Charcot-Marie-Tooth disease is the mostfrequently occurring inherited peripheralneuropathy. CMT is classified in two

main groups1,2: (i) demyelinating CMT,associated with reduced nerve conduc-tion velocities and segmental de- and

remyelination and onion bulb forma-tions, and (ii) axonal CMT, characterizedby axonal degeneration without demyeli-nating lesions and the presence of clustersof regeneration. We diagnosed an axonalCMT phenotype associated with hoarsevoice and vocal cord paresis in two smallfamilies, LF20 and LF249, and one largeinbred family, LF38, with Spanish ances-try. The disease segregates as an autoso-mal recessive trait. The clinical picture ischaracterized by onset at childhood withweakness and foot and hand wasting,leading to disability at the end of the firstdecade3. Sensory-nerve action potentialswere decreased or absent in all affectedindividuals. In families LF20 and LF249,we did not register median, ulnar, andperoneal compound motor action poten-tials (CMAPs). However, we obtained

The gene encoding ganglioside-induced differentiation-associatedprotein 1 is mutated in axonalCharcot-Marie-Tooth type 4A disease

Published online: 17 December 2001, DOI: 10.1038/ng798

We identified three distinct mutations and six mutant alleles in GDAP1 in three fami-lies with axonal Charcot-Marie-Tooth (CMT) neuropathy and vocal cord paresis, whichwere previously linked to the CMT4A locus on chromosome 8q21.1. These resultsestablish the molecular etiology of CMT4A (MIM 214400) and suggest that it may beassociated with both axonal and demyelinating phenotypes.

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22 nature genetics • volume 30 • january 2002

(Fig. 1). The first nonsense mutation is ahomozygous [G92A] mutation in familiesDUK 1404 and DUK 1405, confirming theshared haplotype over this region. Thesingle–base pair mutation in the first exonconverts a codon specifying tryptophan(amino acid 31) to a nonsense codon andis predicted to result in a truncated pro-tein. Family DUK 1402 has a secondhomozygous nonsense mutation in exon 5[C581G], which converts a codon specify-ing serine (amino acid 194) to a nonsensecodon and is also predicted to result in atruncated protein. This family does notshare any haplotype with the other fami-lies over the entire CMT4A region.

The third mutation is a missense muta-tion in DUK 1403. This is a homozygous[G482A] mutation in exon 3 that causes a[R161H] change. Although this is a con-servative amino-acid change, the 204 con-trol chromosomes tested did not show thebase-pair change (for sequence tracingsand molecular methods, see Web Figs Band C and Web Note A) .

The observation of three differentmutations in these four geographicallyand ethnically close Tunisian families wasunexpected. We do not know whetherthese mutations are attributable tochance, to a region with a high mutationrate or to a frequent gene defect.

Little is known about the function ofGDAP1. It gives rise to a 4.1-kb transcriptand encodes 358 amino acids. The mouseand human proteins have 94% homology.The carboxy-terminal region contains aglutathione S transferase (GST) domain,along with two or three transmembranehelices. Liu et al.5 used α-2, 8-sialyltrans-

ferase (GD3 synthase) activation to initiatecholinergic differentiation with neuritesprouting in a Neuro2a neuroblastoma cellline. GDAP1 was highly expressed duringthis activation. It seems to be developmen-tally regulated in mouse brain, with maxi-mal expression in adult brains, and wasfound to be specifically expressed in neuraltissue. Notably, GDAP1 is expressed in bothSchwann cells, the primary source of cDNAin the cauda equina, and the central ner-vous system. This suggests that GDAP1 mayhave a role in the nervous system other thanits involvement in cell differentiation, thedisruption of which leads to CMT4A.

A new locus was recently reported6 forautosomal recessive axonal CMT inTunisian families, between markersD8S1807 and D8S548. This region over-laps GDAP1 and raises the question ofwhether mutations in GDAP1 can causeboth axonal and demyelinating neuro-pathic phenotypes. The answer is yes: onpage 22, Cuesta et al.7 report that Spanishfamilies with axonal neuropathy familiesdo have GDAP1 mutations. This findingsupports reports that a known CMT1gene, MPZ (P0) can also cause axonal(CMT2) phenotypes3. The finding ofGDAP1 mutations in these two reportsshows that this axonal and demyelinatingdelineation8,9, although useful, does notimply unique etiologies.

Our finding that mutations in GDAP1lead to CMT4A suggests a potentiallyunique mechanism leading to thisdemyelinating neuropathy. This workshould continue to provide insight intothe normal and disease mechanisms ofperipheral nerve biology.

