Cell, Vol. 67, 423-435, October 18, 1991, Copyright © 1991 by Cell Press

The Candidate Gene for the X-Linked Kallmann Syndrome Encodes a Protein Related to Adhesion Molecules Renaud Legouis,* Jean-Pierre Hardelin,* Jscqueline Levilliers,* Jean-Michel Claverie,t Sylvie Compain,* V6ronique Wunderle,*$ Philippe Millasseau,$§ Denis Le Paslier,§ Daniel Cohen,§ Dominique Caterina,§ Lydie Bougueleret,§ Henriette Delemarre-Van de Waal,lJ Georges Lutfalla,# Jean Weissenbach,* and Christine Petit* *lnstitut Pasteur Unit~ de G~n~tique Moldculaire Humaine (CNRS URA 1445) 75015 Paris France tNational Center for Biotechnology Information National Library of Medicine National Institutes of Health Bethesda, Maryland 20894 SGenethon 91002 Evry France §CEPH 75010 Paris France IIFree University Hospital Department of Paediatrics 1007 MB Amsterdam The Netherlands #CNRS Unitd d'Oncologie Virale 94801 Villejuif C6dex France

Summary Kallmann syndrome associates hypogonadotropic hypogonadism and anosmia and is probably due to a defect in the embryonic migration of olfactory and GnRH-synthesizing neurons. The Kallmann gene had been localized to Xp22.3. In this study 67 kb of genomic DNA, corresponding to a deletion interval containing at least part of the Kallmann gene, were sequenced. Two candidate exons, identified by multiparameter computer programs, were found in a cDNA encoding a protein of 679 amino acids. This candidate gene (ADMLX) is interrupted in its 3' coding region in the Kallmann patient, in which the proximal end of the KA/. deletion interval was previously defined. A 5' end deletion was dete(:ted in another Kallmann patient. The predicted protein sequence shows homologies with the fibronectin type III repeat. ADMLX thus encodes a putative adhesion molecule, consistent with the defect of embryonic neuronal migration. Introduction Kallmann syndrome is defined by the association of hypogonadotropic hypogonadism with anosmia (deficiency of

the sense of smell). It was originally described by Maestre de San Juan (1856). Because agenesis of the olfactory bulbs has been reported in several cases (de Morsier, 1954; Males et al., 1973; Klingm~iller et al., 1987), the syndrome is also referred to as olfactogenital dysplasia (de Morsier, 1954). The first familial cases were reported by Kallmann et al. (1944). Later, segregation analysis revealed several transmission modes: X-linked, autosomal recessive, and autosomal dominant (Sparkes et al., 1968; Santen and Paulsen, 1972; White et al., 1983; Chaussain et al., 1988). The incidence of Kallmann syndrome has been estimated at 1 male out of 10,000 (Jones and Kemmann, 1976). The approximately 5- to 7-fold excess of affected males versus females (Jones and Kemmann, 1976; Bouloux, personal communication) suggests (assuming a non-sex-limited expression) that the X-linked form (McKusick, 1990; cat. no. 308700) is the most frequent. In this form, carrier females may be affected by hyposmia. The association of the two symptoms has long remained enigmatic. However, recent reports have revealed a common developmental pathway of both olfactory neurons and neurons synthesizing gonadotropin releasing-hormone (GnRH) (SchwanzeI-Fukuda and Pfaff, 1989; Wray et al., 1989; Ronnekleiv and Resko, 1990). Both types of neurons eriginate in the olfactory placode. The central processes of olfactory neurons cross the ethmoid bone to reach the anlage of the forebrain. Contact between the ingrowing olfactory nerves and the forebrain is essential for development of the olfactory bulbs (Pearson, 1941). The GnRH neurons also migrate to the brain, crossing the ethmoid bone with branches of the nervus terminalis and entering the brain with the central roots of these nerves. Ultimately, they reach the septal preoptic area and the hypothalamus. These results suggested a possible involvement of the Kallmann gene (KAL) in this common embryologic pathway. The finding by SchwanzeI-Fukuda et al. (1989) that GnRH neurons were present outside the brain tissue only in an X-linked Kallmann human fetus strengthens this hypothesis. Genetic linkage analysis and deletion studies have localized the X-linked KAL gene proximal to the STS locus in the Xp22.3 region (Ballabio et al., 1987, 1990). Previous analysis of DNA from two individualswith Xp terminal deletions, one affected by Kallmann syndrome and the other not, allowed us to map at least part of the gene within an interval spanning less than 350 kb (the KAL interval; Petit et al., 1990a). This interval is located between 8.6 and 8.95 Mb from the Xp telomere. We report here the isolation and the characterization of a candidate gene for the X-linked Kallmann syndrome. After an accurate size determination of the KAL interval, the entire interval was sequenced. A search for coding exons by several computer programs led to the identification of 19 putative exons, among which only 2 turned out to be conserved among mammals. One of them was used to isolate cDNA clones from fetal libraries. From the cDNA sequence, one can predict an extracellular protein of 679

Cell 424

amino acids, containing a whey acidic protein (WAP)-type motif and exhibiting significant homology with adhesion molecules. Because of this latter homology, we have named this gene ADMIX, for "adhesion molecule-like from the X chromosome."

