Partial Dominance of a Keratin 14 Mutation in Epidermolysis Bullosa Simplex - Increased Severity of Disease in a Homozygote

ZhiLan Hu, * Lynne Smith;! Sarita Martins,:j: Jecumette M. Bonifas,* Hua Chen,* and Ervin H. Epstein, Jr* *Department of Derma to logy, Unjvc rsity of Califo rnia San Francisco, San Francisco, C ali fo rnia. !'Departments o f Dermato logy and Bio logic Structure , University of Washjngton, Sea ttl e, Washington, U.S. A.; and ~Depa r tment of Dermato logy, Federal Uni versity of Pernambuco, R eci fe, Brazi l

Epidermolysis bullosa simplex is a disease in which keratin gene mutations cause the production of de­fective intermediate filaments, which leads in turn to epidermal basal cell fragility and blistering. The inheritance in nearly all kindreds is autosomal dom­inant, most kindreds have missense mutations, and the encoded proteins appear to exert a dom.inant negative function. One previously reported patient with generalized blistering had a fully dominant

E pide rmolys is bull osa simplex (EBS) is characte rized by non-scarrin g blisters o f the skin that arc caused by basa l cell fragili ty . A half decade ago, three gro ups identified underlying heritabl e mutati o ns in genes encoding th e keratin s ex.1Jressed specifically in basa l cell s o f stratified

squ am o us epithelium: ke ratins 5 and 14 (Bo nifus el a/, 199 1; Coul ombe el a/ , 1991; Lan e e/ a/ , 1992). Keratins fonn the intermedi ate filam ents th at make up the major componen t o f the epiderm al cytoskele ton, and mutatio ns in suprabasal kera tins more r ecentl y have been fo und to unde rlie o th er skin diseases: ke ra tin s 1 and 10 in genera.lizcd congenital epidermolytic hyperkeratosis, ke ratin 9 in epiderm oly ti c hyperke ratosis limited to th e palms and soles, keratin 2e in ichthyos is bullosa o f Siemens, keratin 6, 16, and 17 in focal no nepid ermolyti c hyperkeratosis o f the palms and sol es wi th or wi tho ut pachyo nychia congenita, and keratins 4 and 13 in white spon ge nevu s (Ch eng el a/ , 1992; C hipev et a/, 1992; Rothnagel el a/, 1992; R eis et a/, 1994; Jlo thnagel ef a/, 1994; Bowde n et a/ , 1995; M cLean ef a/, 1995 ; Richard e/ a/ , 1995 ; Rugg et a/ , 1995; Sham sher el a/ , 1995) .

As m o re mu tati ons of the basall y expressed ke ratins have been iden tified in patients with EBS, som e gen otype-phenotyp e corre­latio ns have begun to e m erge. The clinicall y m ost severe form is the D o wlin g-Mea ra (DM) type , in which blistering is generalized and frequent, bUsters often are g rouped (he rpe tifo rm) , and ke ratin inte rmediate fil am ents are seen to be clumped o n ultrastru ctural examin ation (Fin e ef a/, 1991 ). One frequ entl y repo rted mutatio n in

EBS-D M is at Arg 125 of keratin 14, with substituti o n by hi stidin e

Manuscrip t received February 26, 1997; accepted fo r publication April 2, l 997.

R eprint reques ts to: Dr. Ervin Epstein , Jr, San Francisco General 1-Iospi ­tal , R oom 269 . Building 100, 1 OO l Potrcru Ave ., San Francisco, C A 94 110.

Abbreviations: O M, Dowling-Mcara rypc o f EBS; WC. W eber-Cock­aync type of EI3S.

mutation of keratin 5; in that kindred a homozygote was affected no more severely than the heterozy­gotes. By contrast we report h ere a keratin 14 muta­tion that causes blistering limited to the hands and feet in heterozygotes, but homozygotes have tnore severe, widespread blistering of the skin and mucous membranes. Thus keratin gene ntuta tions tnay be not only fully recessive or fully dominant but also partially dominant as well.] Iuvest D ermatol 109:360-364, 1997

o r cys tein e (Coulombe ef a /, l 99 l a, 1991 b; Stephens e/ a/, 1993 ) . T hi s arginin e residu e is in the mid st of the he lix initiati o n regio n; it is hig hl y conserved in other type I (ac idic) keratin s; and pathogeni c mutati o ns o f the sam e codon arc fo und fi·equ ently in di seases o f o the r type f keratin genes (C he ng et a/ , 1992 ; C hipe v et a/ , '1992; R o thnagel cf a/, 1992; Bonifas et a/ , 1994; McLean ef a/ , 1994; R e is e/ a/, 1994). A C -terminal keratin 5 mutatio n al so was iden tified in o ne fa mil y with severe di sease (Lan e e/ a/, 1992).

