Journal of Medical Genetics 1986, 23, 46-51

Prenatal diagnosis of bullous ichthyosiformerythroderma: detection of tonofilament clumps infetal epidermal and amniotic fluid cellsR A J EADY, D B GUNNER, L DORIA LAMBA CARBONE*,F DAGNA BRICARELLI*, C M GOSDENt, AND C H RODECKtFrom the Department of Electron Microscopy and Cell Pathology, Institute of Dermatology, HomertonGrove, London; *Centro Regionale di Genetica Umana, Ospedali Galliera, Genoa, Italy; tMRCClinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh; andtHarris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London.

SUMMARY The prenatal diagnosis of bullous ichthyosiform erythroderma (BIE) has beenachieved at 20 weeks' gestation by electron microscopic identification of a pathognomoniccytoskeletal abnormality within fetal epidermal cells obtained by fetoscopic skin biopsy. Thesame abnormality was also observed in skin derived amniotic fluid cells. The question whetheramniocentesis might be used instead of fetoscopy for future prenatal detection of BIE isdiscussed.

Bullous ichthyosiform erythroderma (BIE) is one ofthe more severe types of congenital ichthyosis'-4 andis transmitted as an autosomal dominant trait. Itsincidence has not been established although it ispresumed to be very low. Clinically, BIE presents atbirth or soon afterwards with widespreaderythroderma, blistering, and scaling. Blisteringtends to improve with age. Warty thickening of theflexural skin usually appears by the third or fourthyear and persists into adult life. There is, inaddition, hyperkeratosis of the palms and soles. Lifeexpectancy is usually normal although the diseasecan be severely incapacitating. Death, often associ-ated with severe infection, may occur in infancy.The diagnosis of BIE depends on the detection of

highly characteristic tonofilament clumps in theepidermal cells, associated with intraepidermalvacuolation and hyperkeratosis.5 6 The conditionhas been diagnosed prenatally by light and electronmicroscopy of fetal skin biopsy samples obtained at19 or 20 weeks of gestation.' x In a retrospectivestudy, Holbrook et al detected the characteristictonofilament aggregates within the amniotic fluidcells (in addition to the fetal epidermis) associatedwith two affected pregnancies and postulated thatprenatal diagnosis of BIE might be possible bymorphological analysis of amniotic fluid cells with-

Received for publication 19 November 1984.Accepted for publication 11 January 1985.

out recourse to fetoscopy and fetal skin biopsy. Wehave succeeded in diagnosing BIE prenatally bydetecting tonofilament clumps in both fetal skin andamniotic fluid cells at 20 weeks' gestation. Ourfindings are reported here.

Case report

The patient was a 37 year old woman who hadsuffered from BIE since birth. She had many of thetypical features of the disease, including widespreaderythema, hyperkeratosis, and scaling. The hyper-keratosis was accentuated in the major flexures andwas particularly severe on the hands and feet,affecting both ventral and dorsal surfaces. When shewas young blistering was a constant problem, but asshe became older it tended to occur mainly inwinter.A skin biopsy specimen taken from a hyperker-

atotic area on the patient's right hip showedcharacteristic histological changes of BIE, namelyhyperkeratosis and vacuolation of the epidermisabove the basal layer, associated with darkly stain-ing intracellular granules or inclusions, many ofwhich were larger than normal keratohyalingranules (fig 1).

FAMILY HISTORYThe patient's parents were not consanguineous andno other family member was affected. There was no

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FIG 1 Photomicrograph ofproband'sskin. Note thickening ofthe epidermis,especially affecting the stratumcorneum (sc) and vacuolation(arrows) of the stratum granulosumwhich contains large granules.(Haematoxylin and eosin. Originalmagnification x 140.)

history of ichthyosis or blistering in her husband'sfamily.

