Genetic linkage of type VII collagen (COL7A1) to
dominant dystrophic epidermolysis bullosa in
families with abnormal anchoring fibrils.
M Ryynänen, … , R G Knowlton, J Uitto
J Clin Invest. 1992;89(3):974-980. https://doi.org/10.1172/JCI115680.
Epidermolysis bullosa (EB) in a group of genodermatoses characterized by the fragility of skin. Previous studies on the dystrophic (scarring) forms of EB have suggested
abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association of the cutaneous basement membrane to the underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we
examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoring fibrils. Strong genetic linkage of EBDD and COL7A1 loci was demonstrated with the maximum logarithm of odds (LOD) score of 8.77 at theta = 0. This linkage was further confirmed with two additional markers in this region of the short arm of chromosome 3, and these analyses allowed further refinement of the map locus of
COL7A1. Since there were no recombinants between the COL7A1 and EBDD loci, our findings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families.
Research Article
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Genetic Linkage
of
Type
VII
Collagen (COL7A1)
to
Dominant
Dystrophic
Epidermolysis Bullosa
in
Families
with Abnormal Anchoring Fibrils
MarkkuRyyninen,JaanaRyynanen,StephanSollberg, Renato V.lozzo, Robert G. Knowlton, and Jouni Ultto
DepartmentsofDermatology, Biochemistry and Molecular Biology, and Pathology and Cell Biology, JeffersonMedicalCollege; and Section ofMolecularDermatology, Jefferson Institute ofMolecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Abstract
Epidermolysis bullosa (EB) is a group of genodermatoses char-acterized by the fragility of skin. Previous studies on the dys-trophic (scarring) forms of EB have suggested abnormalities in anchoring fibrils, morphologically recognizable attachment structures that provide stability to the association ofthe
cutane-ousbasement membrane tothe underlying dermis. Since type VII collagen is the major component of the anchoring fibrils, we examined the genetic linkage of dominant dystrophic EB (EBDD) and the type VII collagen gene (COL7A1) locus, which we have recently mapped to chromosome 3p, in three large kindreds with abnormal anchoringfibrils. Strong genetic linkageof EBDDandCOL7A1loci was demonstratedwith the maximum logarithm of odds (LOD) score of 8.77 ata =0. This linkage was further confirmed with two additional markers in thisregion of the short arm of chromosome 3, and these
analy-sesallowed further refinement ofthemaplocusofCOL7A1. Sincethere were norecombinants between theCOL7A1 and
EBDDloci,ourfindings suggest that type VII collagen is the candidate gene that may harbor the mutations responsible for the EB phenotype in these three families. (J. Clin. Invest.
1992.89:974-980.)Key words:basement membranezone* bul-lousdermatosis * genetic skin diseases
Introduction
Epidermolysis bullosa (EB)' is a heterogenous group of
me-chano-bullousdiseases characterized by easyblisteringas a re-sultof minortrauma.EB canbedivided into threemajor
cate-goriesonthebasis of the level of tissueseparationwithin the
cutaneousbasement membranezone atthedermal-epidermal
junction. Inthesimplex(nonscarring) forms,theblisters
de-velop within the epidermisatthe level ofthe basal layer of
keratinocytes; in thejunctional forms, the tissue separation
occurswithin the basement membraneatthelevelof the lam-inalucida; andin the dystrophic (scarring)
forms,
the blister formation occursbelow thebasement membrane withintheAddresscorrespondencetoJouniUitto,M.D.,Ph.D.,Thomas
Jeffer-sonUniversity, Department ofDermatology,BluemleLife Sciences Building,233 South 10thStreet,Suite450,Philadelphia,PA19107.
Receivedfor publication 19August 1991 andinrevisedform24
October1991.
1. Abbreviations usedinthis paper:COL7A1,type VIIcollagengene;
EB,epidermolysisbullosa;EBDD, dominantdystrophicEB;EBS,
sim-plexEB;LOD,logarithmofodds; RFLP,restrictionfragmentlength
polymorphism.
papillary dermis. The inheritance pattern can be either autoso-mal dominant orautosomal recessive (for reviews see refer-ences 1-3).