T he mildes t form is th e W cber-Cockayne (W C ) vari an t, in w hi ch bli sters essentiall y arc limited to the hands and feet. both do rsal and ven tral Sllrfaces. Man y W C patients ha ve missense mutatio ns o f Il e 161 o f keratin 5 (C han el a/, 1993; Ehrli ch et a/, 1995); o th er mu tations have been re p01·ted in W C patients, each in lower fregu ency (C han el a/ , 1993; C hen el a/ , ·1993 ; Rugg ef a/. 1993; C han e/ a/, 1994; C hen et a/, 199 5) .

T he third nu~j o r type of EBS, th e Koebner variant, is of in te r­mediate severity, with blisters that are m ore wid espread th an those of the W C subtype but less se ve re th;m those of th e OM subtype. Underlyin g mutati o ns ha ve been fo und in th e ke ratin 14 he lix initiatio n region as w e ll as scatte red across th e keratin 14 helix and th e regio n linking tb e 1B and 2A he lices (Bo ni f:1s ef a/, 199 1; D o ng e/ a/, 1993; Humphri es ei a/, 1993; Stephens eta/, 1995; Yamani shi et a/ , 1994).

N ea rly all repo rted f.1mili es with EBS have segregation consistent wi th an autosomal dominant inh eritan ce. Several kindrcds, how­e ver, have been described in which inhet·itance appears to be autosomal recessive (Weerd t and Castelc in, 1972; Salih ef fl l , 1985 ; Ni emi e/ a/, 1988; Fin e ct a/ , 1989a, ·1989b; Kle tter cf a/ , l 989) . Pati ents in some of th ese famili es ha ve di sease o f a sevc1·ity similar to that of pati ents in th e m o re commo n autosomal domin ant kindrcds. w hil e o thers have se vere blistet·in g and diseases o f o th e r o rgans includin g muscl e. Som e o f th e latte r kindreds ha ve been

0022-202X/97 /$10.50 • Copyri ght 1997 by Th e Society fo r In vestigative Dermato logy. In c.

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VOL. I 09 . NO. 3 SEPTEMBER 1997

n

m

IV

v

VI

EBS·WCTa

.~ .:;.k~

Figure 1. Pedigree of Ta kindred. • indicate bListering

shown to have abnormalities of plectin, a protein associated with the ker·atin inte rmedia te filament. ' - '1

One of the autosomal recessive kind reds with disease limited to the skin was reported to ha ve a missense mutation of the 1 A coil regio n of keratin 14 (Giu 144---'> Ala) (1-Iovnanian eta/, 1993 ), and others ha ve had "knock o ut '' mutations (Chan ef nl, 1994; Rugg el nl, 1994) . T h e lack of clinical abnormalities in the heterozygotes for "knock out" mutations , both in humam and in mice (Lloyd et nl, 1. 995), is consistent with the idea that k eratin gene null mutations do not interfere with functiorting of the intact allele and h en ce are recessive ~ whereas keratin gene missense mutations en code sub­units that can act as dominant negatives by interfering with the assembly of the multimeric filaments. Further eviden ce favoring that idea includes, first, the disruption of the normal murine ker:1tin intermediate filament network by a transgene encoding mutant hum an keratin 14 and similar· results of i11 uitro transfection studies (Albers and Fuchs, 1989; Vassar et nl, 1991). Second, in on e kindred with severe Koebner EBS and an underlying keratin 5 Lys 1.73 ---'> Asn mutation, a ch ild of a consang uin eous mardage was homozygous for the mutant allele and had clinical disease no more severe than that in the heterozygotes (Stephens el n/, 1995). We report h e re that unlike the jitll)' recessi11e keratin 14 knock out mutations and the j11ll)' do111innnl keratin 5 Lys 173---'> Asn Koebner mutation, a keratin 14 Met 119-> li e mutation is pnrtinlly do11ri11nW:

1 Eady RA , Leigh LM , McMillan JR. Geddes JF, Kirtschig G, Kdscll DP, Spurr NK, ct al: Epidermolsis bullosa simplex with muscular dystrophy: loss of plectin express ion in skin and musclc. J lnl'c.<l Dennntol 106:842, 199(> (abstr).

2 Gachc Y. Chaanas S. Lacour JP, Wiehe G, Owaribe K, Meneguzzi G, Ortonne JP: Defective expression of plccrin in epidermolysis bullosa simplex with muscular dstrophy. J ln11e.<t D cr111nlol 106:824, 1996 (abstr).