In 1978, her first child, a boy, was born witherythema and widespread blistering (fig 2). He diedat 6 days of age with severe generalised candidiasis.Skin biopsy showed changes of BIE similar to thosepresent in the mother's skin. In 1983, she had a

spontaneous abortion at eight weeks' gestation.Later in the same year, she became pregnant againand, knowing that there was a 50% chance of havinganother affected child, sought prenatal diagnosis.

Investigations

FETOSCOPY AND FETAL SKIN BIOPSY

Using the procedure we have previouslydescribed,"1'2 fetoscopy and fetal skin biopsy were

carried out under real time ultrasound guidance at

FIG 2 Proband's first child who died soon after birth withwidespread blistering. (Reproduced with permission fromProspettive in Pediatria, 1981.)

an estimated gestational age of 20 weeks. The fetalbiparietal diameter was 51 mm and the femur length32 mm, confirming the gestational age of 20 weeks.The amniotic fluid was cloudy and the fetal scalpwas scaly, giving the appearance of 'dandruff'. Noblisters or erosions were seen. One skin biopsy was

taken from the left hip. It was not possible to obtainfurther skin samples because the patient began tovomit and the fetoscope had to be withdrawn. Thefetal skin sample was immediately immersed infixative and processed for light and electron micro-scopy using a rapid technique. '3 Semithin sections ofthe plastic embedded fetal skin were stained withbasic fuchsin and methylene blue or with methyleneblue and azure II for light microscopy. Ultrathinsections were stained with uranyl acetate and leadcitrate and examined in a JEOL 100CX transmissionelectron microscope.

AMNIOTIC FLUID CELL ANALYSIS

During fetoscopy, amniotic fluid was withdrawn formorphological and cytogenetic studies.

Morphological studiesA total of 10 ml amniotic fluid was centrifuged at1500 rpm for five minutes. The resulting pellet ofcells was resuspended in 0-1 mol/l cacodylate bufferwith 7% bovine serum albumin. After a secondcentrifugation, the pellet was fixed for one hour inhalf-strength Karnovsky fixative'4 in 0-1 moVlcacodylate buffer containing 5% sucrose and 0-05%CaCl2. After several washes in buffer, the pellet waspostfixed in 1% osmium tetroxide in distilled waterfor one hour on ice. After dehydration in gradedalcohol, the cells were embedded in TAAB resin.Sections were prepared for light and electron

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microscopy using similar techniques to those usedfor fetal skin samples (see above).

Total cell counts, viable cell counts, and estimatesof numbers and origins of red blood cells in theamniotic fluid were made after vital staining of thecells with trypan blue.'> t Size class analysis wasalso carried out. Scanning electron microscopypreparations were made according to the method ofRodeck et al. "' Cells were layered onto alcian bluetreated coverslips and examined in a CambridgeS180 scanning electron microscope.

Chromosome stludiesUsing the methods of Gosden et al, 17 chromosomestudies were carried out on cultured amniotic fluidcells because of the risks of autosomal trisomy withthe advanced maternal age.

Results

SKIN MICROSCOPYThe single skin specimen was sufficiently large andwell preserved for detailed morphological examina-tion. The microanatomy of the epidermis wasgrossly altered: there was widespread vacuolationassociated with dense cytoplasmic inclusions affect-ing all cell layers between the stratum basale andperiderm (fig 3). The epidermal vacuolation wasmore marked than might be expected (probably as aresult of biopsy artefact) in fetoscopic samples ofnormal fetal skin of similar gestational age (seebelow).

Electron microscopy revealed a variable degree ofintracellular oedema and cytolysis within the

epidermis. especially in the superficial part of theintermediate layer. The normal filamentous cyto-skeleton was grossly altered. Instead of coursingthrough the cytoplasm, the tonofilaments tended toaggregate in tight intracellular clumps with relativelyregular borders (fig 4). Desmosomes appearedgenerally normal. The clumps were not detected ineither periderm or basal cells. The ultrastructure ofthe periderm appeared normal for 20 weeks'gestation. There were focal zones of regressingperiderm with early corneocyte formation. In theseareas, small keratohyalin granules were alsoevident.