The patientswith dystrophic EB have the most severe form
ofthis condition.Inautosomalrecessive EB, the tissue
separa-tioncanresultfrom minor trauma, and the healing occurs with
eitheratrophic or hypertophic scarring. In somewhat less
se-vere, dominantlyinherited forms of dystrophic EB (EBDD), the clinicalmanifestations are primarily present at thepressure
points, hands, and feet; and the cutaneous changes are often associated withcharacteristic naildystrophy. Adiagnostic
ultra-structural feature of the dystrophic formsofEB is altered
mor-phology and marked reduction in the number of anchoring
fibrils,attachmentcomplexesatthedermalside of the cutane-ousbasementmembrane zone (4-6). In severe recessive dys-trophicforms ofEB, theanchoring fibrils can be entirely absent (7), whereas inEBDD some anchoring fibrils may be present,
buttheyareoftenhypoplastic and appear abnormal (4). Type VII collagen is the major component of anchoring
fibrils(8, 9). Thiscollagen isahomotrimer,
lal(VII)J3,
and the individual molecules have been suggested to form antiparallel dimers,whichaggregatelaterallytoformanchoring fibrils (10, 11). Because thenumber of anchoring fibrils (which are re-strictedintheirdistributiontothestratifying squamous epithe-lia) is extremely loweven innormalhumanskin, elucidation of abnormalities in typeVIIcollagenat theprotein level isnotyet feasible. However,recentcloningof human typeVIIcollagencDNAshasprovideda means tostudyanchoring fibril involve-ment in thepathogenesis ofEBthroughgeneticlinkage
analy-ses(12).Mapping of the typeVIIcollagen gene(COL7A1) has revealedasinglelocus in theshort armof humanchromosome
3 (12). Using a PvuII restriction fragment length polymor-phism (RFLP)in theCOL7A1 gene,werecentlydemonstrated
linkage between COL7AIand EBphenotype inafamilywith
EBDD (13).Inthisstudy,weexaminedthegeneticlinkageof the EBphenotypeto theCOL7A1locus andtwo otherpolymor-phic markersontheshortarmof chromosome 3in three
fami-lies with EBDD, demonstrating abnormal anchoring fibrils.
Thesegregationanalyses,whichdefined thegenetic map loca-tions ofEBDDandCOL7A1 onchromosome3, provided evi-dence forstronglinkagebetweenCOL7A1and the EBDD phe-notype,suggestingthatCOL7AI harbors the mutation in these threefamilies withEBDD.
Methods
Clinical.ThreeFinnishpedigreeswith EBDDweresubjectedtostudy.
One of them(FEB33)consisted of 13affected and 16 unaffectedliving individualsintwogenerations(Fig.1A).Theinheritance ofEBDD in
thisfamilywasconsistent withautosomaldominantpattern with
com-plete penetrance. Thediagnosis ofEBDD(theCockayne-Touraine
type)wasmadeonthebasis ofclinicalobservations,includingmarked
tendencyforblisteringand erosions notedatbirthorshortlythereafter. Theerosions healed withextensivescarring,and theaffectedpatients
J.Clin. Invest.
© TheAmerican Society forClinicalInvestigation,Inc.
0021-9738/92/03/0974/07 $2.00
A. FEB 33
I
14
II
YNZ86.1 -_
COL7A1
--EFD145.1 -*
III
YNZ86.1 -*
COL7A1 -_
EFD145.1 -*
B. FEB 37
YNZ86.1 _-*
COL7A1
_-EFD145.1 _
II
YNZ86.1
--COL7A1 --*
EFD145.1 -_
In
C.
FEB32II
YNZ86.1 -*
COL7A1 -.
EFD145.1 -*
III
YNZ86.1 -_
COL7AI _-*
EFD145.1 -_
IV
YNZ86.1 -* COL7A1 -_ EFD145.1
--V
YNZ86.1
--COL7A1 -*
EFD145.1
--Figure1.SegregtionofD3S30,COL7A1,andD3S32lociin three kindreds withEBDD. The allelesateach locusareindicated under thesymbol
hadcharacteristic nail dystrophy.Anotherfamily(FEB37)with5
af-fected and 10 unafaf-fected members was alsostudied(Fig. 1 B). The
clinical featuresweresimilartothosefoundinFEB33,and the diagno-sisofEBDD wasconfirmed by histopathology.Theinheritancewas
also consistent withanautosomaldominantpattern.Thethird family (FEB32), withan EBDDmutation previously shown to be linked to
COL7A1locus(13),wasalsoexamined with additionalRFLPs onthe
chromosome3p (Fig. I C).
Skin biopsy specimenswereobtained for routine histopathological examinationbyhematoxylin-eosin stain, for indirect
immunofluores-cencewitha monoclonalanti-typeVIIcollagen antibody L3D (14),
andfordiagnosticelectronmicroscopy.
Southern analyses.DNA was isolated from peripheral blood leuko-cytes andsubjectedtodigestions with restrictionenzymes. The South-ernanalyseswereperformedasdescribedpreviously (15).The DNA probes usedfor hybridizationsof the Southern blots are described in Table I. Thetwo-pointlinkage data were analyzed by the LIPED pro-gram (16), and the multipoint linkage analyses were performed with theLINKAGEprograms(17).
