1 1 1U40268 1 1 1

WI-12098 1 1 1

CASP10-Ca 1 1 1CASP8-Ea 1 1 1zx51a03.s1b 1 1 1WI-6694 1 1 2WI-14342 1 1 1

stSG2450 1 1 1stSG27924 1 1 1

sts-AA015930 1 6 2stSG2739 1 6 1

WI-22275 1 1 6A007E47 1 1 1N38735 1 1 2

SGC33812 1 1 2

SGC30128 1 1 1sts-AA019054 1 1 2stSG30101 1 1 1H96813 1 1 1N40208 1 1 1stSG22618 1 1 1sts-AA004596 1 1 2stSG31733 1 1 1N63597 1 1 1WI-17495 1 1 1U31089 1 1 1WI-19869 1 1 1SGC32154 1 1 1stSG16472 1 1 2stSG9022 1 1 2WI-7843 2 2 1

stSG8520 1 1 2WI-11579 1 1 2

LE

LS

and a poly(A) tail have been identified. The nucleotide

207ALS2 CANDIDATE GENES

FIG. 2. Nucleotide sequences and predicted amino acid sequences of ALS2CR1 (a), ALS2CR2 (b), and ALS2CR3 (c) cDNAs. Intron–exonboundaries are shown. The positions of putative polyadenylation signals are underscored. The positions of the 59 ends of six independent

59-RACE clones for ALS2CR3 are shown as inverted triangles.

s

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208 HADANO ET AL.

sequence of ALS2CR3 has been deposited with theDDBJ, EMBL, and GenBank Data Libraries underAccession No. AB038951.

The amino acid sequence predicted from theALS2CR3 ORF was subjected to a sequence homologysearch using the BLAST server. We have identifiedsignificant homology (P 5 3.1 3 10214) between part ofALS2CR3 (108–384 residues) and the huntingtin-as-ociated protein HAP1 (Li et al., 1995), a protein pos-

sibly mediating a role in retrograde vesicle trafficking(Block-Galarza et al., 1997). Prediction of a coiled-coil

omain using the COIL program indicated that thisomologous region clearly overlapped with the puta-ive coiled-coil region at residues 133–352 in its trans-ated sequence.

Two transcripts of approximately 7.0 and 4.5 kb ofhe ALS2CR3 gene were detected in various tissuesFig. 3). Both transcripts showed a similar pattern ofxpression. The large 7.0-kb transcript was expressedt a slightly higher level than the 4.0-kb transcript andhowed the highest level of expression in the heart.he sizes of the two forms of transcripts were wellatched with those predicted from the position of pu-

ative polyadenylation signals in the cDNA sequencenalysis.

enomic Organization

We determined the genomic organization of six genesncluding ALS2CR1, ALS2CR2, ALS2CR3, CFLAR,ASP10, and CASP8.The ALS2CR1 gene was found to contain six introns

nd seven exons and resided within 14.5 kb of genomicNA close to the polymorphic DNA marker D2S116

Fig. 1). The transcriptional polarity of the ALS2CR1

FIG. 3. Northern blot analysis of ALS2CR1, ALS2CR2, andALS2CR3. Northern blot containing 2 mg of poly(A)1 mRNA fromvarious adult human tissues was hybridized with each cDNA clone.The bottom panel represents the same blot hybridized with thehuman b-actin cDNA to confirm RNA quality and relative loading.The size of the transcripts are shown on the right.

ene was in the centromere to telomere direction.

The ALS2CR2 gene comprised 11 introns and 12xons and was predicted to span approximately 30 kbf genomic DNA close to the STS marker WI-10756Fig. 1). Polarity for the ALS2CR2 gene was in theentromere to telomere direction. In the course of DNAequence analysis and mapping of the ALS2CR2 gene,e identified the intron-less ALS2CR2 gene sequence

howing significant identity (99%) to the ALS2CR2DNA. Somatic cell hybrid mapping of this intron-lessLS2CR2 revealed that this sequence mapped to bothhromosomes 1 and 9 in the human genome, but not tohromosome 2 (data not shown), suggesting that ateast two pseudogenes for ALS2CR2 exist in the hu-

an genome.The ALS2CR3 gene was found to contain 15 introns

nd 16 exons and mapped close to the STS markerI-6694 (Fig. 1). The size of the ALS2CR3 gene re-ains to be determined. Transcriptional polarity of theLS2CR3 gene was in the telomere to centromere di-ection. Analysis of the genomic DNA sequences of the9 flanking sequences for the ALS2CR3 and ALS2CR2enes revealed that the transcription start site of theLS2CR3 gene was located 262 bp upstream from the9 end of the ALS2CR2 cDNA in the reverse orienta-ion. The 262 bp of this 59-flanking sequence comprised

