Linkage of Monilethrix to the Trichocyte and Epithelial Keratin Gene Cluster on 12qll-q13

H oward P. Stevens, David P . Kelsell, * Steven P. Bryant, '" D. T imothy Bishop, t Rodney P. R. D awber,:j: N igel K. Spurr, * and Irene M. Leigh Department of Experimcnta l Dcnnato logy. The Royal London Hospita l. London: "Human Gcncri c Resourccs. Clan, Hall Laboratories, Impcrial Cancer Research Fund, Hens; 'I'Gcneti c Epidemiology LaboratOl'Y, Imperial Cancer Rcsearch Fund , St. James Hospital, Leeds: and :j:Department of Dermatology, Churchill Hospital. Oxford. Uni ted Kingdol11

Monilethrix is characterized by beaded or moniliform hair, which results from the periodic thinning of the hair shaft. The beaded hair thus produced is subject to excess weathering and premature fracturing at the internodes. Clinically, Inonilethrix presents with short, fragile, broken hair. The follicular abnormali­ties range from subtle perifollicular erythema and hyperkeratosis to horny follicular papule formation . At the ultrastructural level, cytolysis and keratin tonofilament clumping (epidermolysis) are seen in the cortical cells of the bulb of the hair follicle. Microsatellite Juarkers flanking the keratin gene clus-

Monile thrix is a rare congeni ta l defect o f hair that is inherited as a fully pene trant, autosomal dom­inant condition with variable expression. C lini­ca lly, .the hair is subj ect to excessive w eath e ring and fi'agility with a tcndency to fi'ac turc prcma­

cu re ly at the narro w er internodes (G umm er c/ ,,/, 1981). A ffected indiv idua ls have no rmal hair at birth , but within th e first few months o f life this hair is rep laced by fragile, brittl e hair, which ten d s to fra cture , produ cing bald patches. In the mi ldest fo rms, it involves only the occipita l regjo ns and the nape of th e neck. UsuaUy onl y the scalp is invo lved , but in its m o re severe fo rm. th e seco ndary sex ual hair, eyebrows, and eyelashes may a lso be in­volved . Fol licular hyperkeratosis with perifo llicul ar erythem a is cha racte ri sti c , with horny folli cul ar papules seen in the most severe fo rnols of the disease .

U ltrastructura lly, moni lethrix is charac te ri zed by uniform e llip­tica l nodes and in tc rmittent constrictions, intern odes, alo ng the hair shaft (Gummer cl ,,/. 1981) . Transve rse sections o f affected hair shaft s at the node dem onstrate a reduced number of cuticle ce ll layers compared with th e intern odal regions (fto el ,,/, '1984) . The cuti cl e ce ll m embrane appears no rmal. with a regular concentric arrangem ent o f endocuticle and exocuticl e. In the inte rn odal regions, the cuticle is seen to degenerate progressive ly along the hair shaft. Distall y along th e hair shaft , the re is increased ridg ing

Manuscript received October 16, 1995: revised November 14, 1995; accep ted fo r publication December 7, 1995 .

R.eprint requests to: Dr. Howard P. Stevens. Departll1ent ofExperil11 ental Dern13tology, 56 Ashfield Street, London E 1 211L. United l{j ngdom.

Abbrev iations: HOI , hard acidic keratin : Hb. hard basic keratin; LO D score, the logarithm (to base 10) of the ratio of the likelihood of the data at a specified vallie of0 di vided by the likelihood of rhe pedigrce data if there were free recombination between the loci (0 = 0.5) ,

ters at 17q12-q21 and 12qll-q13 were used to perform linkage analysis in a monilethrix pedigree. This study demonstrates linkage of lDonilethrix in a pedigree to lDicrosatellite . DNA loci lDapping to the region on chron'losome 12 containing the type II keratin clus­ter. A major group of structural hair proteins, the basic type II trichocyte keratins, lDap within this epithelial cytokeratin gene cluster. This study impli­cates a lDutation in a trichocyte keratin gene in the pathogenesis of a structural hair disorder. K ey wo,.ds: Irai,. disease/iraI'd kevatills/jolliCIIlal' Irypel'kel'a tosis/epide,.­IIIolysis. ] In vest De,.,natol 106:795-797, 1996

and Auting of the cuticle w ith cuticul ar ce ll breakage and shedding, until distally th e cortex is completely e xposed (Gummer et ,, /. 198 '1). With the light microscope, the hair ma trix has a pal e and edem atous appearan ce; wi th e lec tron mi crosco py. the cortex of the hair bu lb has an amorphous :lppea rance w ith areas of tri chocyre degeneration . In o ther areas of the cortex. cytolysis and tonofi la­m ent clumping are seen (Ito c/ ai, 1990) . T he keratin tonofilam ent ne two rk appears re latively w e ll preserved in the cuticul ar cells. w ith only the occasional to no fil am ent aggregate described (Ito ct ,, /, 1990) .