Results
Demonstration oftype VIIcollagenandanchoringfibril defects
infamilies withEBDD. Alarge kindred(FEB33) withEBDD
with 13affectedand16 unaffectedfamilymemberswas
exam-ined (Fig. 1A). Routine histopathology of the apparently nor-malskin into which mildtrauma wasappliedrevealed
subepi-dermal blisterformation, apparentlywithin the upperdermis (Fig.2A). Indirect immunofluorescence oftheunaffected peri-lesional skin withaMAb L3D demonstratedthe presenceof
type VIIcollagenepitopesin the dermal-epidermal junction
(Fig.2 B). However, thestainingpatternwasweak,irregular,
anddiscontinuouscompared withthestainingpatternof con-trol skin from an unrelated healthy individual examined in
parallel (Fig.2C),suggestingalterations in type VIIcollagen.
Diagnostic transmissionelectron microscopy oftheclinically
unaffected skin fromapatientinthisfamilyrevealedpaucityof
anchoring fibrils, andseveralareasalongthe basement
mem-branezonewerecompletelydevoid ofrecognizableanchoring
fibrils(Fig.3,A-C).Therelativelyfewanchoringfibrils present werelargelyhypoplasticand demonstratedamarkedlyreduced diameter(Fig.3A)comparedwithhealthycontrol skin froma
normalperson(Fig.3D).Thus,theanchoringfibrilswere ab-normal both inmorphology and innumericaldensity inthe
skin of affected individualsinthisfamily.Similar
histopatho-logicaland electronmicroscopicobservationsweremadein the twootherfamiliesexamined inthisstudy(resultsnotshown).
Thus,COL7A1 could serve as acandidategene in these fami-lieswithEBDD.
Geneticlinkage ofEBDD and COL7AI loci. The families
withEBDD were then analyzed for segregation ofRFLPs at the
COL7A1 locus and nearby on the short arm of human chro-mosome 3 (12, 18-20; Table I). Since type VII collagen is the major component of anchoring fibrils(11), COL7A1was first testedasacandidategene in the FEB33 family by using a
two-allelePvuIIRFLP,whichwehavepreviouslyshown to have an
allelicfrequencyof 0.65/0.35 in a normal population with the
polymorphism informationcontentof0.35 (13). Southern
hy-bridizations of genomic DNA revealed cosegregation of the PvuII polymorphism with the EB phenotype in pedigree
FEB33 (Fig. 1 A),witha peak logarithm ofodds (LOD) score of
2.10atO=0(Table II).
Cytogenetic mapping has recently localized COL7A1 to
3p21 (12).Furtherevidencefor linkage ofthe EBDD mutation tothisregion ofchromosome 3 wasobtainedwith analysis of twoanonymous markers (see Table I). Specifically,theprobe pYNZ86 (D3S30) yielded Z = 2.1 at 0 = 0, and the probe pEFD145.1 (D3S32) gave a maximum LOD score of 3.74 at
O
=0.
RFLPanalysisindicated the same chromosome 3 map
lo-cation for theEBDDmutationsin twoadditional families.In
family FEB37 (Fig. 1 B),theCOL7A1 andD3S30RFLPs were
uninformative forlinkageto EBDD, but D3S32 cosegregated
withthe disease,withamaximumLOD score of 1.51 at 0 =0 (TableII). Thethird family (FEB32, Fig. 1 C), inwhich we havepreviously demonstrated linkage ofEBDDandCOL7A1 (13), was also analyzedfor segregation ofD3S30 andD3S32.
The resultsfrom FEB32alsosupported linkage ofEBDD tothe chromosome 3p markers. The Z for linkage ofEBDD to D3S32 was 2.27 at 0 =0, and toD3S30,Z =0.88at 0= 0.17
(TableII). Withtheaddition ofnewfamilymembers,the maxi-mumZforCOL7A1 andEBDDin thefamilyis now6.66at 0 =0.
Thecombined LODscorefromthe threefamilies for
link-ageofEBDDtoCOL7A1 locuswas8.77at0=0.Multipoint
analysis withthe LINKAGE programplacedthe EBDDlocus
closertoD3S32thantoD3S30, with maximum likelihood(Z
=8.2)atD3S32(Fig.4). Thus, thelinkagedata indicateasingle
locus on chromosome3pasthesite of theEBDDmutation in
all three ofthese families. The absence of recombinants be-tweenthe COL7A1 and EBDDlociand thedemonstrated
an-choring fibril defectsin theaffected individualsstronglysuggest thatCOL7A1 harbors theunderlyingmutationinthese
fami-lies.