GC-rich nucleotide sequences containing several puta-tive binding sites for Sp1 and other transcriptionalfactors as well as several GC-box sequences (Hadano etal., unpublished results). This result suggests thatboth transcription start sites and promoters for theALS2CR3 and ALS2CR2 genes are located close toeach other or overlap each other.

The CFLAR, CASP10, and CASP8 genes were foundto contain 14, 11, and 12 exons, respectively. Thesethree genes are tandemly located within 200 kb of theALS2 region and have identical transcriptional polar-ities in the centromere to telomere direction as de-scribed previously (Hadano et al., 1999) (Fig. 1). Arecent study reporting the structure of the humanCASP8 gene (Grenet et al., 1999) was consistent withour data. Several isoforms or variants of the CFLAR,CASP10, and CASP8 genes have been reported. Thusfar, at least nine representative isoforms of CFLAR(usurpin-a, usurpin-b, usurpin-g, FLAME-1g, FLIPl,FLAME-1b, FLAME-1d, MRITa2, and clarp2), twoforms of CASP10 (Mch4 and FLICE2), and eight formsof CASP8 (Mch5, MACHa1, MACHa2, MACHa3,MACHb1, MACHb2, MACHb3, and MACHb4) areknown (Rasper et al., 1998; Shu et al., 1997; Han et al.,1997; Inohara et al., 1997; Irmler et al., 1997; Sriniva-sula et al., 1997; Fernandes-Alnemri et al., 1996; Vin-cenz and Dixit, 1997; Boldin et al., 1996). Character-ization of the DNA sequences of these isoforms andcomparisons to the intron–exon structures revealedthat all junctions of cDNA sequences in these isoformsmatched the sites of intron–exon boundaries (datanot shown). These data suggest that isoforms ofCFLAR, CASP10, and CASP8 might arise by alterna-

tive splicing.

flGmtA

atgbD

209ALS2 CANDIDATE GENES

All exons, not including the 59 and 39 end exons, wereanked by splice junctions, which conformed to theT-AG rule (Shapiro and Senapathy, 1987). The sum-ary of the DNA sequences of intron–exon boundaries,

he size of exons, and the size of introns for the

TAB

Intron–Exon Organizations

39 Splice acceptor site Exon (b

ALS2CR1 59UTR .....TGTAGTTGTG .. Exon 1 (2..gtcatcttgcgtacgcccag/TCCCCACGAC .. Exon 2 (411)..tcccacaatattttctctag/GAGCTTGTAC .. Exon 3 (163) ....gagtgatttttcccccctag/GACTGGTAAT .. Exon 4 (127) ...taacttctgtttctcctcag/CCTTTGCTTC .. Exon 5 (139) ....ttgtttcctctgttttgaag/AGTCTCAAGT .. Exon 6 (84) ....tgctcttccctttttggtag/GTGAGATGTC .. Exon 7 (430) ..

ALS2CR2 59UTR .....TCGCGGCCTC .. Exon 1 (1..tttgcttttgttttttatag/TAGGATATAT .. Exon 2 (108) .. GT..aatgcatttctgacttccag/GATTGCTTCT Exon 3 (81)..atctttaaaaacaaatgcag/GTTGATGAGC .. Exon 4 (100)..agttttatgtctgattacag/GAAGAGGATT .. Exon 5 (122) ....ctcttgtgtgtttatcctag/AAAGCCGTGA .. Exon 6 (109) ....attttaggggtttctttcag/GTTCAGCAAG .. Exon 7 (124) ....attgtttctttttgttctag/GAGTATTAAA .. Exon 8 (172) .. A..aaattaattgtgtcctacag/GATTTACATG .. Exon 9 (105) ...gaaagttctgtttcttttag/ATGCTGTTAC .. Exon 10 (245) ....aactttcttaaaaatttcag/GCCATCAGCA .. Exon 11 (43) ...attttgagtttctttaacag/ATGAAAGAAG .. Exon 12 (814) ..