AMNIOTIC FLUID CELLSCell countsThe total number of amniotic fluid cells was71 400/ml of which 16 700/ml were viable (23%viability). Fewer than 1% of the viable cells wererapidly adherent to glass in the short term (20 hour)cultures. It was, however, interesting to note thatthe only adherent cells were those in the clumpswhich were very similar in appearance to those seenin the scanning electron microscope (see below).

Light atid transinission electron mnicroscopYSeveral semithin sections taken from differentblocks of the same pellet were examined by lightmicroscopy. About 10% of cells contained intra-cytoplasmic dense inclusions. Many cells with theinclusions were still attached to periderm derivedcells (fig 5). Electron microscopy revealed that theinclusions were tonofilament aggregates similar tothose seen in fetal epidermis (fig 6).

FIG 3 Photomicrograph offetoscopicskin sample showing gross disruption,especially vacuolation, of the fetalepidermis at 20 weeks' gestation.Dense cytoplasmic inclusions(arrows) are evident in all epidermal

^* '. cell layers, except the periderm (p)and basal layer. (Basic fuchsin andmethylene blue. Originalmagnification x 710.)

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Prenatal diagniosis of blilloiis ichtlhvosiforin erythrodern7a

khfa

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FIG 4 Electron micrograph showingnumerous tontofilament aggregates (fa)in the fetal epidermis. Earlykeratohyalin (kh) and corneocvte (c)formation is evident. n=nucleus.Bar=1 .m.

Scanning electron microscopyNormally, individual cells are seen (fig 7a and b),each characteristic of the fetal cell surface fromwhich the cells have been shed (for example, fetalskin, trophoblast, respiratory tract). In this case,many cellular clumps were observed containing cellswith variable surface characteristics (fig 7c and d).

p

FIG 5 Photomicrograph of I p.m plastic embedded sectionshowing a clump ofamniotic fluid cells. Darkly staininggranules (arrows) can be seen in cells thai are attached toperiderm derived cells (p). (Methylene blue and azure IL

Original magnification x280.)

CHROMOSOME ANALYSIS

Analysis of the cultured fetal cells showed them tohave a 46,XX apparently normal female karyotype.The fetal chromosomal fluorescence heteromorph-isms were compared with those of the mother.These confirmed that the fetal cells were distinctfrom those of the mother and that no maternalcontamination was present.

FIG 6 Electron micrograph ofanamnioticfluid cellcontaining numerous filamentaggregates. Bar= I Mm. Inset showsdetail of an aggregate withcharacteristic banding (arrow) wherefilaments are aligned. Bar= I nm.

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FIG 7 Scanning electronmicrographs of amniotic fluidcells. (a) and (b) show normalsingly dispersed cells of varyingdimensions with characteristicsurface features. (c) and (d) showclumps of cells found in bullousichthyosiform erythroderma.Bars= 15 [tm.

'I'ERMINATION OF IPREGNANCYThe fetus was aborted at 21 weeks' gestation byintra-amniotic prostaglandin injection. At deliverythe skiin looked generally normal with the exceptionof a few small superficial erosions. There were no

blisters. An hour after delivery, the skin appearedvery dry and wrinkled, especially on the elbows,knees, and ankles. The skin on the palms and soleswas abnormally thick and scaly. A skin sample takenad acent to an eroded area showed characteristichistological features of epidermolytic hyperkerato-sis. Electron microscopy demonstrated clumping oftonofilaments in the epidermis which was lessmarked compared with that seen in the fetoscopicskin biopsy.

