Am. J. Hum. Genet. 59:625-632, 1996

Genetic and Physical Mapping of the Chediak-Higashi Syndromeon Chromosome 1q42-43Franck J. Barrat,' Laurence Auloge,1 Elodie Pastural,1 Remi Dufourcq Lagelouse,1Etienne Vilmer,2 Andrew J. Cant,4 Jean Weissenbach,5 Denis Le Paslier,3 Alain Fischer,'and Genevieve de Saint Basile1

'INSERM U 429, H6pital Necker-Enfants Malades, 2H6matologie-Immunologie, H6pital Robert Debre, and 3Centre d'6tude dupolymorphisme humain CEPH, Paris; 4Department of Paediatric Immunology and Infections diseases, Newcastle General Hospital, Newcastleupon Tyne; and 'Genethon and CNRS URA 1922, Evry

Summary

The Chediak-Higashi syndrome (CHS) is a severe au-tosomal recessive condition, features of which are par-tial oculocutaneous albinism, increased susceptibility toinfections, deficient natural killer cell activity, and thepresence of large intracytoplasmic granulations in vari-ous cell types. Similar genetic disorders have been de-scribed in other species, including the beige mouse. Onthe basis of the hypothesis that the murine chromosome13 region containing the beige locus was homologousto human chromosome 1, we have mapped the CHSlocus to a 5-cM interval in chromosome segmentlq42.1-q42.2. The highest LOD score was obtainedwith the marker D1S235 (Zmax = 5.38; 0 = 0). Haplo-type analysis enabled us to establish DlS2680 andD1S163, respectively, as the telomeric and the centro-meric flanking markers. Multipoint linkage analysis con-firms the localization of the CHS locus in this interval.Three YAC clones were found to cover the entire regionin a contig established by YAC end-sequence character-ization and sequence-tagged site mapping. The YACcontig contains all genetic markers that are nonrecombi-nant for the disease in the nine CHS families studied.This mapping confirms the previous hypothesis that thesame gene defect causes CHS in human and beige pheno-type in mice and provides a genetic framework for theidentification of candidate genes.

Introduction

The Chediak-Higashi syndrome (CHS) is an autosomalrecessive condition characterized by partial oculocuta-neous albinism, a predisposition to pyogenic infections,

Received May 9, 1996; accepted for publication June 19, 1996.Address for correspondence and reprints: Dr. Franck J. Barrat, IN-

SERM U 429, H6pital Necker-Enfants Malades, 149, rue de Sevres,75743 Paris Cedex 15, France. E-mail: barrat@ceylan.necker.fr© 1996 by The American Society of Human Genetics. All rights reserved.0002-9297/96/5903-0018$02.00

and the presence of abnormally large granules in manydifferent cells (Beguez-Cesar 1943; Sato 1955; Blumeand Wolff 1972). The susceptibility to infection may beexplained by the defect observed in T-cell cytotoxicity(Baetz et al. 1995), in natural killer cell activity (Haliotiset al. 1980; Targan and Oseas 1983) as well as in chemo-taxis (Clark and Kimball 1971) and bactericidy (Rootet al. 1972; Clawson et al. 1991) of patients' granulo-cytes and macrophages. In the absence of bone-marrowtransplantation (Fischer et al. 1994; Haddad et al.1995), patients with CHS die at an early age because ofso-called accelerated phase, which consists of infiltrationof most organs by lymphocytes and histiocytes, pancyto-penia, and a coagulation disorder (Bejaoui et al. 1989).At the cellular level, the excessively large vesicles orgranules are easily detected in the cytoplasm of variouscells types. Large granules are particularly well detectedin cells that are normally engaged in a controlled secre-tion process such as polymorphonuclear leukocytes,platelets, or cytolytic lymphocytes (Blume and Wolff1972). These structures are mostly perinuclear, have ly-sosomal-like characteristics, and are formed by the fu-sion of normal sized granules. In each case, these en-larged secretory granules retain their characteristicultrastructure and protein composition (White 1966;Holcombe et al. 1994). However, unlike normal cyto-lytic T-cell clones (CTL), the CHS CTL clones are unableto secrete their giant granules, in which the lytic proteinsare stored. (Baetz et al. 1995). In nonsecretory cell types,such as fibroblasts, the enlarged organelles belong to thelate endocytic and lysosomal compartments (Burkhardtet al. 1993). Thus, mislocalization and/or abnormalfunction of these large granules is believed to be respon-sible for the symptomatic and diagnostic features of thisdisorder.