1

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3

4X X 4 Y

A

X

Results

X

Isolation and Characterization of a YAC Spanning the KAL Interval

The previously defined KAL interval (Petit et al., 1990a) was limited on its distal end by the breakpoint in patient LIL155 and on its proximal end by the breakpoint in patient AM. LIL155 has a terminal deletion ef the short arm of the X chromosome (Petit et al., 1990a); AM has an Xp22.3; Yql 1 translocation (Tiepolo et al., 1980). Both patients are affected by all the diseases that have been mapped distal to the Kallmann locus (Ballabio et al., 1989; Petit et al., 1990a): statural defect, recessive chondrodysplasia punctata, mental retardation, and X-linked ichthyosis. However, patient AM is affected by Kallmann syndrome (Ballabio et al., 1990) while patient LIL155 is not (Petit et al., 1990a). This interval, first estimated to be and an extracellular domain of N-CAM/L1 (p = 0.014) and between segment < 2 2 3 - 2 7 0 > and twitchin type I motifs (p = 0.07) are most significant. Segment < 3 0 4 - 3 4 0 > displays similarities with extracellular regions of various receptors (Figure 9B). Again, these

similarities involve sequences themselves related to the fibronectin type Ill repeat. However, within the < 1 7 6 - 6 6 0 > region, the segment < 4 6 0 - 6 0 0 > does not reveal any clear similarity with a known protein. This region, encoded by at least 3 exons and containing 4 cysteines, might constitute an original domain. A final characteristic of the ADMLX ORF is a hydrophilic C-terminus, in which 10 basic residues, histidine, lysine, and arginine are clustered in the 19 residues encoded by the last exon. Expression of the ADMLX Gene When Northern blots of poly(A) ÷ RNAs from an M. fascicularis fetus (used for the cDNA library) and human adult brain and NT2/D1 teratoma cells (Andrews, 1984) were hybridized with a 2 kb coding cDNA probe, no transcript was detected in these tissues. The expression of the ADMLX gene was therefore studied by reverse transcrip-

X-Linked Kallmann Syndrome Candidate Gene 429

B

2041 2101 2201 2301 2401 2501 2601 2701 2801 2901 3001 3101 3201 3301 3401 3501 3601 3701 3801 3901 4001 4101 4201 4301 4401 4501 4601 4701 4801 4901 5001 5101 5201 5301 5401 5501 5601 5701 5801 5901 6001 6101