Note: Supplementary information is avail-able on the Nature Genetics web site (http://genetics.nature.com/supplementary_info/).

AcknowledgmentsThis work was supported by grants from theNational Institutes of Health. We would like tothank S. Stinnett at GlaxoSmithKline, RTP, fortechnical assistance in helping construct theshotgun sequence.

Rachel V. Baxter1, Kamel Ben Othmane1,Julie M. Rochelle1, Jason E. Stajich1,Christine Hulette1, Susan Dew-Knight1,Faycal Hentati2, Mongi Ben Hamida2, S.Bel2, Judy E. Stenger1, John R. Gilbert1,Margaret A. Pericak-Vance1

& Jeffery M. Vance1

1Center for Human Genetics, Institute ofGenomic Sciences and Policy, Research ParkBuilding II Room 105, Box 2903, DukeUniversity Medical Center, Durham, NorthCarolina 27710, USA. 2Institut National deNeurologie, La Rabta, 1007 Tunis, Tunisia.Correspondence should be addressed to J.M.V.(e-mail: jeff@chg.mc.duke.edu).

Received 23 July; accepted 12 November 2001

1. De Jonghe, P., Timmerman, V., Nelis, E., Martin, J.J.& Van Broeckhoven, C. J. Peripher. Nerv. Syst. 2,370–387 (1997).

2. Ben Othmane, K. et al. Hum. Mol. Genet 2,1625–1628 (1993).

3. Vance, J.M. The many faces of Charcot-Marie-Toothdisease. Arch. Neurol. 57, 638–640 (2000).

4. Ben Othmane, K. et al. Neurogenetics 18, 18–23(1998).

5. Martinotti, A. et al. Hum. Mol. Genet. 1, 331–334(1992).

6. Barhoumi, C. et al. Neuromuscul. Disord. 11, 27–34(2001).

7. Cuesta, A. et al. Nature Genet. 30, 22–25 (2001).8. Dyck, P.J. & Lambert, E.H. Arch. Neurol. 18, 603–618

(1968).9. Dyck, P.J. & Lambert, E.H. Arch. Neurol. 18, 619–625

(1968).

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CMAPs in axillaris nerves with distallatencies within the normal range (3.7 msand 3.2 ms, respectively; the normal limitis <5.3 ms). In family LF38, motor-nerveconduction velocities in distal medianand ulnar nerves were slightly reduced(41 m s–1, with a CMAP of 1.4 mV, and42 m s–1, with CMAP of 0.8 mV, respec-tively) and were normal in proximalmedian nerves (58 m s–1). Notably, suralnerve biopsies of two probands showedloss of myelinated fibers and axonaldegeneration, with no signs of demyeli-nation and remyelination.

We obtained cumulative lod scores of6.2 and 4.6 at θ=0.00 with STR markersD8S286 and D8S164, respectively (WebTable A), originally associated with theCMT4A locus, suggesting that the candi-date gene mapped to chromosome 8q21.1(refs 4–6). We further analyzed the largeinbred family for ancestral recombinationevents by homozygosity mapping (Fig. 1)7.Loss of homozygosity was observed distalto marker D8S541 in patient V.1 and proxi-mal to marker D8S551 in patients IV.12and IV.14, placing the genewithin the 2-cM interval flankedby these two markers.

The gene GDAP1 (ref. 8) mapswithin the genetic candidateregion. The homolog Gdap1 ishighly expressed in mouse brainand was identified after ganglio-side-induced differentiation ofthe mouse neuroblastoma cellline Neuro2a8. This gene may beinvolved in a signal transductionpathway in neuronal develop-ment. GDAP1 spans 13.9 kb ofgenomic DNA, with the codingsequences split into 6 exons. Thesequence of GDAP1 contains anORF of 1,077 nt and encodes a358-aa protein8. A 2,505-bp EST(AL110252) located 344 ntdownstream of the stop codon ofGDAP1, shows two putativepolyadenylation signals. We pos-tulated that this sequence may bepart of GDAP1 (Fig. 2a). Weamplified a 754-bp productfrom total brain cDNA usingnested primers based on theGDAP1 exon 6 and AL110252sequences (Fig. 2b), suggestingthat the EST is an untranslatedpart of exon 6.