ALS2CR3 59UTR ....CGCAGAACCG .. Exon 1..acttgatgtttttgttttag/GTCATGACTG .. Exon 2 (290) ....acatagactctcctctttag/ATGTCTGCTC Exon 3 (195)..gttgtttttaactatattag/TTCTAGGCAC Exon 4 (77) ...tgtatttggtattttaacag/AGGGATCGTG .. Exon 5 (117) ....caagcattttatatggacag/GTTAATCAGC .. Exon 6 (210)..tactgatttttttcttgaag/GCTTGTCACA .. Exon 7 (79) ....aaattttttttgcttccaag/GTGAAACAAA .. Exon 8 (131) ....cttttttaatatgttgttag/CATGTGATTG .. Exon 9 (75) .. G..actaccatcctttcttttag/CTGCACGAGT .. Exon 10 (138) ....tctcaaactctttttcttag/GAATCTTTGG .. Exon 11 (80) ....aacacttccttttttgacag/AGCCCAACAG .. Exon 12 (204) ...tgaacgcgattttcctatag/GAGCTCCCAG .. Exon 13 (299)..cctttgttttgcatattcag/GATCACAAAC .. Exon 14 (267) ....tccctaaaaatctatttcag/TTGCAACCGC .. Exon 15 (106)..tttttttttttttttttaag/CTCTGGGTTC .. Exon 16 (4012) .. TGA

CFLAR FLIP 5 9UTR ..CAAGCCATAG .. Exon 1Usurpin 5 9UTR ..GACCACGCAT .. Exon 2alternative 5 9UTR(EST)..ATTCTGAAAA .. Exon 3 (..aaattcccttttaaccacag/AACTCCCCCA .. Exon 4 (418)..tgaacttgtctggtttgcag/AGTGCTGATG .. Exon 5 (106) ...tttgttggtggttctcttag/AGTTTCTTGG .. Exon 6 (136) ....cctcccttctgcttttatag/TTCAAGGAGC .. Exon 7 (83) ....tgcttttttctcttctacag/ATGATAACAC .. Exon 8 (225) .. G..cttattctttgtatttgaag/CTCCATAATG .. Exon 9 (55) ....ttctctctctcatcccccag/AAGAACCAGT .. Exon 10 (50) ...tttcttgtcttcctttccag/AGCATACCTG .. Exon 11 (82) ....gtttcccttttgttgcccag/GGTTGGAGTG .. Exon 12 (271) ....ttgttttttgttttccccag/AGCTTCTTCG .. Exon 13a (511) .. G..CAAAGATGTTTTTTCTTCAG/AACTATGTGG .. Exon 13b..tatccttaattgttttctag/GGGGACAATT .. Exon 14a (839) ....GCCTTTCTTGTTTTCTCCAG/AAAACGCCCA.. Exon 14b (

CASP10 59UTR ....ATTCAGTTTT .. Exon 1 (1..cacctctctgtccctttcag/GCTGGCCATG .. Exon 2 (354)..attttgggtgtgtgtcttag/AAACCTGCTC .. Exon 3 (94) ....ctctattgattctcttgtag/ACCTCCCTAA .. Exon 4 (136) ....gtctgtgattttattttcag/CTATCCAGAT .. Exon 5 (107) ....cctcctttcttttttctcag/CAGGAGTCCT .. Exon 6 (37) ....ctgagtacctctctttctag/GTAACAGAGC .. Exon 7 (92) ...ggctctatctattcttcaag/AGGGCAGCTG .. Exon 8 (109)..ttttttttgtttttaaacag/AGATCCTGAG .. Exon 9 (493) .. T..tgttttcttctttgttgcag/ACATGAAGAC .. Exon 10 (2103) .. T..tttctctttgtgctcagtag/GATGCTGAAA .. Exon 11 (2772) ..