Discussion

In this study there was no difficulty in identifying thediagnostic tonofilament clumps in both fetalepidermis and amniotic fluid cells. Our findingstherefore strengthen the proposal9 that BIE mightbe detected in utero by electron microscopy ofamniotic fluid cells obtained by amniocentesis.However, we feel that the amniotic fluid cellabnormality needs further confirmation before we

could consider recommending amniocentesis as a

preferable alternative to fetoscopic skin biopsy inthe prenatal examination of pregnancies at risk forBIE. The patchy clinical expression of the diseaseindicates that, where possible, fetal skin biopsiesfrom several different sites should be examined. Wecould obtain only one sample and were fortunate to

be able to make the diagnosis on the basis ofexamination of this limited material. The amnioticfluid cell abnormality therefore provided us withvaluable diagnostic confirmation.

Tonofilament clumping has not been reported innormal amniotic fluid cells,)9 2' nor has it beenobserved in any of our own sections of amniotic fluidcells from normal 18 to 21 week pregnancies (R A JEady, unpublished observations). Demonstration ofthe tonofilament abnormality by indirect immuno-fluorescence using monoclonal antikeratin anti-bodies might prove a useful alternative or additionto transmission electron microscopy in the future.Preliminary results with this technique in the presentcase2' were not sufficiently clear cut for the basis ofa diagnosis.

In our experience of examining over 60 fetoscopicskin samples taken at 18 to 21 weeks' gestation, wehave noted a tendency for vacuoles to form withinthe epidermal cells, associated with apparent extrac-tion of glycogen. These changes, therefore, maymimic the vacuolation which also occurs in BIE. Wetherefore suggest that the diagnosis of BIE shoulddepend on the demonstration of the tonofilamentclumps rather than epidermal cell vacuolation.No abnormality was observed by fetoscopy on

gross appearance of the fetal skin except for scalingof the scalp. The cloudy appearance of the amnioticfluid noted during fetoscopy might be attributed tothe desquamation of high numbers of cells inaddition to the presence of cell clumps. The latterwere readily demonstrated by light microscopy ofsemithin sections and by transmission and scanning

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electron microscopy. The total amniotic fluid cellnumbers are very close to the upper limit fornormality, while the viable cell numbers and propor-tion of viable cells are relatively low22 indicating thatthe cell dynamics seem disturbed in this case. Anincreased production of amniotic fluid cells by skinmight result from excessive epidermal cell prolifera-tion and rapid transepidermal cell transit, as demon-strated in adult patients with BIE.23

It is possible to diagnose other forms of ichthyosisbefore birth. Both harlequin ichthyosis24 andSjogren-Larsson syndrome25 have been detected bymicroscopy of fetoscopic skin biopsies. The diagno-sis of X linked ichthyosis has been achieved bybiochemical analysis of amniotic fluid.26

This work was supported by a grant (to RAJE)from the Herbert E Dunhill Trust.

References

Brocq L. Erythrodcrmie congcnitale ichthyosifornic avcc

hyperepidermotrophie. Ann Dermatol Sy'phil (Paris) 1902;3:1-3 1.

2 Simpson JR. Congenital ichthyosiform erythroderma. Trans StJohn's Hosp Dermatol Soc 1964;50:1-31.

3 Wells RS, Kerr CB. Genetic classification of ichthyosis. ArchDerinatol 1965;92:1-5.

4 Williams ML. The ichthyoses-pathogenesis and prenataldiagnosis: a review of recent advances. Pediatr Derinatol1983;1: 1-24.

5 Weibel ER, Schnyder UW. Zur ultrastruktur und histochemieder granulosen degeneration bei bulloser erythrodermic con-

genitale ichthyosiforme. Arch Klin Exp Dermatol 1966;233:124-38.

h Ishibashi Y. Klingmuller G. Erythrodermia ichthyosiformiscongenita bullosa Brocq. Uber die sogenannte granulose degen-eration. Arch Klin Exp Derinatol 1968;233: 124-38.

7 Golbus MS, Sagebiel RW, Filly RA, Gindhart TD. Hall JG.Prenatal diagnosis of congenital bullous ichthyosiformcrythroderma (epidermolytic hyperkeratosis) by fctal skinbiopsy. N Engl J Med 1980;302:93-5.Anton-Lamprecht I. Prenatal diagnosis of gcnctic disordcrs ofthe skin by mcans of electron microscopy. Hlum Geniet1981 ;59:392-405.