actgttcaaa aagattttgt gaaattgcac agatgtgtaa gcttgttgaa cttcggccac gagacatgca cacttccaga ggcagtggga actgctcaga ggcCCGGact ctcctatgtg actttagtgc aggaagaact tctgtcaatc atggacgcat ctggagacaa gtgagaaaca gtagattggt gaagacagac accagttccc tacaagcatg gagaaaatga agaataggcc tgtttaatgc taaattttgt tttcatgtat ggtgtcgctc atttctattg aattacaaca gaactcagtt ttccctgaat ttggagcacc aaactccgcc ccaaaaagga gagtaacaaa tacacaattc acacataaca ctaagcgtaa atctaatca%A TAAAatatat ttttgactaa attattgatt cgatatgaaa aatcaactaa gattacacag ctttgttttt ttgaatcttt cctaagatca tttttatcct aggtgatttt taaatgaaaa tgtgtaatct aaaatatacc agcgaattta aatctaaaaa tgctcctact ttaagtacct tgtgctgctc tttatgcaaa ggtaaatcaa agttccctct ataaattatg atttacaaaa gacacccaag ccagaggaac tcaatgaaat aagctgctaa tcagatttta ccttggagaa atgaaaatta tttcttgggg atgcctttta atatttgatc ctattatgtg agagattttc ctgatatgtt atcttattta tattttccct tattttcctc aatgcagata atagcttttg gtgcactttt gtttcaccat ctgaaaattc acaaaacttc ttgcttcaaa tgaaaaaatc ccaactattg agcatgttta aatctttgca gagatttgcc ttttcttaat caaagaaagg tctttgtgtg ctagaatatt attggtaatg ttttaaaaat tcctttgatt gatagagaag gacagttatt tgcatttaat tcacccatat gctttcaaat ctagtatatc ttactttttg gaaatgtttt atgctacaaa ttagtgcctt gtagcatgaa cttaagtcaa aacgtgttat caatatagag tgttgcagtg tatattgtaa caacctaaaa cgcagagaag tttaatttaa tactgttttt tttcttgaag gaatactcac atacatggtt tgaaatgtgc atagatatgc atgtctatat aattataaat gcatgtgtat atatatgcaa atatatgtac atataoatgt atatacacac agacacatgc atatacatga atataccttg agcatgaatc cctggagaaa tcgttttogt aggctcacca atggtgagta aagatacagc tcttttaaag gtcataagga taatatattt tccccatcaa tgctgattct gagaaaagag caatttatca aaattaaaca ctgtaaaaga aaggtgtcca tatgtcttta cctacctaag taaaacagga agaaaatcag taacattatc cttaggtttt gacaatggta cttgcttctt gttgttttat tgtttcotga attcatgcag atgcctggcc attcctggga agagtggata actcagaagt cactgtactc cacagagcct cactgcagtg tctaaaggta gatgcaaatt aaaatgcagg gaaaataact tttctgatgt tgatgcatgt ctttgggaaa cacatttata aacatggata cctgataata gatattgaaa cccatttcct gtgtgttaaa atatttaaaa agtggatatt ccaggaatgt tttgcagctt tgtacaagta acataaattg gacacctcag aatgaaagtt catgttggtt ctgaatggtt cactgcagct cctgtcacaa gctgggatgg atttatcaca ttgagttatg aaattacctg gttctaagaa tttttgagtg gcaaaaatag aaaacaatct tcatttgaaa acatccctaa gcttgAATAAAtggatacca tagatagctt ctctttttta ttctggtgtc attaccagca tctgaatttc aagttcttaa aatttcaaaa attaaaattt ttcattatta gctatccatt tatcttttac atgaacttgt catgaacaaa ttcaaatgtt tatgccagca aatttttgta ctgttgcata gttaaaaatg ctgggagtct ctgcatagat acaaaatatt attaaattat tacataaatt taattttata aaatttaatc atgcttcttt tgtctggtaa tagacattgg acagatattt ttagttcaga tggtgattct gaagcttaca tctcccttaa aaaaatctaa agcagctctt atgggcttct aattttaata taAATAAAta atttaaattt tattggtgtt attggaagaa aaatgctatt aatgggctAA TAAAaaacat gtgtttctct tatggatttt aataagctcc agtattattc aaatgatcaa aaatatagtt ataatttttt gaattttaaa aatgtgattg ctctAATAAA gAATAAAatc tatgcttttt aacaaacata gttttggtgc ctaattctgt aatatgtttt attgaaatta gattcatttc tctaatgtga gaaaaatata tccagtaata gtattgactg tttaaaaaat tgagctcatc aaaaatattg tcatcaaata caggtggtta atctgacata cattgcagtt acatgcatta tttttattta caacatttgc tccttaatga tgaatttatc tgtgttaccc tgtttttcta cctggaactc catagaatga tgtttgcaaa ccaacatgtg ctcttttcag tcattcactg ttttaatatg acatggtaga gaagataagg tttatggcag gtaatttttt gtaatgtgta ttaaacgaag ttcaaagatt agaaatacat ctgtgtcctg aaaaccttag atacatagcc gactgtatac agaggttcat ctcaacctca acactattga cttttggggc tggatagttc tctgttgtgg gggtttgtct tgtgcactgt aggtttttag tagcatccac actttctcct caccagatgc cagttgcacc ctcccccaag ttgagacaac caaaaatgtc tccagatatt gccagctacc ccttgaggga tggtacctct ggttgagaac cattgctaga gaatgatctt tactgaattt gccctttata agaaacccag tgaatttcta gagcaagtcc caaaaactaa gggacagcta agaagttatt atggttgact tcaaaggcct aaactgtgtt ttttatgtcc actaaacaac ttgattaaaa gacggaattt tgactcgtgt ctgtatcata caagtacaaa tactaatttt gccctatgta tccgtaaatg tcatttgtga ttttgactta tttatttaat gccctttctt atgccgtggg ttttcaagtt tactcatttc tatggttgca aataactcta aaacttatta tataaacttt catattatag gcagaacaca atggctaaat atctgttgca tgtactttaa agtttattat aaaatataaa cagatatata aagatgttga ctcttacctg tgattttgca tggtcagact cggtgtcagg tacggagagg attctcatga ctgtcttacc tctactgaat attctagtga gttatatgat ttacggagtg attaacagag gtctatataa agttactttt cccctttact taattatatt gtagtgtgca gataacaaaa ctgctacctt ctcatccaag tggtctgtag aattcatgtc ccttacagtg gtcatttaaa gtcaatattt atttatgtat gtAATAAAaa aagttggatt tttgtgtatg tctgtcacat tatttagaga gaagtaatct tgtaaaaatg ttttgtaaaa aacaaaaaag tattgtaaat agtcttgata ttctgtgact cattattttc atgttagagt ttgtacatac tggttcaatA ATAAAgtatc cttaaaccag a (poly A) 6161

tion of the RNAs from various cell types and tissues, followed by PCR amplification using primers derived from the cDNA sequence. Three sets of primers along the coding region of the cDNA (between positions 100 and 394, 1060 and 1380, and 1560 and 1850) were used to analyze the expression of the gene. These primers belonged to 6 different exons (as shown by PCR on genomic DNA for the two first sets, and as deduced from the genomic sequence for the last one). PCR products were detected in all tested tissues: adult brain, liver, kidney, skeletal muscle, lymphoblastoid cells, and NT2/D1 teratoma cells (Figure 10) and fetal kidney and muscle (data not shown). However, a more efficient amplification was frequently observed in adult brain and teratoma cells, as can be seen in Figure 10C. The PCR products are the expected sizes and hybridize to internal oligonucleotide probes (data not shown). Discussion