Northern-blot analysis showedthe greatest GDAP1 expression inwhole brain and spinal cord. Weidentified a 3.9-kb major tran-script, in agreement with the sizepredicted from the AL110252-containing mRNA (Fig. 2c). We

confirmed, by RT–PCR, ubiquitous expres-sion in several human (Fig. 2d) and mousetissues (Fig. 2e), with apparently predomi-nant expression in nervous system tissues.Amplification of human sural nerve andmouse sciatic nerve transcripts suggeststhat GADP1 expression does not occur justin neurons but also in Schwann cells. Whenexpression levels of different nervous tis-sues are compared, however, GDAP1expression is higher in central tissues thanin peripheral nerves.

We PCR-amplified and directlysequenced the entire coding region ofGDAP1, including exon–intron bound-eries, in probands of the three familieswith CMT (Web Table B). We identified(i) six mutant alleles and three distinctmutations; (ii) two nucleotide substitu-tions; (iii) a C487T transition in exon 4and a C581G transversion in exon 5 thatgenerate two nonsense mutations, Q163Xand S194X, respectively; and (iv) inser-tion 863insA in exon 6, leading to aframeshift mutation that generates twoabnormal amino acids after threonine 288

and terminates the protein at codon 290(T288fsX290). Family LF38 proband(IV:1) was homoallelic for the Q163Xmutation (Fig. 2f). Using HaeIII restric-tion analysis, we observed complete seg-regation between the mutation and thedisease (Fig. 2g). The proband from fam-ily LF249 was a compound heterozygotewith respect to Q163X and S194X muta-tions (Fig. 2f). The index individual fromfamily LF20 was heteroallelic with respectto Q163X and T288fsX290 (Fig. 2f). Weconfirmed mendelian segregation of thedisease in these two families by HaeIIIrestriction analysis, SSCP analysis andASO analysis (data not shown). Thesenucleotide changes were not observed in134 normal chromosomes.

The amino-acid sequence of GDAP1shows strong similarity to glutathione S-transferases (GSTs), enzymes that have arole in the detoxification of cells. By sec-ondary structure analysis we detected theβαβαββα topology of the glutathione(GSH) binding site of the N-terminalthioredoxin-like fold domain of GSTs in

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nature genetics • volume 30 • january 2002 23

Fig. 1 Pedigree and haplotype segregation of fam-ily LF38. Affected individuals are indicated by thefilled symbols, unaffected individuals by unfilledsymbols. Below the symbol for each individual aregenotypes for 15 markers at 8q21.1. The shared dis-ease-associated haplotype is in black.

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24 nature genetics • volume 30 • january 2002

Fig. 2 Molecular analysis of GDAP1. a, Diagram showing exon–intron structure. Exons are indicated by black boxes; exon 6 coding sequence is in black and thenoncoding sequence is in white. The position of AL110252 EST is indicated as a solid bar. b, Nested PCR between exon 6 and AL110252 (lane 2). c, Northern-blotanalysis with a 1,015-bp cDNA probe shows both the major 3.9-kb transcript and a minor 2.9-kb transcript that are more abundant in brain (whole), spinal cordand thyroid gland. β-actin gene ACTB was used as an internal control for the amount of RNA in each sample. d,e, RT–PCR analysis of GDAP1 and GAPDH (control)transcripts in human (d) and mouse (e) tissues. RT(–), no reverse transcriptase was added to the first-strand reaction of the brain RNA. PCR(–), no first-strand tem-plate was added to the PCR reaction. f, Direct sequences of GDAP1 mutations identified in the three probands. Mutation changes for exons 4, 5 and 6 are repre-sented on the left side and normal controls are shown on the right. g, Segregation of the Q163X mutation in family LF38 revealed by restriction analysis. PCRamplification product of wildtype DNA produces a 288-bp band. A C487T substitution destroys an HaeIII restriction site that normally generates two fragmentsof 148 and 140 bp in the wildtype. Affected individuals (filled boxes or circles) show a unique undigested band of 288 bp, indicating that they are homozygouswith respect to the Q163X mutation. Obligate carrier parents and carrier sibs show one undigested 288-bp band and the two digested 148- and 140-bp bands.Noncarrier individuals only show the two digested fragments. M, 1-kb plus DNA ladder weight marker. C, undigested control PCR product.