Similar genetic disorders are found in other species(Windhorst and Padgett 1973), including mice (beigemouse), mink (Alentian), rats, cats, cows, and whales.In all species examined, the mutants share similar mor-phological features, such as altered size and distributionof lysosomes (Windhorst and Padgett 1973). However,manifestations of the accelerated phase have never been

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reported in animal models of CHS (Windhorst and Pad-gett 1973). The bg locus has been localized on mousechromosome 13 linked to the T cell receptor (TCR) y-chain gene (Owen et al. 1986; Justice et al. 1990). How-ever, linkage between CHS and TCR-y on human chro-mosome 7p has been excluded (Holcombe et al. 1987).Further analysis in mice demonstrated a tight linkagebetween the bg locus and the Nidogen entactin gene,which encodes for a basement membrane protein (Jen-kins et al. 1991). The human Nidogen gene has beenlocalized on human chromosome lq42-43 (Olsen et al.1989), defining a new region of homology betweenmouse chromosome 13 and human chromosome lq. Wehave taking advantage of this new region of conservedsynteny between human and mouse to localize in thisregion the CHS gene, by using polymorphic markers. Inan effort to isolate the gene responsible for CHS, wehave also constructed a YAC contig-covering the entireCHS candidate region.

Patients, Material, and Methods

CHS FamiliesPatients were diagnosed as having CHS when they

demonstrated the association of pigmentary dilution, in-creased susceptibility to infections, and azurophilic in-tracytoplasmic giant granulations in leukocytes. The gi-ant granulations were detected on blood smear stainedwith May-Grunwald-Giemsa coloration. Most of thenew cases were diagnosed when they entered into an

accelerated phase.After giving informed consent, nine families partici-

pated in this study consisting of 71 individuals tested,among whom 10 were affected patients (fig. 1). Six fami-lies were known to be consanguineous. They were fromvarious ethnic origins: France, England, Portugal, Israel,Turkey, Algeria, and Mali. Among the 10 affected indi-viduals, 8 were treated by bone-marrow transplantation(Haddad et al. 1995).

Genotype AnalysisDNA was isolated from blood samples by using stan-

dard procedures (de Saint Basile et al. 1987). DNA ofeach individual was genotyped by use of nine polymor-phic microsatellite PCR markers. All markers are refer-enced at Genethon (Dib et al. 1996). PCR primer pairswere purchased from Genset. The forward primer (FP)of each primer pair was end-radiolabeled with 32P-yATP(Amersham) using polynucleotide kinase. The amplifi-cations were performed in a final reaction volume of17.5 ,ul containing 100 ng template DNA, 0.2 mM de-oxynucleotides, 3 pmol unlabeled reverse primer, 1.5pmol unlabeled FP, 1.5 pmol end-labeled FP, and 0.07U Taq polymerase (ATGC; Noisy-le-Grand).

PCR conditions consisted of 30 cycles with 45 s at940C, 1 min at Tm - 50C, and 45 s at 720C for each cycle.The PCR products were analyzed by electrophoresis ondenaturing 5% polyacrylamide gels and autoradio-graphic revelation. Scoring was performed by visual in-spection, and allele sizes were assigned by comparisonwith CEPH pedigree member 134702 as a standard.Each experiment with markers defining the CHS haplo-type was confirmed by repetition.