ADMLX: A Candidate Gene for KAL This paper reports the identification and characterization of a candidate gene (ADMLX) for the X-linked form of Kallmann syndrome. Very strong arguments already support the. hypothesis that the ADMLX gene is the X-linked KAL gene. First, among the putative exons found in the entire KAL interval sequence using multiparameter computer analy-

sis, the only 2 that detected conserved sequences on zoo blots belonged to the same transcript. Thus, although the presence of another gene in the KAL interval cannot be excluded, exhaustive exon screening failed to provide any suggestion for it. Second, the breakpoint of a Kallmann patient interrupts the open reading frame of ADMLX gene more than 10 amino acid residues upstream from the carboxyl terminus, and a deletion of the 5' end of this gene was found in another Kallmann patient. Structure and Putative Function of ADMLX Protein The protein sequence deduced from the ADMLX gene can be divided into two regions. The N-terminal domain of 175 amino acid residues has a high density of cysteines, and the C-terminal two-thirds displays similarities with the socalled fibronectin type ill repeats (FNIII domains). These results are in agreement with the I}-sheet structure predicted for the ADMLX protein. These FNIII domains are known to be present not only in fibronectin, but in a number of other molecules also involved in adhesion: N-CAM (Cunningham et al., 1987), L1 (Moos et al., 1988), Nr-CAM (Grumet et al., 1991), contactin ( F l l , F3; Ranscht and Dours, 1988; BrSmmendorf et al., 1989), fasciclin II (Harrelson and Goodman, 1988), TAG-1 (Furley et al., 1990), and tenascin (cytotactin; Jones et al., 1989). While cytotactin has been identified only as a component of the extracellular matrix, the others were first recognized as expressed

Cell 430

1

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3

123

1

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A

I

-.-X

~X A

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C I 1085

3347

Hind III

H i n d III 3307

3250

I codon

I

1987

~Y

~047

II

=

gB

1

IOA

last exon

exon

Figure 7. Characterizationof the Breakpointof PatientAM Threesetsof primers,4A-6B(A),4A-8B(B), and 10A-9B(C),wereused for PCRon genomicDNAof patientAM (lane1).A 46,XXnormalfemale (lane2) and a 46,XYmalewith an interstitialdeletionincludingthe KAL interval(lane3) (patientPAR252)were usedas positiveand negative controls, respectively.The deletionin PAR252,who is affectedonly by Kallmannsyndrome,was detectedduringsystematichybridization with probesfrom the KAL interval.In patientAM, both (A) and (B) PCR productsare present,whereasPCR product(C) is absent,allowingus to map the breakpointbetweenprimers 9B and 10A at least 31 bp upstreamfromthe stop codon.The rightsideof the figurecorresponds to the centromeredirection.

at the cell surface and subsequently as possibly released (reviewed in Rutishauser and Jessell, 1988, and in Edelman and Crossin, 1991). ADMLX protein exhibits not only similarity with FNIII repeats, but even stronger segmental homologies with some of these related adhesion molecules. Among the five subregions (Figure 9) similar to FNIII, two appeared even more closely related to the neural cell-cell adhesion molecules TAG-l, contactin ( F l l ) , and L1. TAG-1 (Furley et ai., 1990) and contactin (Rathjen et al., 1987; Gennarini et al., 1991) promote neurite outgrowth, and L1 and F11 mediate axon-axon interactions (Fischer et al., 1986; Chang et al., 1987; Lemmon et al., 1989). These data suggest that the ADMLX protein is a new type of adhesion molecule. The ADMLX sequence contains an N-terminal hydrophobic leader peptide, but no transmembrane helical segment, nor a hydrophobic C-terminus for CA phosphatidyl inositol anchorage. This protein may therefore be viewed as a substrate adhesion molecule. However, since some glycoproteins expressed on the surface of developing neurons, N-CAM (Gower et al., 1988) and axonin 1 (Ruegg et al., 1989), are found in both multiple membrane-bound and soluble forms, we cannot exclude the possibility that the ADMLX cDNA studied here is a specific transcript either lacking a membrane association domain or exhibiting another type of anchorage to the membrane. The ADMLX protein could interact with the cell membrane via a specific integrin. In this respect, it can be pointed out that the most significant similarity found in the region is centered on the a5/131 integrin binding motif of fibronectin Arg-Gly-Asp-Ser (RGDS; Fogerty et al., 1990).