TableL RFLPs inChromosome 3LociUtilizedforLinkageAnalysis
Allele
Locus Map DNA
symbol location probe Enzyme Symbol Size Reference
kb
D3S30 3p pYNZ86.1 MspI A 2.0 Nakamuraetal.
(19)
B 1.9
COL7A1 3p21 K-131 PvuII C 3.6 Parenteetal.(12)
D 1.9,1.7
D3S32 3p pEFD145.1 RsaI E 2.4 Fujimotoetal.(20)
A -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A
.0
*
< n;,,.
%Vv.,>.
2 _rOf* t.
/§.A;?,
d i ,^a.
'
e v aim E
K SS f < r a; OfI §
Figure2. Histopathologyandimmunofluorescencewithanti-type
VIIcollagen antibody.(A) Histopathologyof theperilesionalskin
intowhich mildtrauma wasapplieddemonstrates the presence ofa
subepidermalblister formation(.). (B)Indirectimmunofluorescence
withaMAb(L3D) recognizingtypeVIIcollagenepitopesreveals weak andirregular stainingin theunaffected skin ofthepatientwith EBDD from kindred FEB33. Notediscontinuitiesin thestainingpattern in theaffectedskin(arrows) comparedwith the strong linearstainingat
thecutaneousbasement membranezoneof control skin(C).e,
epi-dermis; d,dermis.Bars, 100 ,um.
Discussion
Inthis
study,
wehavedemonstrated stronggenetic linkageoftheEB
phenotype
and theCOL7A1 locusonchromosome3pin three families with the dominant dystrophic form ofthe disease. The
diagnosis
ofEBDDin thesefamilieswasbasedoncharacteristicclinical
presentation
andwasconfirmedhistologi-cally by thepresenceof subepidermal blistersand byelectron microscopic demonstrationof abnormal anchoring fibrils.
Abnormalities in theanchoring fibrilsandthe paucityof type VII collagen have been demonstrated in the dystrophic
forms ofEB(4-6), and the anchoring fibrils andtype VII colla-gen areabsent insome caseswithsevere recessivedystrophic EB(7). Previouscellbiological investigations concerningthe pathogenesis of recessive dystrophicEBhave centeredaround proteolytic degradation oftheanchoring fibrils(21). It has been demonstrated thattype VII collagen canbedegraded by the interstitial (fibroblast) collagenase (22); and it hasbeen sug-gested that enhanced levels of collagenase and/or other matrix metalloproteinases,such asstromelysin(MMP-3), cause degra-dationofanchoring fibrils and result in the clinical phenotype (23-25). However,our presentresultsraise the possibilitythat structural and/or biosyntheticabnormalitiesin COL7A1 may betheprimarygenetic defect in patients with recessive dystro-phicEB. In supportofthis interpretationis a recent
demonstra-tion that the collagenase locus on chromosome 11 was ex-cludedasthecandidategeneinafamily with recessive
dystro-phicEBbygenetic linkage analysis (26). These observations raiseatleast the possibility ofgenetic heterogeneity of recessive
dystrophicEB.
Asindicated above,EBisagroupofheritable diseases, and
different kindsofmutationsareexpectedtobetheunderlying
cause ofclinically distinct phenotypes (27). Previous linkage analyses have suggested thepresenceofthe mutated locuson thelongarmofchromosome 1 insomefamilieswith the
sim-plex form ofEB(EBS) (28, 29). Furthermore,astronggenetic linkagetotheGPTlocusonchromosome 8 hasbeen demon-strated inafamilywith therareOgnavariantofEBS (30).In thesecases, no candidategene hasbeenidentified, however, and nidogen atthe chromosomal locus 1q43 (31) has been excludedasthecandidategeneinafamilywith EBS linkedto chromosome lq (32). More recently,we(33) and others (34) demonstratedastronggenetic linkage ofEBStoepidermal ker-atingeneclusterseitheronchromosome12qor 17q,suggesting
thatkeratin mutationsmayunderliesomeformsof EBS. This interpretation is supported by recent immunocytochemical demonstrations ofalteredkeratin geneexpressioninpatients
with EBS (35). Furthermore, development oftransgenicmice thatexpressed truncated human keratin14resulted ina
pheno-typereminiscentofthe human EBS(36).