CASP8 Mch5 59UTR ....TGAAGGCTGG .. Exon 1 (..tcatctctccttccttccag/GAGCAGCCCC .. Exon 2 (97) .MACHa1 59UTR ....GATTCTGCCT .. Exon 3 (2MACHa2 59UTR ....GTAGTGGATA .. Exon 4 (..taccatttattttgacttag/ATTATATTCT .. Exon 5 (331) .. C..accatcttggtcctttgaag/GTTCCACTTC .. Exon 6 (96) ...gtctccatttcccaccacag/GGTCATGCTC .. Exon 7 (106)..tttatttctcctcctcttag/AACCTGCTGG .. Exon 8 (139) ....ggtttccccttttaattcag/AGAGAAGCAG .. Exon 9 (45) ...tgtctcttcgttgtttgcag/GGGAGGAGTT .. Exon 10 (65) ....gttgggtttttgttttccag/ACTTTGGACA .. Exon 11 (142) ....gttcaaatttcacttttcag/GGGCTTTGAC .. Exon 12 (502) ....tgttatgtgatgtatttcag/AGGCGATGAT .. Exon 13 (1250) ..

Note. ND denotes not determined.

LS2CR1, ALS2CR2, ALS2CR3, CFLAR, CASP10, A

nd CASP8 genes are shown (Table 4). Nucleo-ide sequences of intron–exon boundaries of theseenes, except for ALS2CR1 and ALS2CR2, haveeen deposited with the DDBJ, EMBL and GenBankata Libraries under Accession Nos. AB0388952–

4

the ALS2 Candidate Genes

59 Splice donor site Intron (kb)

.. CATAGATGAG/gtaacgtcgagggagcggtt.. 2.32AAGGGCTTG/gtgagaagcctctttcatat.. 0.87

TCTGCTAG/gtacaatttatttttctctt.. 1.86TGGAGAAG/gtaataagaatattttgtat.. 1.69ACCTTAG/gtaaatatagctccttcatc.. 2.17

CTCACAG/gtaggacagactttggatct.. 4.03TTTACTTaaaaaaa...... CGCCGCTAGG/gtaaagacgggaggcaggcc.. 2.81

TTTTG/gtaagtttttgtaaatatag.. 3.98CAATACTTG/gtaagaaaatggttaggctg.. 11.09TAGAAATAG/gtaataatcctgaatttcta.. 2.91

CTTTACAG/gtaacaatatgaagaaaatg.. 1.58GCCTATG/gtaagaatgctggttttaaa.. 0.87TTCACAG/gtatttacttatttgtattt.. 2.10

AGACAG/gtcaggtgtggccgttggat.. 0.30GAACTCAG/gtaagtgctgctaaaatccc.. 0.17AGAAAAG/gtaatattgatctcttttaa.. 0.89TCAAACAG/gtgagctgatctatcatccg.. 0.50AATTGAAaaaaaaa....

) .. AACCACTCCA/gtgagtaagctgtccgcgca.. NDATCACTG/gtaagtccagtttaataaat.. 12.00GTTACATGA/gtgagtattttttttactct.. 6.30TGGCAGAG/gtaggaatatcttttctttc.. 1.53TTGATCAA/gtaagcctttgatcaaatgt.. 1.02CGATCCAAG/gtacgttcaacctaattgcc.. 0.57GAACTTC/gtgagtattaacatattctc.. 2.06TAAAGAA/gtaggtttattcattcattc.. 0.44

AATGGAG/gtaatgaaatctttatcttg.. 1.75TACTGGG/gtaagcaactaagaaagatg.. 3.03

AACAAAA/gtaagtacaggtcacatgtg.. 0.30GTTGCAGG/gtaggaatccgatgataaca.. 1.30

CCCTTGAAG/gtaataaatcagtaagggcc.. 1.22GTTACAG/gtgagaagagtgtctggttg.. 2.00ACCCCCAG/gtaagagtgcctgggaaatc.. 2.89TTCAaaaaaaa....) .. CGACAGGACG/gtacgtgccccgcgcttgac.. 2.07) .. CTGCCCGGGC/gtgagtagaccgagaatcct.. 10.55