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amino-acid residues 26–119, followed bythe detection of an α-helical domain II forrecognition of xenobiotic substrates9–11.By analyzing the secondary structure, wedetected the two domains of GSTs.Amino-acid residues 26–119 show theβαβαββα topology of the glutathione(GSH) binding site, and amino-acidresidues 210–287 show the α-helicaldomain II that may recognize xenobioticsubstrates9–11. The carboxy-terminalsequence of GDAP1 has two putativetransmembrane domains. Phylogeneticanalyses showed that GDAP1 belongs to anewly discovered and probably mono-phyletic group of GSTs that includes themouse protein and others from Drosophilamelanogaster, CG4623, and Arabidopsisthaliana, T14N5.14.

In this issue, Baxter et al. have alsoidentified mutations in GDAP1 in theTunisian families with CMT4A12

described as a severe demyelinating neu-ropathy of childhood4,13. Genetic dataconfirm that mutations in GDAP1 may beassociated with both axonal and demyeli-nating phenotypes, as reported for otherinherited peripheral neuropathies14. Wesuggest a putative role for GDAP1 in the

interaction between Schwann cell andaxon that, when interrupted, may causeeither axonal degeneration or demyelina-tion in peripheral nerve. Mutated GDAP1might prevent the correct catalyzing S-conjugation of reduced GSH, resulting inprogressive attrition of both axons andSchwann cells.

GenBank accession numbers. GDAP1 cDNA,Y17849; GDAP1 protein, CAA76892.

NCBI reference sequences (RefSeq) for GDAP1.Genome contig, NT_008209; model nucleotide,XM_005273; model protein, XP_005273.

Note: Supplementary information is avail-able on the Nature Genetics web site (http://genetics.nature.com/supplementary_info/).

AcknowledgmentsWe thank the members of the three families fortheir collaboration. We also thank G. López-Carballo and D. Barettino for help withnorthern blot experiments, A. Díez-Juan forpreparing mouse tissues, J. Carmona forcollaboration in the artwork design and P.González-Cabo and R. Vázquez-Manrique fordiscussions. A.C. is the recipient of a predoctoral

fellowship from the Instituto de Salud Carlos III,and L.P. is the recipient of a fellowship from theFundació Karl Faust, Estació Internacional deBiologia Mediterrànea, and a predoctoralfellowship from the Spanish Ministry of Scienceand Technology. This work was supported bygrants from the Comisión Interministerial deCiencia y Tecnología and the Fondo deInvestigación Sanitaria. E.L. and F.P. aremembers of the European CMT Consortium(Biomed 2 concerted action CT961614).

Ana Cuesta1, Laia Pedrola1, TeresaSevilla2, Javier García-Planells1, MaríaJosé Chumillas3, Fernando Mayordomo4,Eric LeGuern5,6, Ignacio Marín7,8, Juan J.Vílchez2 & Francesc Palau1,7

1Laboratory of Genetics and MolecularMedicine, Instituto de Biomedicina, ConsejoSuperior de Investigaciones Científicas (CSIC),46010 Valencia, Spain. Departments of2Neurology, 3Clinical Neurophysiology and4Pathology, Hospital Universitari La Fe,Valencia, Spain. 5INSERM U289 and6Departement de Génétique, Cytogénétique etFoetopathologie, Hôpital Pitié-Salpêtrière,Paris, France. 7Department of Genetics and8Institut Cavanilles de Biodiversitat i BiologiaEvolutiva, Universitat de València, Burjassot,Valencia, Spain. Correspondence should beaddressed to F.P. (e-mail: fpalau@ibv.csic.es).

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brief communications

nature genetics • volume 30 • january 2002 25

Much of the phenotypic variation amongclosely related organisms is due to changesin gene expression rather than to alter-ations in protein sequence1. Conse-quently, it might be expected thatvariations in disease phenotype would fre-quently be caused by changes in expres-sion levels rather than structuralalterations of genes. However, few exam-ples of genes in which small changes inexpression result in severe disease havebeen documented2. Here we show thatslightly lower levels of APC expression areassociated with a pronounced predisposi-tion to hereditary colorectal tumors.

Notably, in a study designed to detectthe causative mutations in individualswith familial adenomatous polyposis(FAP)3, tentative evidence was obtained4

for a partial reduction in APC proteinexpression in one affected person (patient1). To search for sequence variations inthe coding region of APC that mightexplain this reduction, we isolated thealleles of patient 1 using Conversion tech-nology5; however, no sequence variations,other than previously described polymor-phisms, were identified in either allele.