ItisconceivablethatastructuralmutationinCOL7A1 in-terferes with thebiosynthesis, secretion, orprocessingof the molecules, manifestingatthesupramolecular levelas abnor-malanchoringfibrils.Thus,COL7A1 defects could be similar tothose identified in othercollagengenesassociatedwith herit-abledisorders of connective tissue(37). Forexample, alarge
numberofmutations have been identifiedin type Icollagen genes in patients manifesting with osteogenesis imperfecta (38); defectsin thetype IIcollagengene,primarily expressedin thecartilaginous tissues,have beenidentified inpatientswith
chondrodystrophies (39);andtype IIIcollagendefectsarethe
likelycauseof vascular andcutaneousfragilityinpatientswith theEhlers-Danlossyndrome type IV(40). Defectsin a5(IV)
chainoftype IVcollagen, which ispredominantly expressedin thekidneybasementmembranes, are themajorcauseof Al-port'ssyndrome (41). Thus, geneticmutations intype VII col-lagen,whichisexpressedexclusivelyin thestratifiedsquamous
epithelia,could wellexplainthemanifestations ofEBDDin the skin and themucousmembranes.
Figure 3. Ultrastructural demonstration of anchoring fibril abnormalities in the skin of a patient from pedigree FEB33. These electron micro-graphs are representative of three different regions ofdermoepidermaljunction and show paucity of anchoring fibrils (A and B) and the presence
ofonly afew fibrils(C). In the most affected areas (A and C, o) the fibrils appear thin and rudimentary compared with normal controlskin fromahealthy individual (D). As expected, boththelamina densa (LD) and the interstitial collagen fibers(Co)areunremarkable.Original
magnifications:AandB, x43,500;C and D, X47,500.
Thepossibilityof a secretion defect in type VII collagen has beensuggested by recent observations thattype VII collagen
epitopes accumulate within the basal keratinocytes in some
individuals with eitherthedominantdystrophic or the reces-sivedystrophic form ofEB(42, 43). Recentstudieshave also
indicatedthat both epidermalkeratinocytesand dermal fibro-blasts arecapable ofexpressing COL7AI in culture, but the basalkeratinocytesappear to be theprimarycell type
responsi-blefor synthesisof typeVIIcollagenduring fetal skin develop-ment (44, 45). Specifically, in situ hybridizations of human skin from fetusof19 wkgestationrevealed anabundanceof
type VIIcollagen mRNAs in the basalkeratinocytes adjacent
to thedevelopingbasement membrane zone, whereas dermal fibroblasts were devoidoftypeVIIcollagenmRNAs(45). Fur-thermore, immunofluorescence studies ofreconstituted hu-man skin equivalent have suggested that epidermal keratino-cytesaretheprimary sourceoftypeVIIcollagen inanchoring fibrils (44).Thus, byanalogy withother heritabledisordersof connective tissue, fibroblasts or keratinocytes cultured from patients with EBDD shouldprovideasystem to identifythe precise mutations and to study the phenotype at the molecular level. Such information will be helpful toward accurate
diagno-z
1-3.]
2.] 1 j
-20 0 20 40 60 80
D3S30 D3S32 MAPDISTANCE (cM)
Figure 4.Multipoint analysis ofthelinkage ofEBDDlocustoD3S30 andD3S32onthe shortarmofchromosome3. Malegeneticmap distances(in cM)fromD3S30areshown. Thesex-specificmap
dis-tances betweenD3S30and D3S32werereported by Leppertetal.
(18).Theverticalaxis represents themultipointLODscore(Z)for
Table II. Maximum Two-Point LOD Scores (Z) forLinkage of Chromosome 3 Loci and EBDD
Loci 0 Z
COL7A1 vs. EBDD
FEB33 0 2.10
FEB37 NI NI
FEB32 0 6.66
Combined 0 8.77
D3S30 vs. EBDD
FEB33 0 2.10
FEB37 NI NI
FEB32 0.17 0.88
Combined 0.10 2.45
D3S32vs. EBDD
FEB33 0 3.74
FEB37 0 1.51
FEB32 0 2.27
Combined 0 8.11
D3S30vs.COL7AI
FEB33 0 2.11
FEB37 NI NI
FEB32 0.17 0.77
Combined 0.11 2.32
D3S32vs.COL7A1
FEB33 0.50 0
FEB37 NI NI
FEB32 0.11 0.82
Combined 0.12 0.66
LOD scoreswerecalculatedforassumed valuesofrecombination
fractions (0) bythe LIPED program(16).a and Zaremaximum
like-lihoodvalues.NI,notinformative.
sis, both pre- and postnatal, in these families, and it would
potentially allow identification ofsubtypes ofEBDD at the molecular level. Identification of such mutations mightalso allow formulation of rational pharmacological approachesto counteractthediseaseactivity,perhapsusingapproaches that would allow enhanced expression of the normal allele. Inthis regard, it isofinterestthattransforminggrowthfactor-,B is able tosignificantly enhance COL7A expression in cultured kera-tinocytes (46).