Keratin mutations have n ow been dem onstrated in a number of disorde rs chara cteri zed by var)ring deg rees o f epide rm al fi'agili ty with secondary h);perke ratos is. Cyto lys is and keratin tono fil am enr clumpin g have been demonstrated in m ost o f th e diseases in w hich hLlm~n epithe lial ke ratin gene mu tations ha ve been fo und: epider­m o lysis bullosa simpl ex (K5; K14) (Coul ombe el ,, /, 199 1; Ishida­Y am am oto cl ,, /, 1991; Dong ('I ,,/ , 1993). ich thyos is bL1.lI os~ of Siem ens (K2e) (M cLean er " I, 1994b) , bull ous congeni ta l ich th yo­sifo rm erythroderma (K1 ; Kl 0) (M cLean el ,, /, 1994a) , epide rmo­lytic palmo planta r ke ratoderma (K9) (Na vsaria cl aI, J 994) . and pachyonychia congenit~ (K 16; KGa) (Bowden et ,,/, 1995; M cLean el a/. 1995) . In two o f th e m ost recentl y described keratin discases­pachyonychia congenita and foca l palmo pl antar ke ratoderma w ith orogenit~1 hyperkeratos is (McLean c/ ,, / , 1995; Sham sher el ,,/,

1995)-fo l1icular e rythema and hyperkeratosis are characteri sti c features.

T he fo llicular changes seen in m onil ethrix (de J3 erke r el ai , 1993 ). combined with the abnormal kerati1l tonofi lamenr ne two rk , sug­ges t a tri chocyre ke ratin gene mutati on in the path ogenesis of thi s disease. In this study, w e foc lI sed on a single r., mily with m oni­lethrix . All the known epi the lial and tri chocyte ke ratins have been sho wn to map to two ke ratin gene clusters: the acidic keratin cl uster

0022-202X/9(,/S10.50 • Copyright © 1996 by T he Society fo r Investigative Dermatology. Inc.

795

796 !iTEVEN!i ET I lL

Table 1. Linkage Analysis in MOllilethrix With Micr-osatellite Markers Flanking the Keratin Gene

Clusters 011 17q and 12q"

l_lecornbiI13[i on Fraction

locus 0.0 0.01 0.05 0.1 0.2 0.3

0 12S361 3.25 3.20 2.96 2.65 1..98 1.24 012S368 2.86 2.80 2.57 2.27 1.64 0.98

0.4

0.49 0.38

KI1...T 9 'CA' Intin . - 6. 19 - 3.45 - 2.30 - 1.19 - 0.59 - 0.22

,I LOD SCores were computed with FASTLlNK version 2.2 . asslm'llng autosomal dOlllinanl" iuI ICril:lIJcc .

(type II) at 17q12-q21 and the basic keratin gene cluster (type II) at 12qll-q13 (Rogers eI aI, 1995) . Lin kage analysis was performed in this pedigree using mic rosa tellite DNA marker loci Ranking the two keratin clusters.

MATERIALS AND METHODS

Scannin g Electron Microscopy T he plucked hair snmples were exam­ined by scann.ing electron microscopy after gold coating.

DNA Analysis DNA was extracted frOI11 the blood from all consenting individw.ls from the pedigree, using a Nucleon \I \cit according to the manufacturer's instructions (Scotlab, Lanark, OK). Microsatelli te DNA marker loci were obta ined and analyzed as described previously (Kelsell ('I

al. 1993 ; Kc\sell ('I ai, 1995). The map order of markers used ill this study waS as fo llows: 12ql l-q13 ccn-O 12S361-012S368-0'12S90-tcl; 17qI 2-q21 cen-KR T' CA' -D17S855-tel.

Linkage Analysis Linkage analysis was performed with the ' MLlNK modu le of the FASTLINK software package version 2.2 (Cottingham ('I al. 1993), ass ull1in g an autosonlal d0111inant inhcritanc~ for n1onilcthrix. w ith a gene frequency of 0.003 and a penctl'al1ce of95'X •. Allele frequencies for the DNA markers wcre eithcr computed from the data Or estimated to be equifrequent.

RESULTS

Follicular and Hair Shaft Abnormalities T he fam ily was traced through four generations with 12 affected indi vid uals. T he same clin ical charnctel'istics were o bserved in all affected indi vidu­als. T h e hair was normal at birth , but follicular e rythema and

Figure 1. Segregation an alysis in a monilethrix pedigree using markers flanking the 12q keratin gene clus­ter . 1-1 aplotypes constructed for the mo­nilethrix pedigree show the consistent inheritance of a 12q haplotype (indicated in Iwld Iype; 4- 1- 1) with the disease for the DNA marker loci 0 125361. D12S368, ond 012S90 .