.. CCTTTACAAT/gtaggttttcttctctgtct.. 0.21GGACTATAG/gtaattcatcaactcttcct.. 2.89AGGAGAAG/gtgagttttcttcttttggt.. 2.84

CAGTCTG/gtaagaattttggcctctca.. 4.25CTTCAGG/gtgagtctggagaaaacata.. 0.94TTCTTGaaaaa; FLIPS last exon 3.78

GCTCAAC/gtaagaccacctttttttaa.. 3.69TGCCTCAG/gtacagaataaatgatctga.. 0.70

AGACAG/gtaggtgtggaagctgcaga.. 4.31TTTTCAAG/gtgaggaagctgagcagtga.. 6.03AGAAAG/gtgagcccccaggaggtggt..0) .. same as exon 13a 2.05

TCTCCAG/connect to exon 14b) .. CTATTATTATaaaaaaa...... TGATCTGAAG/gtatgtaattaacaaccctt.. 2.46CTGTTTAG/gtgaggacgggtctgtggtg.. 1.58TGAAATG/gtgagtgggtcatacagaat.. 5.19GAGAAAG/gtaagtagcttgtgattgct.. 2.72CTTACCG/gtaggttcagtggtgtgctg.. 7.78AGTAATG/gtaggtatttctaatgtact.. 2.12GCACAAAG/gtctggatggtttcttttat.. 2.10AGATGCTG/gtaagaaagtctggaacagt.. 0.89CCCAAG/gtgagagctcttttttttct.. 8.03AAGAATaaaaa..; casp10a last exon 8.85AGTTATGaaaaa..; casp10b last exon

.. TCTGCTGTCT/gtgagattaagcactatgta.. 0.39ATATTGAG/gtaagcctgggatttccaga.. 24.00.. GGTGGAGCGG/gtgagtgcctgttgccaagg.. 2.18.. TGAGAGTAAG/gtaaggatttgcctctttgt.. 5.90

CCTACAG/gtgggtggaaactcccattg.. 2.72TTAATAAG/gcaggatctctcttaaaatc.. 1.91GATGACATG/gtaagacctggtatcttact.. 1.02AGCAAAG/gtagaaacaacctgacagcc.. 0.12TTCAAATG/gtaatgcttggagatacatt.. 1.95AATCACAG/gtagacggaaacctccaaat.. 1.87GATGCAG/gtacagtagaacccaaaaga.. 5.00TGTCCTCG/gtaagttttgcctactcagc.. 1.14TGATCTTaaaaaaa....

LE

of

p)

29).. ATT

. ACAGATACCTG46)CTC.. T.. GAG

ATGGTA

CTG. TACTG. CTCTG

(182AGC.. C

. CCCT

.. TAAAACTACTTT

TTA. AG.. CCCA.. TT

ATT(315(356

66).. C. CAAAGTAATAC

GGC. TTAATG

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46)TG

. TA.. T

TTC. TT

TGTTG

GAATT

B038985.

sa

c3ikiat(aim(

ta

tmrit

210 HADANO ET AL.

Mutational Analyses

We next sought to identify the genetic cause of ALS2,which would be a recessive mutation in the gene in theALS2 candidate region.

To detect gross deletions, we have analyzed 72 DNAmarkers including all polymorphic DNA markers,STSs, and nonoverlapping transcriptional units withinthe 3 Mb of the ALS2 region (Fig. 1) by amplifying thegenomic DNA of affected individuals and normal con-trols. Further, 73 exons from 6 genes, ALS2CR1,ALS2CR2, ALS2CR3, CFLAR, CASP10, and CASP8,were also analyzed. All DNA markers, STSs, ESTs,and exon fragments in affected individuals and con-trols were of the expected sizes (data not shown). Thisfinding suggests that ALS2 patients do not have exten-sive DNA deletions across or within this region.

The ALS2CR1, ALS2CR2, ALS2CR3, CFLAR,CASP10, and CASP8 genes were evaluated for muta-tions by sequencing exons as well as intron–exonboundaries in three affected individuals in the Tuni-sian ALS2 family and three normal control individuals.Although several single-nucleotide polymorphisms(SNPs) were observed in these genes (Table 5), noALS2-specific sequence alterations were identified.