Linkage AnalysisTwo-point LOD scores were computed using MLINK

and ILINK programs of the FASTLINK 2.2 package(Cottingham et al. 1993). Multipoint linkage analysiswas performed using HOMOZ, a specific software usinga fast algorhythm for computing multipoint LOD scoresin consanguineous families (Kruglyak et al. 1995). TheCHS was considered to be autosomal recessive, with anestimated frequency of 10-6. The allele frequencies forCEPH markers were as reported in public databases.

YAC Library ScreeningThe screening of the YAC libraries from CEPH have

been performed using a PCR-based method. The YACclones were then analyzed for the presence of the ninemarkers previously described and of the eleven othermarkers obtained from Genethon (DlS2712), theWhitehead Institute (WI-1 mot 11392, WI-5672, WI-9317, WI-8489, WI-12765, WI-2822, WI-7199, andWI-10762), and the Cooperative Human Linkage Cen-ter (D1S1680). The amplifications were performed asdescribed above, with DNA from the YAC/genomic tem-plates.Identification of YAC Terminal SequencesThe terminal sequences of the human insert of three

YACs were determined by using of a ligation-mediatedPCR derived from the method described by Kere et al.(1992). In summary, genomic/YAC DNA was digestedwith three blunt-cutting restriction enzymes for each endand fragments were self-ligated. The terminal insert se-quences were then specifically amplified by PCR withYAC-derived primers. After purification, the productswere directly sequenced by PCR with a 32[P-yATP] end-labeled primer. End-fragment sequences were comparedto the public databases of GIS INFOBIOGEN, and valu-able oligonucleotide sequences were then chosen withthe aid of the computer program Oligo4. Because mostYAC clones are chimeric, chromosome origins of thesequence-tagged sites (STS) developed in this study werechecked on a panel of somatic hybrids (the Human Ge-nome Mapping Project [HGMP] resource center).

ResultsA total of 10 affected individuals were analyzed. They

belonged to nine families with CHS, six of which were

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Barrat et al.: Mapping of the Chediak-Higashi Syndrome

A

G

6 0

B C

H

Figure 1 Pedigrees of the CHS families used in this study. DNA was available from all alive individuals except for those identified withblack ovals.

consanguineous (fig. 1). All patients underwent clinicaland immunological examinations. All patients fulfilledthe three diagnostic criteria for CHS: partial oculocuta-neous albinism, susceptibility to infections, and presenceof large intracytoplasmic azurophilic granulations,which were detected in the hematopoietic cells (see Pa-tients, Material, and Methods).The niologene gene, which is linked to the bg locus

on the murine genome, has been assigned to humanchromosome 1q43 by in situ hybridization (Olsen et al.1989). Markers of the telomeric region of chromosome1 were thus selected and studied in the nine families fortheir possible linkage with the CHS locus. Evidence forsignificant linkage was first observed with locusDlS235, which provides a cumulative LOD score of5.38 at 0 = 0. This positive finding was confirmed bytesting flanking markers (table 1). In addition, in theconsanguineous families, the seven patients were homo-zygous for the haplotype defined by the markers DlS235and DlS2649.Haplotype analysis was used to establish the smallest

cosegregating region in the nine families. The haplotypeswere constructed on the assumption of the most parsi-monious linkage phase. The telomeric and centromeric

limits were defined by markers D1S2680 in family Aand DIS163 in family C (see fig 2), respectively. Thus,CHS is likely to be localized to a 5-cM region betweenD1S163 and DlS2680. We failed to observe a commonhaplotype in the nine pedigrees analyzed. The YAC699C3 described in the CEPH database to contain theD1S235 locus was localized by in situ hybridization tolq42.1-lq42.2 (data not shown). Multipoint analysiswas performed with the eight markers used in the haplo-type analysis of the families. The intermaker distanceswere taken from the CEPH data and from the data ofthe physical map defined in this study (see followingresults). The highest location score peak value (Zmax= 7.37) is obtained at 3 cM from D1S2800, which corre-sponds exactly to marker D1S2649 (fig. 3). This resultconfirms the location of the CHS locus between markersD1S163 and D1S2680 in the region found homozygousfor the markers tested in the affected individuals fromconsanguineous families.We then constructed a YAC contig of the CHS candi-

date region. YACs were isolated from the CEPH library.Several YAC clones were identified by data availablefrom CEPH YAC data repository. The other clones wereidentified by a PCR-based method using the primers of