9 Holbrook KA. Dalc BA. Sybert VP. Sagcbicl RW.Epidermolytic hypcrkeratosis: ultrastructure and biochcmistryof skin and amniotic fluid cclls from two affected fetuses and a

newborn infant. J Invest Dermnatol 1938;80:222-7.Rodeck CH. Eady RAJ. Gosden CM. Prcnatal diagnosis ofepidermolysis bullosa letalis. Latncet 1980;i:949-52.

Rodeck CH, Nicolaides KH. Fetoscopy and fetal tissue sam-

pling. Br Med Bull 1983;39:332-7.2 Eady RAJ, Rodeck CHi. Prenatal diagnosis of disorders of the

skin. In: Rodeck CH. Nicolaides KH. eds. Proceedings of the11th Studv Group of the RoYal College of Obstetricians anldGynaecologists. London: Royal College of Obstetricians andGynaccologists. 1984:147-58.

'3 Eady RAJ, Gunner DB, Tidman MJ. Nicolaides KH. RodeckCH. Rapid processing of fetal skin for prenatal diagnosis by lightand electron microscopy. J Clini Pathol 1984:37:633-8.

4 Karnovsky MJ. A formaldehyde-glutaraldehyde fixativc of highosmolality for use in clcctron microscopy. J Cell Biol1965:27: 137-8A.Gosden CM. Brock DJH. Morphology of rapidly adheringamniotic fluid cells as an aid to the diagnosis of neural tubcdefects. Latncet 1977;i:919-22.

16 Gosden CM. Brock DJH. Combined usc of alphafetoproteinand amniotic fluid ccll morphology in cirly prenatal diagnosis offetal abnormalities. J Med Geniet 1978:15:262-7(1.

'7 Gosden CM, Buckton K, Fctheringhatm Z. Brock DJH.Prenatal karyotyping and maternal serum alphafctoproteinscreening. Br Med J 1981;282:255-8.

x Holbrook KA. Human cpidermal cmbryogencsis. Itot J Der-mnatol 1979;18:329-56.Hoyes AD. Ultrastructurc of the cclls of the amniotic fluid. JObstet Gvnaecol 1968;75:164-71.

20) Nieland ML. Parmley TH. Woodruff JD. Ultrastructuralobservations on amniotic fluid cclls. Amt1 J Obstet Gvnecol1970;:108:103(1-42.

21 Eady RAJ. Gunncr DB, Lcigh IM. Dctcction of tonofilamentclumping in cpidermal and amniotic fluid cells cnables prenataldiagnosis of bullous ichthyosiform crythroderma (BIE). J CutanlPathol 1983;11:212.

22 Gosdcn CM. Amniotic fluid cell typcs and culture. Br Med Bull1983;39:348-54.

23 Frost P. Weinstcin GD. Van Scott EJ. The ichthyosiformdermatoses. II. Autoradiographic studics of epidcrmal prolif-cration. J Invest Dertnatol 1966;47:561-7.

24 Elias A. Mazur M. Sabbagha R. Estcrly NB. Simpson JL.Prenatal diagnosis of harlequin ichthyosis. Cliti Genet1980;:17:275-80.

25 Kousseff BG, Matsouka LY. Stccn KS. Hobbins JC. MahoneyMJ, Hashimoto K. Prenatal diagnosis of Sj6igren-Larssonsyndromc. J Pediatr 1982;101:998-1(001.

25 Hahncl R. Hahnel E. Wysocki SJ. Wilkinson SP. Hockey A.Prenatal diagnosis of X-linkcd ichthyosis. Clini Chi,n Acta1982;120:143-52.

Correspondence and requests for reprints to DrR A J Eady, Department of Electron Microscopy andCell Pathology, Institute of Dermatology. Homer-ton Grove, London E9 6BX.

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