" McLean WHI. Smith FJD. Rugg EL, Storey A, Leigh IM. Eady I~J. Pulkkinen L, ct al: Cloning and sequencing of the human pl cctin gene. J h wesr Dcnnnto/1 06:843, 1996 (abstr).

·I UittoJ , Amana S, McGrath] , Nishiyama T, Pulkkinen L, Owaribe K, Burgeson R_E, et al: Absent expression of the hemidesmosomal inner plaque prol'cin HD- '1 and its ce ll b1ological consequences in epidermolysis bullosa with muscular dystrophy.) lni'CS I D<'mwro/ 1 06:842, '1996 (abstr).

361

h eterozygotes b ave mild we disease , and a homozygote has disease that is more severe clinically and ultra struc turally.

MATEIUALS AND METHODS

Tissues Blood samples were anticoagulated with acid citrate and sent at mnbicnt tc1npcraturc to San Francisco ,.vherc the DNA \.vas extracted within 48 h after venesection (Bonifits e/ nl, 1991 ). Four millimeter punch biopsies were taken of clinica ll y norma l skin fi·om the posterior calf of patients TV-3 and IV-17 without rubbing or other purposeful preceding skin trauma. Biopsies were transported in 2.5% glu tara ldehyde, 2'Y,, para-formaldehyde. 0. ·1 M cacodylate buftcr, pH 7. and were processed as described elsewhere (Smith and Sybert, 1990).

Mutation Detection .Exon 1 of keratin 14 was amplified and sequenced as described previously (Chen <'I nl 1995). In brief. primers corresponded to keratin 14 (bp 274-303 and 627-607) . and amplifica tion was for 35 cycles of1 min at 94°C . 2 min at 55°C. and 2 min at 72°C. T he PCR product was sequenced directly 3 '-45' ming the primer 5 ' -TTGATCTCAG CAGGC­CGCTG-3' . which is complementary to bp 539-520.

RESULTS

Clinical Features The Ta f.,mily (Fig 1) is of mixed F re nch­Portuguese ancestry, and m ost members live in Brazil. B listerin g is known to have occurred in ;tt least four generations. Blisters begin around 1 )' of age, typicall y starting when children b egin to wal k , and occur only on the hands and feet. Bliste rin g is markedly mon! severe during tbe summer than the winter. Although it b eco m es less severe with maturation , adults may continue to h ave limitati on of th e ir activities by th e blisters. Milia and scarri ng do not occur.

C li11ical features in patient IV-1 7 have been con siderably more severe. Patient IV -17 is the daughter of a con sanguin eous uni on of first cousins, both of whom had EBS of a sevet;ty simiJar to other affected family m embers. Blistering was n oted at age 3 mo and during childhood was widespread with blisters not onl y of the skin but also of the oral, vaginal , and anal mu cosa. Since age 14, blistering has been limited to the hands and feet. The distal skin is scarred , all ten toenails are missi ng, and small areas of palmar hyperkeratosis are readily apparent (Fig 2). This patient li ves in Recife, where there is little seasonal variation in th e clim ate, and her fi·agility is little changed during the yea r. A U three of her childre n have EBS, and their phenotype is simi.lar to that of other afFected m embers of the kindred with blisters limited to the hands and feet . One siste r (lV-18) had hydrocephalus and severe blister­ing, and she died at age 1.

Histopathology In skin from patient IV-3 , basal and lower spino us layer keratinocytes were distended and had diflirsely orga­nized k e ra tin filaments and cytoplasmic holes (Fig 3) . T h ese findings are typical for the Weber-Cockayne variant of EBS. In con trast, the skin biopsy of patient IV-17 showed dense keratin filament bundl es in basal cells and larger aggregates in spinous and granular cells. T he epidermal morphology was similar . to tha t of epidermolytic h ype rkeratosis with the additional involvement of the basal cell layer. Alo ng one edge of the biopsy the epidermis was more norm al in appea ran ce. The cells were intact with only a few dense keratin bundl es limited to the basal cell layer. This supports

Figure 2. Clinical features of patient IV-17: (A) Atrophi c skin over the toes and absence of toenail. (B) Patchy hyperkernto­sis of palms.