B

2 3 4 5 6 7 8 ---X ---X

~X ~-y ~-y ..-y ----X Figure 8. Deletionof the 5' End of the ADMLXGenein PatientRM254 PCR probesderivedfrom the cDNA, betweenpositions306 and 505 (A)and positions399 and 965(B),werehybridizedto the genomicDNA of Kallmannpatientsdigestedby Pstl (A) and Hindlll (B). Lanes1 and 3: normalfemales.Lanes2 and 4'8: Kallmann-affectedmales.Probe (A) revealsX- and Y-specificbands.TheX-specificbandis polymorphic (lane 6) and is deleted in patient RM254(lane 7). Probe (B) reveals several X- and Y-specific bands. Only the upper X-specific fragment is deletedin patient RM254.

However, the protein encoded by ADMLX differs trom previously described adhesive molecules containing FNIII r~peats by a striking feature: ADMLX lacks the C2 immunoglobulin-like domain present in N-CAM, L1, Nr-CAM, contactin, and TAG-1 and lacks the EGF-like repeats present in tenascin (cytotactin) (see Edelman and Crossin, 1991). The ADMLX protein exhibits the original WAP 4-disulfide core signature in the N-terminal part of the molecule. Previously, WAP-type motifs had been described only in short (60-140 residues) polypeptides with protease or ATPase inhibitor activities (Dear and Kefford, 1991). The strong similarity between this domain and protease and/or ATPase inhibitors suggests a related function. Both putative inhibiting functions might be involved in cell adhesion. The adhesion molecules termed "repulsins" (Edelman and Crossin, 1991) modify the shape of the cell (Cox et al., 1990; Davies et al., 1990; Raper and Kapfhammer, 1990) in a way evoking the action of proteases. Extracellular ATP is also involved in several adhesion processes (Jones, 1966), and the cell adhesion molecule cell-CAM 105 has recently been reported to be identical to an ecto-ATPase (Aurivillius et al., 1990).

X-Linked Kallmann Syndrome Candidate Gene

431

A

ADMLX WDNM ELAF SPAI

B76

127 21 I0 14

I, II,

LVKQG~apekasgfaa~v ..... E~V~N~SGVK~SN~H~VPK L E K P G ~ C ~ k n p p r s igt - ~ V . . . . . E ~ S G ~ Q S ~ N I Q ~ C q S N ~ C ~ H ~ C ~ S P V S TKGP ~C~i i i i r ..... ~aml nppN~LK~T~C~GI K~CC~ GS~GM~C~VP Q

~,S~RGC~!~ril~r ..... ~ I ~ n p ~ N ~ F % Y ~ G V ~ _ ~ G ~ K ~ Y P K

TLYKGVPLKPRKE~--~LQSGQLEVKWSS TL ++ P + ~ F T ~ + + + V W++ 1350 TLTQQTAVPPPTD~R~_~NIGPDTMRVTWAP

205

ADMLX

1379

Rat fibronectin

223 N Y G I H P S E D H A T ~ T V A Q T T D E R V Q L T D I R P S R ~ +Y I + + +~I + +++ D ++ D+ ++ 2287 H Y V I E K Q D A A T G ~ W F A C G E S K D T N F H V D D L T Q G H E ++ + ~W I ++T+D + + + ++ 2588 QEDGGR~PCGETSDTSLKVNKLSEGH~ + E Q+ + P 666 EGNAETAQVLGLMPWME E ++ D+ P 655 ESARVIDLIPWM~

304 678 419

(QFRVAAVNVHG FRV AVN H6 {KFRVKAVNRHG FRV AVN +6 (KFRVKAVNRQG ~+FRV+A N+ G {EFRVSASNILG {+FR+ A N G ~EFRIIATNTLG

270

ADMLX

2333

C.elegans

Twitchin

2628

C.elegans

Twitchin

695

Rat Axonal

681

Chicken

SDGSVTVTIVWDLPEEPDIPVHHYKVFWSWMVSSKSL 340 S++S + + W P +P+ + HY V F W + L SNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSEL 714 T+ + W+ PEEP+ V+ Y+V+ + TMLVQWEPPEEPNGLVRGYRVYYT 442

gp TAG-I

Contactin/Fll

ADMLX human

insulin

human

350 T T D G F Q N S V I L E K L C ~ D C [ ~ W E L Q A I T Y ~ R L K S A K V S L H F T S 395 T G ++ ++++ P [Y + L A+T I+G+~ S VS+++ + 1585 T V P G S K S T A T I N N I } P 3 A [ Y r I T L Y A V T RGCt~_~PASSKPVSINYQT 1630

receptor

LAR receptor

ADMLX Rat fibronectin

610 I L P S D H Y V L T V P N L ~ P S T I Y R L E V Q V L T P G G E G P A T I K T F R T P E L P P S S A H R S H L 664 ++P++ ++ L~P + Y÷+EVQ G GPA+ TF TPE P+ + HL 871 W P A N T T S A I L S G L ~ P Y S S Y H V E V Q A F N G R G L G P A S E W T F S T P E G V P G H P E A L H L 925 ++++ Y + NL4P+T Y+++V ++G GP++ 850 V S N Q E Y S T K L E N L ~ P N T ~ Y H I D V S A F N S A G Y G P P S 884 TV L P+ Y + V L GGE+++T T +T+ PP+ 1323 TVTGLEP3IEYgISVITLINGGESAPTTLTQQTAVPPPT 1361