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Am. J. Hum. Genet. 59:625-632, 1996

Table 1

Pairwise LOD Scores for the CHS Locus with Microsatellites on Chromosome 1

LOD SCORE AT 0 =

Locus NAME .000 .001 .048 .091 .130 .165 .225 .275 Zmax (Omax)

DlS2800 AFM360zgl -0c -.19 1.26 1.28 1.18 1.06 .83 .63 1.29 (.070)D1S179 D1S179 -00 -1.63 1.27 1.42 1.36 1.26 1.01 .79 1.42 (.091)D1S163 D1S163 -6.30 -3.84 .39 .80 .88 .86 .72 .57 .88 (.132)D1S2649 AFM176xfl 3.86 3.85 3.28 2.79 2.38 2.04 1.52 1.15 3.86 (.0)D1S235 AFM203xg9 5.38 5.37 4.70 4.09 3.56 3.09 2.35 1.78 5.38 (.0)D1S2680 AFM247wg9 -X -.45 2.27 2.25 2.06 1.84 1.42 1.07 2.27 (.035)D1S2678 AFM245wdS -3.20 .04 1.37 1.33 1.21 1.07 .82 .61 1.38 (.060)DlS2850 AFM88xe5 -mc -2.58 1.82 2.08 2.02 1.88 1.53 1.21 2.08 (.097)

the polymorphic markers encompassing the CHS locusinterval. All the YACs identified are now part of theCEPH database. The presence of one to several of thegenetic markers of the region in each YAC clone allowedthe construction of a YAC contig, except between mark-ers D1S235 and DlS2649, since no YAC clone wasfound to contain both markers. To complete the contigassembly, the terminal sequences of the human inserts ofthe YAC on each side of the gap contig were determined.Because of the high frequency of chimeric clones inYACs, we confirmed the chromosome 1 origin of theYAC end sequences isolated on a panel of somatic cellhybrid DNAs. The most telomeric end of YAC 775 B11was found to be contained in YACs 906 H7, 699 C3,and 786 A4, thus linking the markers DlS2649 andD1S235. Confirmation was obtained by the presence ofthe most centrometric end of YAC 906 H7 in the YAC775 B11.

Thus, a minimum of three YAC clones cover the entireCHS candidate region. Several genetic markers raised atthe Whitehead Institute have been recently assigned tothis chromosome region, because of their genetic local-ization or their position in the same known radiationhybrids as genetic markers already localized. Each ofthese new markers was tested in the nine CHS familiesand on the YAC clone contig established from the candi-date region. Combined results, presented on figure 4,determined their respective localization on chromosome1. However, none of these markers were found to beuseful to better refine the minimum genetic interval con-taining the CHS locus.

Discussion

On the basis of the hypothesis that the homologybetween human chromosome lq and mouse chromo-some 13 includes the bg locus and that the bg mutationis the mouse homolog of CHS, we were able to map theCHS locus to a 5-cM region on the distal end of the

long arm of chromosome 1. Strong evidence for linkageto CHS was obtained with two polymorphic markersthat mapped to this region and displayed a LOD score>3 and supported by the finding of homozygosity forthis region in the patients from consanguineous families.On the basis of recombination events and homozygousmapping in the consanguineous families, data from hap-lotype analysis defined D1S2680 and D1S163 as thetelomeric and centromeric flanking markers for the can-didate region of the CHS gene. Multipoint linkage analy-sis placed the CHS locus on marker D1S2649 in thesame genetic interval. Thus, our results confirm previouscomplementation analysis that suggested that the samegene could be responsible for the defect in CHS patientsand in beige mice (Perou and Kaplan 1993).