Acknowledgments
We thank Debra Pawlicki for secretarial help and BodilTumsfor
tech-nical assistance.Drs.MattiKero and Jaakko Karvonen participated in
clinical evaluation ofthepatients.Wealso thank Dr.LairdJacksonfor hissupportand encouragement.
Thisstudy was supported in part by the U. S. Public Health Service,
National Institutesof Health grants RO1-CA-3948 1, RO1-CA-47282,
PO1-AR-38923, andT32-AR-07869; the Dermatology Foundation; the Paulo Foundation; and the Ella and Georg Ehrnrooth Foundation. M. Ryynanen was a postdoctoral fellow supported by the Division of Medical Genetics, Jefferson Medical College; R. V. Iozzo was recipient ofaFacultyResearch Award from the American Cancer Society (FRA-376);andS. Sollberg was supported by the "Deutsche Forschungsge-meinschaft" grant So239/1-1.
References
1.Kero,M. 1984.Epidermolysisbullosa inFinland: clinicalfeatures,
mor-phology and relationtocollagenmetabolism. Acta Dermato-Venereol.Suppl.
110:1-51.
2.Gedde-Dahl, T.,Jr. 1981. Sixteen types ofepidermolysisbullosa. On the
clinicaldiscrimination, therapyandprenataldiagnosis.ActaDermato- Venereol.
Suppl.95:74-78.
3.Fine, J.-D.,E. A.Bauer,R. A.Briggaman,D. M.Carter,R.A. J.Eady,N. B. Esterly,K. A. Holbrook,S.Hurwitz,L.Johnson,A.Lin,etal. 1991. Revised clinical andlaboratorycriteriaforsubtypesininheritedepidermolysisbullosa:
consensusreportbythe subcommitteeonDiagnosisandClassification of the NationalEpidermolysisBullosaRegistry.J. Am. Acad.Dermatol.24:119-135.
4.Ghadially,F. N. 1988. UltrastructuralPathologyof theCell and Matrix. Vol. 2.3rd ed.Butterworths,London.1228-1230.
5.Tidman,M.J.,and R. A. J.Eady.1985.Evaluation ofanchoringfibrils and other components of thedermal-epidermal junctionindystrophicepidermolysis bullosabyaquantitativeultrastructuraltechnique. J.Invest.Dermatol. 84:374-377.
6.Briggaman,R. A.1985. Is there anyspecificitytodefects ofanchoringfibrils inepidermolysisbullosadystrophica,and what does thismeanintermsof patho-genesis?J.Invest.Dermatol.84:371-373.
7.Bruckner-Tuderman, L., Y. Mitsuhashi, U. W. Schnyder, and P. Bruckner. 1989.Anchoringfibrils and type VIIcollagenareabsent from skin insevere
recessivedystrophic epidermolysis.J.Invest.Dermatol.93:3-9.
8.Sakai,L.Y.,D. R.Keene,N. P.Morris,and R. E.Burgeson.1986.TypeVII
collagen isa majorstructural component ofanchoring fibrils. J. CellBiol.
103:1577-1586.
9.Keene,D.R.,L.Y.Sakai,G. P.Lunstrum,N. P.Morris,and R. E. Burge-son. 1987.TypeVIIcollagenformsanextended network of anchoring fibrils. J. CellBiol. 104:611-621.
10.Morris,N.P.,D. R.Keene,R.W. Glanville, H.Bentz,and R. E.
Burge-son.1986. The tissue form of type VII collagen isanantiparallel dimer. J.Biol.
Chem.261:5638-5644.
11.Burgeson,R.E.,G. P.Lunstrum,B.Rokosova,C. S.Rimberg,L. M.
Rosenbaum,and D. R. Keene. 1990. Thestructureandfunction of type VII collagen. Ann. NYAcad. Sci. 580:32-43.
12.Parente,M.G.,L. C.Chung,J.Ryynanen,D.Woodley,K. C.Wynn,E.
Bauer,M.-L.Chu,andJ. Uitto. 1991. Human type VIIcollagen:cDNAcloning andchromosomal mapping of the gene. Proc.Natl.Acad. Sci. USA. 88:693 1-6935.
13.Ryynanen, M.,R. G.Knowlton,M. G.Parente,L. C. Chung, M.-L.Chu, and J. Uitto. 1991. Human type VII collagen: genetic linkage of the gene
(COL7A 1)onchromosome 3todominantdystrophic epidermolysisbullosa. Am. J.Hum. Genet. 49:797-803.
14.Paller, A. S., L. L. Queen, D. T. Woodley, E. J. O'Keefe, W. R.Gammon,
and R. A. Briggaman. 1985. A mouse monoclonal antibody against anewly
discovered basement membrane component, the epidermolysis bullosaacquisita antigen.J.Invest.Dermatol.82:215-217.