To determine whether a reduction inmRNA levels might account for thereduced protein levels, we quantified therelative levels of mRNA transcripts fromeach APC allele using Digital-SNP6. Thistechnique involves dilution of template(genomic DNA in past studies, reverse-transcribed cDNA in this study) so thatthere is on average less than one tem-plate molecule per well in a multi-wellplate. PCR products are scored with flu-orescent probes that discriminatebetween the two alleles, and the data isrigorously analyzed using likelihoodmethods6.

Using Digital-SNP, genomic DNA fromthe proband yielded the expected 50%allelic ratio, but cDNA from lymphoblas-toid cells showed a skewed distribution,with a ratio of approximately 66% (Fig.1). Linkage analyses with this SNP showedthat the allele whose mRNA was expressedin lower relative amounts was the onelinked to disease (lod score of 3.84 atrecombination fraction θ of zero). Lym-phoblastoid-derived RNA from four otheraffected members of the kindred each hadallele ratios of approximately 66% incDNA, with the linked allele alwaysexpressed less, whereas the cDNA from

lymphoblastoid cells of 24 unrelated indi-viduals without FAP showed normal alleleratios (Fig. 1).

We found additional evidence for thepathogenic significance of this allele bystudying loss of heterozygosity (LOH) ofAPC in benign tumors from this kindred.DNA from the non-neoplastic fractions ofthese tumors had balanced (50%) allelicratios, whereas DNA from the neoplasticfractions of 23 of 28 tumors had allelicloss (Fig. 2). In 22 of the 23 tumors show-ing LOH, the allele lost in the tumors wasthe normal allele that is, the one thatwas expressed at relatively higher levelsand not linked to disease (P<0.0001).

To determine whether similar smalldecreases in expression could beobserved in other individuals with FAP,we examined four people with FAP whohad no APC abnormalities evident uponIVSP or sequencing of Conversion-sepa-rated alleles. One (patient 2) of these fouraffected individuals had an abnormal,71% allelic ratio of alleles in cDNA (Fig.1). No affected relatives were available

Small changes in expression affectpredisposition to tumorigenesis

Published online: 17 December 2001, DOI: 10.1038/ng799

We have used quantitative measures of gene expression to show that constitutional50% decreases in expression of one adenomatous polyposis coli tumor suppressorgene (APC) allele can lead to the development of familial adenomatous polyposis.

40%

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Fig. 1 Reduced expression of one allele in affected individuals with FAP. Dig-SNP analysis of cDNA wascarried out to quantify the relative levels of transcription products of the two APC alleles. The SNPs usedfor these analyses were at APC codons 486 or 1756 (ref. 9). The five blue triangles represent patient 1 andfour of his affected relatives, the green triangle represents patient 2 and the pink diamonds representcontrols that did not have FAP. Points above the red curve provide strong evidence that one allele isexpressed at lower levels than the other6,10.

Received 2 August; accepted 19 November 2001.

1. Dyck, P.J., Chance, P., Lebo, R. & Camey, J.A. inPeripheral Neuropathy (eds Dyck, P.J., Thomas, P.K.,Griffin, J.W., Low, P.A. & Poduslo, J.F) 1094–1136(W B Saunders, Philadelphia, 1993).

2. Lupski, J.R. & Garcia, C.A. in The Metabolic andMolecular Bases of Inherited Disease (eds Scriver,C.R. et al.) 5759–5788 (McGraw-Hill, New York,2001).

3. Sevilla, T. et al. Acta Myol. 20, 49–52 (2001).4. Ben Othmane, K. et al. Hum. Mol. Genet. 2,

1625–1628 (1993).5. Ben Othmane, K. et al. Genomics 28, 286–290

(1995).6. Ben Othmane, K. et al. Neurogenetics 2, 18–23

(1998).7. Lander, E.S. & Botstein, D. Science 236, 1567–1570

(1987).8. Liu, H., Nakagawa, T., Kanematsu, T., Uchida, T. &

Tsuji, S. J. Neurochem. 72, 1781–1790 (1999).9. Salinas, A.E. & Wong, M.G. Curr. Med. Chem. 6,

279–309 (1999).10. Hayes, J.D. & Pulford, D.J. Crit. Rev. Biochem. Mol.

Biol. 30, 445–600 (1995).11. Armstrong, R.N. Chem. Res. Toxicol. 10, 2–18 (1997).12. Baxter, R.V. et al. Nature Genet. 30, 21–22 (2001).13. Hentati, F. et al. Acta Myol. 20, 25–28 (2001).14. Lewis, R.A., Sumner, A.J. & Shy, M.E. Muscle Nerve

23, 1472–1487 (2000).

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