RT-PCR Analyses of the Coding Regionsof the Candidate Transcripts

To detect aberrant transcripts that might resultfrom mutations that we were not able to detect byexon-PCR, RT-PCR was performed on lymphocyteRNA isolated from affected and unaffected individualsin the Tunisian kindred as well as brain RNA derivedfrom normal control individuals. RT-PCR productswere evaluated for abnormal sequences in the codingregion of each transcript. However, no-ALS2-specificsequence alterations were observed in the coding re-gions of the ALS2CR1, ALS2CR2, ALS2CR3, CFLAR,CASP10, and CASP8 genes (data not shown). Repre-entative RT-PCR results for ALS2CR1, ALS2CR2,

TABLE 5

Single-Nucleotide Polymorphismsin the ALS2 Candidate Genes

Transcript Location Position PolymorphismCodingchange

ALS2CR1 No polymorphismALS2CR2 Exon 5 487 nt CCCAC{G/A}GGA None (Thr)

Exon 8 850 nt GCCCT{G/T}TCC None (Leu)ALS2CR3 Exon 1 141 nt GGAA{A/G}CGCG 59-UTR

Exon 5 805 nt CAT{A/G}TCTTA Ile/ValExon 13 1964 nt CCGA{C/T}AGAG Thr/IleExon 16 3934 nt TTTA{G/A}AATA 39-UTR

CFLAR No polymorphismCASP10 No polymorphismCASP8 Exon 12 1144 nt CAC{C/G}ATGAC His/Asp

nd ALS2CR3 are shown (Fig. 4). s

DISCUSSION

We are focusing on the identification of the gene forALS2 by using positional cloning and candidate geneapproaches. Detailed characterization and mapping ofgenes are a prerequisite for the identification of caus-ative genes and their mutation. This process includesprecise mapping of genes within the candidate inter-val, identification of the primary structure of the gene,and analysis of expression of its transcript in the brainor spinal cord. We have previously generated a YAC-based physical map of the 3 Mb of the genomic regioncovering the complete candidate region for ALS2(Hadano et al., 1999). Using this physical map in con-junction with extensive analyses of cDNA/EST se-quences, we have successively mapped 54 nonover-lapping transcriptional units including 12 knowngenes, KIAA0005, CLK1, ORC2L, NDUFB3, CFLAR,CASP10, CASP8, UBL1, BMPR2, AIP-1, CD28, andCTLA4, within the ALS2 critical genomic interval. It isunknown how many genes are represented by thesenonoverlapping transcriptional units. However, it isclear that any transcribed DNA sequence mapped inthis region represents a candidate gene for ALS2.

In this study we have selected three novel tran-scripts, ALS2CR1, ALS2CR2, and ALS2CR3, as pri-mary candidates for the ALS2 gene, as these genes areexpressed in the brain and spinal cord. ALS2CR1 en-odes a protein similar to yeast ngg1-interacting factor, whose function has not been determined. ALS2CR2s likely to encode a novel serine/threonine proteininase, and ALS2CR3 encodes a novel protein contain-

ng coiled-coil domains. ALS2CR3 is considered to ben especially good candidate because it encodes a pro-ein similar to a huntingtin-associated protein, HAP1Li et al., 1995). HAP1 binds to dynactin P150Glued, anccessory protein for cytoplasmic dynein, which partic-pates in microtuble-dependent retrograde transport of

embranous organelles, as well as to huntingtinBlock-Galarza et al., 1997; Li et al., 1998). It is possi-

ble that ALS2CR3 can also mediate axonal transport,and mutations in the ALS2CR3 genes cause malfunc-tion of transport leading to neuronal cell death.