15.Knowlton, R. G., E. J. Weaver, A. F. Stryuk, W. H. Knobloch, R. A.King,
K.Norris, A. Shamban, J. Uitto, S. A. Jimenez, and D. J. Prockop. 1989. Genetic
linkage analysisofhereditaryarthro-ophthalmopathy (Strickler syndrome) and the type II procollagen gene. Am. J. Hum.Genet.45:681-688.
16. Ott, J. 1974. Estimation of the recombination fraction in human
pedi-grees: efficientcomputation of the likelihood for human linkage studies. Am.J.
Hum.Genet. 26:588-597.
17.Lathrop,G.M., J.-M.Lalouel,C.Julier,andJ. Ott. 1984.Strategiesfor multilocus linkage analysis in humans. Proc. Natl. Acad. Sci. USA. 81:3443-3446.
18. Leppert, M., P. O'Connell, Y. Nakamura, P. Cartwright, M.Lathrop,
J.-M.Lalouel, and R. White. 1987. Two linkage groups on chromosome 3.
Cyto-genet. Cell Genet. 46:648.
19.Nakamura,Y., M.Culver,S.Gillian,P.O'Connell, M. Leppert, G. M. Lathrop, J.-M. Lalouel, and R. White. 1987. Isolation andmappingofa polymor-phicDNA sequence pYNZ86.1 on chromosome 3 (D3S30). Nucleic Acids Res.
15:10079.
20.Fujimoto,E., Y.Nakamura,J.Gill, P. O'Connell, M. Leppert, G. M. Lathrop, J.-M. Lalouel, and R. White. 1988. Isolation and mapping ofa
polymor-phicDNA sequence (pEFD145) on chromosome 3 (D3S32).NucleicAcids Res. 16:9357.
21.Bauer,E. A. 1984.Collagenasein recessive dystrophic epidermolysis bul-losa. Ann.NYAcad. Sci. 460:311-320.
22.Seltzer, J. L., A. Z.Eisen,E. A.Bauer, N. P. Morris, R. W. Glanville, and R. E. Burgeson. 1989. Cleavage oftype VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human skin. J. Biol. Chem. 264:3822-3826.
23. Takamori, K., K. Naito, A. Taneda, and H. Ogawa. 1983. Increased neutral protease and collagenase activity in recessive dystrophic epidermolysis bullosa. Br. J. Dermatol. 108:687-694.
24.Winberg, J.-O.,T.Gedde-Dahl,Jr., and E. Bauer. 1989. Collagenase
ex-pressionin skinfibroblastsfromfamilieswithrecessive dystrophic epidermolysis bullosa. J. Invest.Dermatot. 92:82-85.
25.Sawamura,D.,T.Sugawara, I.Hashimoto,L.Bruckner-Tuderman,D.
Fujimoto,Y.Okada,N.Utsumi,and H.Shikata.1991. Increasedgeneexpression
severerecessivedystrophicepidermolysisbullosa. Biochem.Biophys.Res.
Com-mun.174:1003-1008.
26. Hovnanian, A., P. Duquesnoy, S. Amselem, C. Blanchet-Bardon, M. Lathrop, L. Dubertret, and M.Goossens. 1991.Exclusion oflinkagebetween the collagenase gene andgeneralizedrecessivedystrophic epidermolysisbullosa
phe-notype. J.Clin.Invest.88:1716-1721.
27.McKusick,V. A.1988. Mendelian Inheritancein Man.Catalogsof
Auto-somalDominant,AutosomalRecessive,and X-linkedPhenotypes.8th ed. The
JohnsHopkins University Press, Baltimore,pp. 232-234.
28. Mulley, J.C.,C. M.Nicholls,D. N.Propert,T.Turner,andG. R. Suther-land.1984.Geneticlinkage analysisofepidermolysisbullosasimplex,Koebner type. Am.J.Med.Genet. 19:573-577.
29.Humphries,M.M., D.Shiels,M.Lawler, G. J. Farrar,P.McWilliam,P. Kenna, D.G.Bradley,E. M.Sharp,M.Young, J.Uitto,etal. 1990. Autosomal
dominantsimplex epidermolysisbullosa: evidence forlinkagetogeneticmarkers onchromosome 1. Genomics. 7:377-381.
30.Olaisen,B., and T.Gedde-Dahl,Jr. 1973.GPT-epidermolysisbullosa
simplex (EBS Ogna) linkagein man.Hum. Hered. 23:189-196.