Since CHS is characterized by the presence of giantintracytoplasmic organelles and displays a variety ofprotein-sorting defects, gene products known to regulateprotein transport are strong candidate genes for thisdisease. The Rab proteins constitute a subfamily of Ras-related GTP-binding proteins that are localized in dis-tinct intracellular compartments and have been shownto play a key role in vesicular trafficking. Rab genes arewidely dispersed throughout the genome, but Rab4a hasbeen localized on human lq42-43 by in situ hybridiza-tion, thus becoming a strong candidate gene for CHS.We sequenced Rab4a gene in two CHS patients but didnot detect any mutation. Recently, localization of Rab4agene in the mouse has excluded this locus as a candidatefor bg (Barbosa et al. 1995). No other obvious candidategenes for CHS appeared to be localized in this chromo-some region.A YAC contig of the region, aiming at the character-

ization of the gene by positional cloning, was thus estab-lished. The YAC contig connects WI-11392 to WI-12396 and three YACs cover the closest flanking mark-ers that define the minimal region containing the CHSlocus. The combined length of the YACs in this regionprovides an estimate of the physical size of the region

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Barrat et al.: Mapping of the Chediak-Higashi Syndrome

=0003 32 12 14 23 31 32

01 32 22 24 4333 130

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001 21 2

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00 00 00 00 001 2 00 22 00 1 21 2 22 22 22 1 21 1 1 11 1i 1 11

33 3 41331332 2 1 2 112 12222 2 1 2 121211

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242 33 16 53 22 21 121

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2

0

2

4

3

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Dl S2800Dl Si 79Dl Sl63 MD1S26491Dl S235Dl S2680Dl S2678Dl S2850

Figure 2 Pedigrees of the parents and siblings of the affected individuals in the consanguineous CHS families, with their haplotypes. 0= undetermined. Haplotypes or parts of haplotypes inferred as being inherited with the gene CHS are boxed in each family. Double-headedarrow indicates the most likely interval for the CHS locus. Numerous additional individuals in these six families were studied for the LOD-score analysis shown in table 1. The haplotypes of the father of family D were deduced and are shown in italics.

with a maximum size of 3,080 kb. This length may beoverestimated, since these YACs are highly chimeric andalso contains several additional genetic markers fromeach side of the candidate region. No internal deletionwas detected by lack of known STS in any of these

YACs. Since the genetic distance between D1S2680 andDiS163 is estimated to be 5 cM, a high rate of recombi-nation events may occur in the telomeric part of thechromosome. The order of the markers and the orienta-tion of the physical map are consistent with the genetic

A B

T00 003 3 3 12 2 2 13 1 3 33 4 3 21 4 1 2

11 1

00 03 1 3 32 1 2 2

3 2 3 311X 11 1 1 1

t

001 32 22 24 43 3313 Z

03A32 222443

313

D

210034312|1120012l

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1 3 230 0

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1 1 1 2

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32004 31 3112210101

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1213

1 2004 32 32 2310031

II I

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Am. J. Hum. Genet. 59:625-632, 1996

10

8

6

4

2

0

-2

-infini

Lod score (Z)

1 2 3 4

_VW "a Up

VW

6 7 8

Figure 3 Multipoint linkage analysis with eight markers in the nine affected families. D1S2800 has been arbitrarily taken as zero.