Among 12 known genes, CFLAR, CASP10, andCASP8 encode caspase/caspase-like proteins contain-ing a death-effector domain and function as activatorsand/or modulators in apoptosis (Rasper et al., 1998;Shu et al., 1997; Fernandes-Alnemri et al., 1996; Vin-cenz and Dixit, 1997; Boldin et al., 1996). Therefore,the CFLAR, CASP10, and CASP8 genes are also po-ential candidates for ALS2, as these might mediatepoptosis and cell death.We consider that the most plausible explanation for

he genetic cause of ALS2 would be a loss of functionalutation in the gene at the ALS2 locus because of its

ecessive trait. As no large deletions in the ALS2 crit-cal region were observed in ALS2 patients, ALS2 mu-ations are suspected to be small deletions or base

ubstitutions in exons or intron–exon boundaries. To

4inti

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211ALS2 CANDIDATE GENES

detect these types of mutations, we have performedexon-PCR and sequencing in the ALS2CR1, ALS2CR2,ALS2CR3, CFLAR, CASP10, and CASP8 genes. Al-though five SNPs were observed in the coding exons ofthree genes (ALS2CR2, ALS2CR3, and CASP8; Table), no ALS2-linked sequence alterations were observedn six genes. Furthermore, RT-PCR analyses revealedo abnormal sizes or sequences in the coding region ofheir transcripts in lymphocytes derived from affected

FIG. 4. RT-PCR amplification of the coding region of ALS2CR1positions of RT-PCR products in the ALS2CR1, ALS2CR2, and ALproducts are shown as boxes and solid bars, respectively. (b) Ampeparated on 2% agarose gels and stained with ethidium bromide. Arain RNA from a normal individual with reverse transcriptase (R

lymphocyte RNA from an affected individual (ALS2) with RT; lanelymphocyte RNA from an unaffected individual with RT; lane 6, lyemplate RNA.

ndividuals. Therefore, it is unlikely that these six g

enes produce aberrant forms of transcripts and pro-eins. We cannot completely eliminate these six geness candidates for ALS2, because we have not analyzedhe expression of each gene in the affected tissues inhe patients and have not searched for mutations inhe whole 39UTR of each transcript. However, detec-ion of the transcripts in lymphocytes of the patientsuggests that the structures of the promoter regionsnd/or regulatory regions including the 39UTR of these

S2CR2, and ALS2CR3. (a) A schematic representation of relativeCR3 transcripts. Coding regions of three transcripts and RT-PCRation of the mRNA sequences by RT-PCR. RT-PCR products were0-bp ladder DNA (Gibco BRL) was used as a size standard. Lane 1,lane 2, brain RNA from a normal individual without RT; lane 3,lymphocyte RNA from an affected individual without RT; lane 5,hocyte RNA from an unaffected individual without RT; lane 7, no

, ALS2

lific10T);4,

mp

enes in ALS2 patients are functionally normal. Taken

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212 HADANO ET AL.

together, the results shown in this study strongly in-dicate that six genes, ALS2CR1, ALS2CR2, ALS2CR3,CFLAR, CASP10, and CASP8, are not causative genesfor ALS2 and could be excluded from consideration ascandidate genes. A recent study reported that theCASP10 gene, one of the genes we analyzed in thisstudy, carried a mutation associated with autoimmunelymphoproliferative syndrome (Wang et al., 1999).

Assuming that the total length of the human genomeis 3 billion basepairs and that the total number ofhuman genes ranges from 30,000 to 120,000 (Ewingand Green, 2000; Crollius et al., 2000; Liang et al.,2000), the number of genes in the 3 Mb of the ALS2region would predicted to be 30 to 120. Thus far, wehave identified 12 known genes and an additional42 nonoverlapping transcriptional units includingALS2CR1, ALS2CR2, and ALS2CR3 in the ALS2 crit-ical region. It is therefore likely that the majority ofgenes in this interval are represented by these tran-scriptional units. To identify the causative gene andmutation for ALS2, further extensive cloning of full-length transcripts and sequencing will be required.

ACKNOWLEDGMENTS

We thank the Medical Research Council (MRC) of the CanadaGenome Resource Facility at the Hospital for Sick Children. Wethank Mr. Claude Marineau at McGill University for his technicalassistance in preparing the genomic DNA samples and the lymphoidcell lines of the ALS2 family. M.R.H. is an established investigator ofthe BC Children’s Hospital. S.W.S. is a Scholar of the MRC. Thiswork was funded by the Canadian Genetic Diseases Network, theCentre for Molecular Medicine and Therapeutics (CMMT), and theInternational Cooperative Research Project (ICORP)/Japan Scienceand Technology Corp. (JST).

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