2 1 34 3 1

D128361 D128368 012890

43 13 1 2

T H E JOURNAL OF INVESTIGATI VE DERMATOLOGY

h yperkeratosis developed over the occiput and the nape of the neck w ithin the first few months of life'. Subsequently, the hair becam!' brittle and was lost, leaving a stubble 1-2 cm in length over the w h ole scalp. Scannin g e lectron microscopy of plucked hairs cou­firmed the features of l11 0njlethrix with node and internode form a­tion and the longitudinal ridging and Ruting. The secondary sexual Iwir, eyelashes, and eyebrows appeared no rmal. T he skin, orogeni­tal mu cosa, na ils, and teeth were all norm al.

Monilethrix Linked to the Keratin Gene C luster on 12q DNA extracted !Tom fiulli ly m embers was screened w ith polymor­phic microsatelli te DNA marker loci m apping to either 12ql1-q13 or 17q12-21, where the two ep ithe lial keratin cluste rs arc located. Informative m ,lJ'kers and LOD scores (Ioga l'ithm to base 10 of tile like lihood of the data) arc listed in Table I.

Linkage was observed to th e d isease phenotype with the DNA m arker D12S361, w ith a m aximum two-point LOD score of3.25 at a recombination fraction of 0.00. H aplotypes for microsatellite markers mapping to 12q1 1-q1 3 were constnl cted with the miJlimal number of recombinants possibl e in the pedigree. C lea r eosegre­g.]tion o f a haplotype with the disease phenotype was o bserved (Fig 1). T h ere W:lS c1e,u evidence against linkage of the d isease to 17q12-21, with a negative LOO score of - 2.30 for the keratin 9 intragenic microsatellite at a recombination fraction of OJ .

D ISCUSSION

There arc 20 known epithe lial " soft " keratins and at least eight trichocyte "bard" keratins . T he keratin s may be d ivided on the b<lsis of the ir mig ration on 2-dimensional e lectrophores is in to two main groups: the smaller, type I keratins, w ith an :lcidic isoelectric point, ,1I1d the la rger, neutra l or slig htl y bas ic type IT keratins (Fuchs el ai, 1981; Moll CI aI, 1982; Schiller C( <1 /, 1982; Sun (' I ai, 1984). Four trichocyte type I keratin s (Hal, Ha2, \-I n3, and I-b 4) are cl ustered w ith the ep ithe lia l type I ke ra tin s at 17g1 2-q2 '\ , w hereas four trichocyte type Il keratins (Hbl, Hb2, \-Ib3, and Hb4) (f-Jcid CI

ai, 1988a; H eid e/ aI, 1988b; I-k id cl ai , 1986) are cl ustered with the oth er type II kera tins on 12qll-q1 3 (Rogers et ai , 1995). In this study, we ha ve demonstrated lin kage of DNA markers mapping to the region 12q l 1-q13 w ith the m onilethri x disease phenotype. A the marker locus 0 12S368 maps to yeast artific ial chromosome, w hich contains a number of type II keratin genes (Yoon d ai, 1994) .

21 34 3 1

1 2 52 1 2

42 1 2 1 2

52 32 42

22 1 2 1 2

24 44 31 31 31 1 1

42 42 31 1 2 41 1 2

42 1 1 1 1

44 1 3 1 2

54 1 3 1 1

44 1 3 1 1

24 31 31

41 1 1 1 3

4 1 1 1 2 3

VOL. 106, NO." APIUL 1996

it is possible that the disease in this family is due to a mutation in one of these keratins. The keratin gene cluster on 12q is relatively large, however, and the possibility of a mutation in a gene other than a keratin must remain. The moniliform changes seen ill tIlls family were restricted to the sca lp , with normal body and secondary sexual hair. This regional distribution of hair involvement could be explained either in terms of a nonkeratin gene mutation or by proposing a regional localization of the trichocyte keratins in a malUler analogous to that seen with the epithelial keratins .

Whereas the basal cells bordering on the apex of the dermal papill a and also in the cuticle may coexpress both trichocyte hard and epithelial soft keratins, there is no coexpression of tIle epithelial keratins within the cortex of the hair bulb (Heid e( aI, 1988a). The site of maximal cytolys is and keratin tonofilament aggregation in mocilethrix is the cortex of the hair bulb (Ito el aI, 1990) . It is therefore probable that a mutation in a type II basic trichocyte keratin, and not an epithelial keratin, is the genetic basis of monilethrix in this pedigree.

We ac/u/oflliedge II,e sl/pporl oj Ih" 11111' erial Cal/cer Research FI/Illi al/d the WeI/collie Tmsl iI/ Ihis I/Iork . T he HI/Illall Gellollle Mappillg Projecl Resol/rce

Centre, Jlllllied b), Ihe UK Medical Research COl/ llcil, prollided exira WIllPllli ll,!! Jacililies.

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