31. Olsen, D. R., T.Nagayoshi,M.Fazio,M.-G.Mattei,E.Passage,D.Weil,
R.Timpl,M.-L.Chu,and J.Uitto. 1989.Humannidogen:cDNAcloning, cellu-larexpression,andmapping of thegene to chromosome lq43.Am. J. Hum.
Genet.44:876-885.
32.Humphries,M., K.Nagayoshi,Y.Sugita,M.-G.Mattei, M.-L. Chu,R. Knowlton,andJ.Uitto. 1990.Humannidogengene:identification ofmultiple RFLPsandexclusionascandidategene inafamilywithepidermolysisbullosa
simplex (EBS2)with evidence forlinkagetochromosome 1. J. Invest. Dermatol.
95:568-570.
33.Ryynanen,M.,R.G.Knowlton,and J. Uitto. 1991.Mappingof epider-molysis bullosa simplex mutation to chromosome 12.Am. J. Hum. Genet. 49:978-984.
34.Bonifas,J.M.,A.-L.Rothman,and E.Epstein.1991.Linkageof epider-molysis bullosasimplextoprobesin theregion ofkeratin gene clusterson chro-mosome12qand17q. J.Invest.Dermatol.96:550.(Abstr.)
35.Ito,M., C.Okuda,N.Shimizu,T.Tazawa, andY.Sato. 1991.
Epidermo-lysis bullosa simplex(Koebner) isakeratin disorder. Ultrastructuraland immuno-histochemical study. Arch. Dermatol. 127:367-372.
36. Vassar,R.,P. A.Coulombe,L.Degenstein,K.Albers,and E. Fuchs. 1991. Mutantkeratinexpression intransgenicmicecausesmarked abnormalities
re-semblingahumangeneticskin disease. Cell.64:365-380.
37. Kuivaniemi, H., G. Tromp, and D. J. Prockop. 1991. Mutations in colla-gen colla-genes: causesof rare and some common diseases in humans. FASEB (Fed. Am. Soc. Exp.Biol.)J. 5:2052-2060.
38. Prockop, D. J. 1990. Mutations that alter the primary structure oftype I collagen: theperils ofasystem for generating large structures by the principle of nucleated growth. J.Biol. Chem. 265:15349-15352.
39.Vissing, H., M. D'Alessio, B. Lee, F. Ramirez, M. Godfrey, and D. W. Hollister. 1989. Glycine to serine substitution in the triple helicaldomainof
proal(II)collagen results in a lethal perinatal form of short-limbed dwarfism. J.
Biol. Chem.264:18265-18267.
40.Kontusaari, S., G. Tromp, H. Kuivaniemi, R.Ladda,and D. J. Prockop.
1990.Inheritance ofan RNA splicing mutation(G+Inw1 in the type III procolla-gen procolla-gene(COL3A1) in a family having aortic aneurysms and easy bruisability:
phenotypicoverlapbetween familial arterial aneurysms and Ehlers-Danlos syn-drome type IV. Am. J. Hum. Genet. 47:112-120.
41. Zhou, J., D. F.Barker, S. L. Hostikka, M. C. Gregory, C. L. Atkin, and K. Tryggvason. 1991. Single base mutation inaS(IV)collagenchaingene converting aconservedcysteinetoserinein Alport syndrome. Genomics. 9:10-18.
42. Fine,J.-D., Y.Horiguchi,D. H. Stein, N. B. Esterly, andI.M. Leigh. 1990.
Intraepidermaltype VII collagen, evidence for abnormal intracytoplasmic
pro-cessing ofamajorbasementmembraneprotein inrare patients with dominant
andpossibly localized recessive forms ofdystrophicepidermolysis bullosa. J. Am.
Acad. Dermatol.22:188-195.
43.Smith,L. T., and V. P. Sybert. 1990. Intra-epidermal retention oftype VII
collagen inapatient withrecessive dystrophic epidermolysis bullosa. J. Invest.
Dermatol.94:261-264.
44. Konig,A.,andL. Bruckner-Tuderman. 1991.Epithelial-mesenchymal interactions enhanceexpression of collagenVIIinvitro.J. Invest.Dermatol. 96:803-808.
45.Ryynalnen, J., S.Sollberg,M.G. Parente, L. C. Chung, A. M. Christiano, and J.Uitto. 1992.Type VIIcollagengeneexpressionbyculturedhuman cells andin fetal skin.Abundant mRNA andprotein levels in epidermal keratinocytes. J. Clin.Invest.89:163-168.
46. Ryynanen, J., S.Sollberg,D. R.Olsen, andJ.Uitto.1991. Transforming
growthfactor-jI up-regulatestype VIIcollagengeneexpression innormal and
transformedepidermal keratinocytesin culture.Biochem.Biophys.Res.