ADMLX N-CAM Chicken

L1 Contactin/Fll

Rat fibronectin

Figure 9. Sequence Comparisons (A) Kallmann ORF WAP motif. Multialignment of the ADMLX ORF WAP domain with WDNM1, a protein associated with the loss of metastatic potential in a rat adenocarcinoma cell line (Dear and Kefford, 1991); human elafin, a human elastase inhibitor (Wiedow et al., 1990); and SPAI-1, a pig ATPase inhibitor (Araki et al., 1989). Amino acids defining the "WAP" motif (Bairoch, 1991) are boxed. Capital letters denote statistically significant multialignments blocks, as computed by MACAW (Schuier et al., 1991). Disulfide bonds are distributed by homologywith known structures. (B) Local similarities of ADMIX ORF with fibronectin type III repeats and related molecules. Only the core of the similarity blocks are shown. Identities and positions with a positive matching score (+) are indicated. Amino acids characteristic of the fibronectin type III repeat or twitchin motif 1 are boxed. Similarities with insulin and LAR receptors concern extracellularregions themselves related to the fibronectin type III repeat. All the alignments are statistically significant either by themselves or as members of a set of paired segments. The ADMIX ORF is most similar to the cell interaction domain of fibronectin, centered on the RGDS sequence (boxed).

Finally, the initial < 3 1 - 1 1 9 > cysteine-rich region, with no significant homology, might interact via disulfide bonds with other protein(s) or form a multimeric structure, as shown for cytotactin (Erickson and Iglesias, 1984).

ADMLX Function and Kallmann Syndrome The ADM LX putative protein exhibits homologies with various adhesive molecules known to be involved in cell migration. Thus, we propose that ADMLX participates in the migration of GnRH neurons and the axonal extension of olfactory neurons impaired in Kallmann patients (SchwanzeI-Fukuda et al., 1989). Additionally, several neurologic

disorders, including mirror m o v e m e n t s , ocular motor abnormalities, sensory neural hearing loss, cerebellar dysfunction, and pes cavus deformity, have also been reported in Kallmann patients (Schwankhaus et al., 1989) and attributed to d e v e l o p m e n t a l abnormalities of central nervous system midline structures. Furthermore, renal aplasia has been described in several X-linked Kallmann patients (Wegenke et al., 1975; Schaison, personal communication). These data suggest that the KAL gene may also be involved in the migration of other neurons and in the d e v e l o p m e n t of nonneuronal tissues. Low-level ubiquitous expression of A D M L X was ob-

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ble recent loss of ADMLY function suggests that ADMLX still escapes X-inactivation. This is further supported by the fact that ADMLX is flanked on its distal side by the STS gene, which at least partially escapes inactivation (see Shapiro, 1985), and on its proximal side by a CpG island u n d e r m e t h y l a t e d on the inactive X (Petit et al., 1990a). The high d e g r e e of sequence conservation between X and Y contrasts with the absence of detectable ADMLX h o m o l o g y in mouse. This situation is reminiscent of other human genes of Xp22.3 (MIC2 and STS) for which no DNA sequence h o m o l o g i e s have been observed in rodents (L. J. Shapiro, unpublished data). Experimental Procedures

C

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Cell Lines Epstein-Barr virus-transformed lymphoblastoid cells from patient LIL155 (Petit et al., 1990a) and fibroblasts from patients AM and FL (Ballabio et al., 1989) were cultured in RPM11640 medium (Flow laboratories) supplemented with 10O/o fetal calf serum (Jacques Boy, Reims, France). PCR Amplification Alu PCR

Figure 10. Expression of the ADMLXGene Three primer sets chosen along the cDNA were used to determine tissue expression of the gene by the cDNA-PCR method (see Experimental Procedures). For each set, both primers belonged to different exons. The position on cDNA of the 5' end of both primers is given for each set: (A) 100 and 394, (B) 1060 and 1340, (C) 1560 and 1850. Lanes 1-6 show the PCR products obtained on reverse-transcribed RNA from human adult brain (1), liver (2), kidney (3), skeletal muscle (4), lymphoblastoid cell line (5), and human embryonal carcinoma cell line NT2/D1 (6), after ethidium bromide staining. Controls lacking reverse transcriptase show no PCR product (lane 7). The two flanking lanes contain Phi X174 Haelll DNA molecular weight marker.

s e w e d in every tissue tested. Since the d e v e l o p m e n t a l defects are limited to a few organs, a specific pattern of expression or activity is expected. This could be achieved either by a transient high expression of ADMLX or by regulated expression of ligand in particular cells during development. GnRH neurons have recently been reported to migrate into the brain along a preformed N-CAM "scaffold" in mouse (SchwanzeI-Fukuda et al., 1990, Soc. Neurosci., abstract); an onset of expression of the ADMLX g e n e might a c c o m p a n y the a p p e a r a n c e of this N-CAM expression. Evolution of A D M L X on the Sex Chromosomes