362 HU ET A L THE JOURNAL OF INVESTIGATIVE DEflMATOLOGY

F igure 3. E lectron microscopy of epidermis in control (a,tl), patie nt IV-3 (!J,e) and patient IV-17 (c,f). Adult control skin (a) shows in basal kc 1:atinocytes keratin fdame nt bundles spa nning the cytoplasmic compartment (---->) with connections to hcmidcsmosomcs •tnd dcsmmomcs. The m elanocyte (m) lacks keratin fi lam ents. In patient IV-3 (!1) , basal and lower sp ino us kcratinocytcs ha ve numerous cyto~la s mic holes ("') "nd ~Inc keratin filament bundles (---->). In patient IV-"17 (c), lower kcratinocytcs show uneven distribution of discrete keratin filament clumps and dense bundl es (_..,) bu t it paucity of kera tin fi lanu:nts in the remainde r of the cytoplasm . In sp ino us ce lls of the adult contro l (d) <md patient IV -3 (e), keratin filaments are evenly distributed. By co ntrast, in the homozygote patient IV-17 0), the keratin filaments arc mostly aggrcg;~ted into large deme bundles o r clumps (-..,>).

the hypothesis that the fornntion of the keratin clumps ca n be induced by trauma. In the present case, the process of taking the biopsy may have produced the physical stress th at resulted in ke ratin filament d umping. Alternatively, th e variability in structure may have been present in the epidermis prior to the taki1ig of the biopsy.

Mutation As prev iousLy reported, the proband (IV-3) is het­erozygous for a G--7 A substi tution :1t bp 417 of keratin 14, ca usi ng the predicted missense substitutio n Met 119--7 .lle (Chen el nl, 1995). T hi s substitution has not been reported in any othe r indi vidu al, with o i· without EBS, and we did no t find the charac­teristic abnorm al single strand confo rmation polymorphism pattern in 50 norm al indi vidu als o r in the 48 other EBS pro bands studied. Patien t IV-17 is homozygous for this substitution (Fig 4).

DISCUSSION

Our finding of clinically mo1·e se vere disease in a patient homozy­gous for a keratin 14 mutation tha t ca uses blis ters limited tu the hands a11d feet in heterozygotes raises several issues . First, this report adds to the compl exity of the rela tio nship be tween keratin gen e mu tatio ns and phenotype, for it now is clear that missense mutations at a clini cal level may be not only compl etely recess ive

(keratin 14 G lu 144->Aia) (Hovnani;m eta/, 1993) or fuil y domi­n ant (ker;ltin 5 Lys 173 --7 Asn) (Steph ens el nl, 1995) but also ma y be partially do minan t. This finding of increased seveJ-ity of disease in the rare patients who are liom bzygo us for a mutation that typ ica ll y ca uses milder, autosom al dominant disease (e.g., fami lial hypercho leste rolemi:1, th<tlasseniia. etc.) is consid erably more com­mon than is the finding of :1 fulJy dominant allele c:lll sing disease that is as severe in the heterozygous as the ho mozygo us state (e.g., Huntington di sease) (Wilkie, 1994) . Further, missense mutations of genes encoding other stru ctu ral proteins also ma y produce domi­nan t or recessive alleles , e.g., in COL7 Al (Christiano and Uitto, 1996).

Second , it is noteworthy that this homozygo us patiei1t ha s blisters not only of the skin hut also of the mucosa o f the mou th , vagi.na, and anus. Mu cosal blisters generally occur in EBS oi1ly in patients with the OM sub type. This usual lack of extra cutaneous bli stering is somewhat puzzling, sin ce kera tin 5 and keratin 14 torm the keratin in te rm ediate fihim en t netwo rk of basal. cells no t only of epidermis but also of othe1: stratified ~quamou s epithelia su ch as that of esophag us and corn ea. The bListering of the eso ph agus in patients with dysti·ophic E13 suggests that the upper gastro.intestinaL tract is an area su bject to trauma of magnitude that would be expected to

VOL. 109. NO. 3 SEPTEMBER I 997

Sequencing Gel

Th,,. c H\ NL HET HOM rn] Thr118 C G GATC GATC G A TC c

[A T ~ -- - T A J Met11 9 T A ..... =· A T tfe11 9 [ill [Q] + -- - - +ITJ[6] .... - - --

[ C G - - - - G C

Glo'~ ~1! -- "''; \l_~J Gfn1 20 Met11 9 Met119f1fe119

[AT [G~A C~J [ T A s· 3' T A AT G C T A

Figure 4. Mutation detection in Ta kindred. Sequence labeled I-lET is from DNA of JV-3; sequence labeled HOM is fro m DNA of IV-17.

be sufficient to cause blisterin g in EBS as well . R ecently, Lloyd and colleagues have speculated that some stabilization of mucosal basal cells migh t be afforded by expression of keratin 15 and the formation of interm ediate fi lam ents composed of ke1·atins 5 and 15 (Lloyd eta/ , 1995).