linkage analysis data that have been reported (Dib et al.1996). Although no genetic recombinants were reportedbetween D1S2850 and D1S2678/D1S2680 nor betweenD1S235 and D1S2649 in the CEPH families, thesemarkers were separated in our study by their presence

on different YAC clones.The CHS gene product is believed to participate in

the transport machinery into cells (Targan and Oseas1983; Baetz et al. 1995). A great number of proteinsthat regulate vesicular transports have been describedand generally belong to large protein families (Rothman1994; Whiteheart and Kubalek 1995). Among these pro-

teins, four major families play a role in vesicle ad-dressing, docking specificity, and fusion: the RabGTPases (Zerial and Stenmark 1993), the SNARE (24)(Rothman 1994), the COP (Schekman and Orci 1996),and the annexins (Raynal and Pollard 1993). TheSNARE proteins, which are supposed to ensure the spec-

ificity of vesicle addressing, and the Rab proteins, whichcheck the fit between the SNARE, are themselves regu-

lated by various factors. Superfamilies related to thecytoskeleton, like myosins, kinesins, and kinectins, alsoplay a major role in the motility of organelles along themicrotubules or microfilaments allowing intracellulartransport (Burkhardt 1996; Hirokawa 1996). All these

proteins are regulated by various factors and cofactors,the biological functions of which are only partiallyknown (Pfeffer et al. 1995). In addition, the chromo-some localization of all these corresponding genes are,

in most cases, unknown. All these proteins are, however,potential candidate genes for the CHS gene. The geneticand physical characterization of the region encom-

passing the CHS locus provides an important frame-work for further efforts to identify the correspondinggene. Study of new CHS families may be useful to furthernarrow the candidate region, since eight polymorphicmarkers within the region do not show any recombina-tion in the nine families studied here. However, thisapproach may be limited by the rarity of this diseasein humans. Because this study strengthens the previoushypothesis of a common-gene defect involved in CHSin human and in beige in mice, a candidate gene ap-

proach may benefit from the combination of a chromo-some localization screening in both species. Only candi-date gene mapping in the critical region of humanchromosome 1 and on the proximal region of mouse

chromosome 13 will be retained in a search for causativemutations. A positional cloning approach of the bg gene

may also provide an alternative approach in the identi-fication of its human homologue. In any case, the precise

cM

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Barrat et al.: Mapping of the Chediak-Higashi Syndrome 631

WI-11392 WI-5672 775B11-R WI-71991 WI-12396

D1S2800 PT§12S71i2 D1S235 DS2680-|DlS163 |1WN-10762

D1 S26780 D 1 ~~~~S1 680

D1S179 D1 S2649 WI-2822 D1S2850

907A9 -----890F7 I

729G10809E8640F3784A9816D3

775B11 |-l699C3

786A4

i 906H7653F2

| 736D4882F1

887D8

Figure 4 Chromosome 1 CHS contig. The YACs are displayedhere as a physical map (not drawn to scale). Polymorphic repeat mark-ers that could not be distinguished are placed on the same locus. Italicsindicate the right-arm end-terminal sequences of the human insertfrom the 775 BI 1 YAC.

localization of the CHS gene that we have identified willpermit an immediate improvement in genetic counselingfor CHS families. Prenatal diagnosis, at 10-12 wk preg-nancy, can be now proposed in informative females witha small risk of recombination (-0.5%). Further identi-fication of the CHS gene will be an important step to-ward understanding the molecular basis of the regula-tion of vesicular trafficking in cells.

AcknowledgmentsWe are indebted to Drs. R. Seger and I. Yaniv and the

physicians of the Department of Pediatric Immunology andHematology from H6pital des Enfants-Malades for their partin the recruitment and follow-up of patients. We are gratefulto N. Lambert and S. Certain for excellent technical assistance,to Dr. J. Peake for revising the manuscript, to B. Neveu fortyping the manuscript, and to the HGMP resource center forproviding cDNA panel of somatic hybrids. This work wassupported by grants from INSERM, the European EconomicCommunity (BMH1-CT-931321), l'Association Vaincre lesMaladies Lysosomiales, I'Association Franqaise contre les My-opathies, le Ministere de lEducation Nationale de l'Enseigne-ment Superieur et de la Recherche, and I'Assistance PubliqueH6pitaux de Paris.

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