ADMLX exons used as probes gave hybridization signals of c o m p a r a b l e intensity on fragments from the h u m a n X and Y c h r o m o s o m e s , indicating high conservation between these sequences. Evolution studies in primates strongly suggest that the ancestral STS gene was pseudoautosomal and has b e c o m e sex-specific consecutively to a pericentric inversion on the Y c h r o m o s o m e (Yen et al., 1988). Because of its proximity, less than 2 Mb from STS (Petit et al., 1990a), ADMLX has probably e v o l v e d similarly. The s e q u e n c e conservation observed here suggests that the Y h o m o l o g of ADMLX, ADMLY, has r e m a i n e d active on the Y c h r o m o s o m e until recently. Since this form of Kallmann s y n d r o m e is X-linked, ADMLY can no longer c o m p l e m e n t a mutated ADMLX gene. However, the proba-

PCR was carried out with 50 ng of YAC clone DNA, 250 nM primers in 10 mM Tris-HCI (pH 8.3 at 20°C) containing 50 mM KCI, 1.5 mM MgCI2, 0.01% gelatin, dNTPs (dATP, dCTP, dGTP, and dTTP) at 100 pM each (Pharmacia), and 2.5 U of Taq DNA polymerase (Cetus) for a total volume of 50 p.I. Initial denaturation, 4 min at 94°C, was followed by 30 cycles of 94°C denaturation (1 rain), 60°C annealing (1 min), and 72°C extension (4 min). Specific primers were derived from Alu consensus sequence (278 and TC-65; Nelson et al., 1989) and from the right and left arms of the pYAC4 vector (4R and 4L; Nelson et al., 1991). cDNA PCR

cDNA was synthesized by primer extension using 5 p.M random hexamers (Pharmacia) and 200 U of Moloney murine leukemia virus reverse transcriptase (BRL) per p.g of total RNA. One microgram of total RNA was incubated at 42°C for 30 min in 20 p.I of 16.6 mM (NH4)2SO4, 1.5 mM MgCI2, 10 mM ~-mercaptoethanol, 6.7 pM EDTA, 67 mM TrisHCI (pH 8.8 at 20°C) containing 20 U of RNasin (Promega) and dNTPs (100 p.M each). Tubes were then heated at 95°C for 5 rain and chilled on ice. PCR was performed on the single-strand cDNA in the reverse transcription mix supplemented with 10% DMSO (v/v) and dNTPs (100 I~M each) in a final volume of 100 p~l.The two primers, 21 bases in length, we~ used at 0.3 pM. After 10 min at 80°C, 2 U of Taq DNA polymerase (Cetus) was added, and mineral oil was layered on top of the solution. Initial denaturation at 94°C (2 min) was followed by 30-40 cycles of 94°C denaturation (45 s), 55°C annealing (1 rain), and 72°C extension (45 s). Southern Analysis Restriction enzyme digests (EcoRI, Hindlll, Taql, and Pstl) were carried out with 10-15 U of enzyme per p.g of genomic DNA. Digestions of DNAs, migrations, and transfers were performed as previously described (Petit et al., 1988). To minimize background, Alu PCR-derived probes were hybridized at 65°C for 6 hr with 1.5 mg/ml sonicated human placental DNA prior to blot hybridization. Membranes were washed at 65°C in 0.1% SDS, SSC at 2 x to 0.1 x, depending on the probe. Zoo blots were prehybridized and hybridized according to Howley et al. (1979). Washings were carried out at 42°C in formamide (25O/o-35o/0), 1% SDS, 1 M NaCI, 5 mM EDTA, 50 mM phosphate buffer (pH 7.2). Pulse-Field Gel Electrophoresls High molecular weight DNA analyses were performed using TAFE (Beckman) and OFAGE (LKB Pharmacia). Inverted field was also used with the DNAstar Pulse program. Conditions for digestion and electrophoresis have been reported (Petit et al., 1988).