Third, .it is also noteworthy that blistering in the homozygous patient improved with maturation just as it does in the usual heterozygous patients. T his is in contrast to the keratin 14 Glu 144-?Ala mutation , in which severity did not decrease in the homozygote, at leas t by the time of the report when the patient was 20 y o ld (Hovnanian et a/, 1993). One possible m echani srn that th eoretically might account fo r the improvement of EBS in the usual , h eterozygous patient would be the relative mechanical stability of any cells in which the mutant allele was inactivated somatically. Such cells consequently might replace the more frag il e cells that continue to express the mutan t alle le . It is consistent with such an explanation th at founder (chimeric) transgenic mice have waning of their blisters coincident w ith replacement in the skin of cells ca rrying the transgene by cells expressing only w ild type keratins (Vassar eta /, 1991). ln this homozygous patient, however, no such replacem ent would be expected , since no cells expressing a wild type allele would be expected to be available unless th ere were a reversion of the mutation. Indeed, basal cells of clin icall y normal skin taken from the adult hom ozygote appear to have few keratin interm ediate filaments , despite the rela tive res istance of those cell s to blistering. Alternatively, the slower turn over tim e of keratinocytes that occurs w ith increasing age m ay allow abnormal molecules (filam ents) to be more effectively substitu ted by no rmal keratins.

The histologic findings of clumping and aggregation of m ost of the keratin intermediate filaments throughout the full epidermis arc unique and somewhat puzzlin g. C lumping of fi lamen ts of basal cells is characte ristic of the DM form of EBS, in whi ch afFected individuals are heterozygous for keratin mu tations that apparen tly are particularly disrup tive. These clumps do not pe rsist in to the suprabasal layers, however, and appear not to in terfe re with the formation of the intermediate fi lam ent network in these cell s. C lumping of supra basal intermediate fLiaments, however, is char­acteristic of patients with mutations of keratins expressed supraba­sa lly, keratins 1, 2e, 9, and 10, but those patients have normal­appearing basal cells. Kartasova and colleagues (1993) have suggested , on the basis of transfection studies of keratin genes in to fibroblasts, tl1at the formation of normal keratin 1-keratin 10 fi laments may requiJ·e a pre- existin g in termediate filament scaffold . The finding of nonnal-appea ring supra basal intermediate fi lam en ts in patients lacking keratin 14 has been cited as evidence against that hypothesis, although , theoretically, a remainin g keratin 5-kera tin 1.5 network might still perform the scaffold function (Chan et a/ 1994; Rugg ef a/, 1994).

The structural differences in epidermal ke ratinocytes fro m the

363

two patients arc consisten t w ith the relative severity of disease in each case. Whereas the basa l cells were affected in patient IV-3, in pati ent IV-1 7 the spinous cell s had the m ost dramatically altered keratin filam ent o rganization. T he distributio n of large keratin clumps in supra basal cells suggests th e re te ntion of the abnormal keratin 14 throughout these ceil layers . It is in teresting that in both cases the biopsy site o n the leg was not norm all y a site of blister formation , but neverthe less, the histology and ultrastru cture re­fl ected the ex tent of disruption consisten t with the overall clinical presentation.

O ne possible explanation for our finding of clumping of fila­m ents throu gho ut the epidermis is that these clumps, either because of the particular amino acid substitutio n and/or because of th e lack of any normal keratin 14, resist degradation and in this instance do act as a scaffold upon w hich suprabasally expressed keratins are deposited . N othing is known about the m echanism of suprabasal "clearing" of the basa l clumps in EBS-DM. One alte rnative expla­na tion is that keratin 14 molecules carrying this substitution i11 1J i11o might pair directly with the suprabasa lly expressed type II keratins , kera tin s 1 and 2e, and thereby cause their aggregation. W hatever the explanation, it is apparent that ho m ozygosity for this keratin 14 mutation deleterio usly affects intermediate filam ent netwo rk for­mation throughout the epidenTtis.

Fin all y, genetic counselin g mu st in clude the warning that in EBS, as in the maj ority of other autosom al dominant diseases, the homozygous state may produce a disease that is f.1r m o re severe than that in either parent.

T his rl'ork ll'ns}illldcd by N IH Grn11ls A R 41120 (E.E.) a11d A R 21557 (L.S.) . I•Vc 111ish ro thn11k li t embers <if rite fn mily Ji•r their help n11d ro thn11k Dr. Clyde Cnpcrtoll, College Srnrio11, Tcxns , jin abrni 11 illg rite ski11 biopS)' .fi'om pniiew ! V-3.

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364 I-IU ET' AL

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