X-Linked Kallmann Syndrome Candidate Gene 433

YAC 376B4 Subcloning Partial digestion with Mbol was carried out on high molecular weight DNA from YAC clone 376B4. Restriction fragments were ligated with a Z EMBL3 vector after sizing (20-40 kb) on a 0.5O/o agarose gel. Screening for human DNA containing clones was performed with an Alu probe. The DNAs of 150 positive clones were extracted (Manfioletti and Schneider, 1988; Coulson and Sulston, 1988) and digested by Taql. Restriction fragments were separated on agarose, transferred, and hybridized with total DNA from YAC 376B4 and with several other probes (Alu, Kpn, poly(AC), poly(AG), CRI-$232, and Alu PCR-derived probes). Overlapping clones were aligned by comparison of their restriction patterns, using scanner analysis (laser scanner, Molecular Dynamics; Visage 4.3, Bioimage) and a computer program (Bellann6Chantelot et al., 1991). Sequencing Reaction and Alignment Sequencing of genomic and cDNA clones was performed on M13 single-strand templates. The DNA was sonicated, size fractionated (600-1200 bp) on agarose gel, and blunt-end ligated to an rap18 M13 vector. Single-strand templates were sequenced with fluorescently tagged M13 primers, using Taq DNA polymerase (NBL), on Applied Biosystems 370A DNA sequencers (Applied Biosystems Inc.). Raw sequence data were processed using the Staden shotgun package program (Staden, 1987), modified so as to handle this genomic project (750,000 sequenced nucleotides). cDNA Libraries Two human fetal brain cDNA libraries were purchased from Stratagene. One, in Z Zap II vector, oligo(dT) and random-primed, was derived from a 17- to 18-week late-abortion normal female (cat. no. 936206). The other, in Uni-Zap XR, oligo(d'l') primed, was derived from multiple 15- to 20-week gestation donors (cat. no. 937201). In addition, a Z library was made from a 36-day M. fascicularis fetus. Total RNA was prepared by the modified hot phenol extraction method (Sambrook et al., 1989). Ground tissues were lysed in 10 mM sodium acetate (pH 5.2), 0.5°/0 SDS, then extracted by phenol at 55°C. Poly(A)+ RNAs were purified, and cDNA synthesis was performed using random hexamers according to manufacturer recommendations (Pharmacia). cDNAs were cloned in Zgtl0 vector. One million independent clones were plated, then transferred to nitrocellulose membranes (Hybond C-super, Amersham). Hybridizations and washings were performed as previously described for Southern analysis (see above).

Protein Sequence Analysis The primary structure analysis of the candidate Kallmann gene ORF was performed with PCgene (Bairoch, 1991) and a variety of Unixbased tools (J.-M. C., unpublished data). Similarity studies were performed with BLASTp and FASTA (Lipman and Pearson, 1985) on a nonredundant protein sequence collection (described in Boguski et al., 1991 ) maintained at the National Center for Biotechnology Information, Bethesda. Additional multialignments were performed with MACAW (Schuler et al., 1991) and Blast3 (Altschul and Lipman, 1990) to ensure the objectivity of similarity domain boundaries as well as statistical significance. Acknowledgments We would like to thank E. Barillot and P. Gesnouin for their active participation in ~. contig analysis. We also thank J. Hazan, N. Vega, L. Baron, C. Discala, and S. Laganier for their help in sequencing and contig alignment. We are indebted to Genethon for access to their sequencing facilities. We are grateful to J. P. Bourgeois and G. Germain for providing and operating upon the macaque monkey and to A. Marchio for valuable advice on cDNA cloning. This work could not have been undertaken without the patients, whom we would like to thank for their contribution, as well as their physicians, especially Dr. M. F. Croquette and Dr. G. Schaison. Several tissues, cell lines, and RNAs were kindly provided by other scientists. We thank M. SchwanzelFukuda, R. M. M~ge, M. Goodhardt, and J. Hill for discussions and critical reading of this manuscript. This work was supported by the Minist(~re de la Recherche et de la Technologie (grant 90. C.0518) and by the Association FranGaise contre les Myopathies (grant C. S.22/01/90, code 3 CA 8103190). R. L. and J.-P. H. were supported by fellowships from the Minist6re de la Recherche et de la Technologie, the Fondation pour la Recherche M(~dicale, and the Association Fran(~aise contre les Myopathies. At completion of this work, parts of the cDNA sequence of ADMLX were compared with sequencing data obtained by Ballabio et al. This indicated that both groups had identified the same candidate gene for KAL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received August 9, 1991; revised September 18, 1991.

References Exon Screening by Multicriteria Automated Procedure The genomic DNA sequence was screened for candidate exons prior to the completion of the contig process. The two orientations of the 67 kb DNA sequence were merged into a single sequence and submitted to a multicriteria automated procedure (Claverie, unpublished data) aimed at the detection of internal coding exons. This procedure integrated the consideration of ORFs, putative splice junctions, differential nucleotide hexamer compositions, and segmental similarities with known proteins. Briefly, all ORFs (defined between two successive stop codons) of minimal length (50 nucleotides) were selected. All potential (even overlapping) exons within these ORFs were further delimited from all couples of putative splice junctions. Acceptable departures from consensus (Senapathy et al., 1990) were fixed at two and four positions for the donor and acceptor sites, respectively. Each of the candidate exons (thus defined as ORFs bordered by acceptable splice junctions) were then ranked according to an "exon potential index" computed from the differential frequency of nucleotide hexamers in exons (Fex)~/ersus introns (F~n)(Claverie et al., 1990). The value used here was the average of (Fox- Fin)/Fmo=~over the segment length. All segments with a positive exon potential index value were then translated into amino acids and tested for significant similarity with known proteins using the BLASTp program (Altschul et al., 1990) and the optimal PAM120 scoring matrix (Altschul, 1991). Best candidates (and cDNA probes) were chosen from the simultaneous consideration 'of similarity score and exon potential index. The top candidate (CS1) corresponded to the exon (Figure 6) with N-CAM/L1 similarity.

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X-Linked Kallmann Syndrome Candidate Gene 435

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