J Med Genet 1994;31:287-292

Detection of de novo mutations and analysis oftheir origin in families with X linkedhypohidrotic ectodermal dysplasia

J Zonana, M Jones, A Clarke, J Gault, B Muller, N S T Thomas

AbstractHypohidrotic ectodermal dysplasia (EDA)has been localised to the ql2-q13.1 regionof the X chromosome by both physicaland genetic mapping methods. Althoughlinkage analysis using closely linkedflanking markers can clarify the carrierstatus for many females at risk for thedisorder, knowledge of the origin of themutation in instances of possible de novomutation is critical for accurate geneticcounselling of families. Two methodshave been used to confirm de novo muta-tion in families with EDA and to tracetheir origin. Direct detection of three denovo molecular deletions, one arisingduring oogenesis and the other two duringspermatogenesis, was achieved by South-ern analyses using cosmids isolated fromthe EDA region as probes. Seven de novomutations arising during spermatogene-sis, and two possible de novo mutationsduring oogenesis, were identified by ananalysis of the cosegregation of the dis-order with polymorphic markers closelylinked to and flanking the EDA locus. Theconfirmation and analysis of the origin ofthe 10 de novo mutations greatly assistedgenetic counselling in these families. Theapparent 3-5:1 excess of male to femaleorigin of mutation in families studiedwith unidentified types of mutation issimilar to other studies of X linked dis-orders, and suggests that the majority ofthese mutations may involve single basepair substitutions.

(J Med Genet 1994;31:287-292)

Hypohidrotic ectodermal dysplasia (EDA), adisorder involving abnormal morphogenesis ofteeth, hair, and eccrine sweat glands, has beenlocalised to the q12-qI3.1 region of the Xchromosome by genetic and physical mappingmethods.'-5 Linkage analysis using closelylinked flanking markers, in combination withphysical examination, can modify the carrierrisk for many females at risk for EDA.26 Al-though fully penetrant in males, clear physicalmanifestations of the disorder are present inonly about two thirds of carriers, and relianceon more subtle subclinical signs, such as sweatpore counts, can be misleading.78 The identifi-cation of de novo mutations and knowledge oftheir origin is critical for proper genetic coun-selling in many families. For example, clinic-ally unaffected mothers of sporadic cases may

have inherited the gene from their mother, be anon-manifesting carrier owing to a new germ-line mutation in one of the maternal grandpar-ents, or be a non-carrier with mutation arisingduring oogenesis. We sought to confirm appar-ent de novo mutations and detect their originin families with EDA by both direct and in-direct methods of molecular analysis. The firstmethod used screened for detectable re-arrangements of genomic DNA by Southernanalysis with cosmids, previously localisedto the EDA region, as probes.' The secondmethod analysed the segregation of the dis-order in families with polymorphic marker lociclosely flanking the EDA locus.

Methods and materialsDETECTION OF DNA REARRANGEMENTSA panel of 80 unrelated families with EDA wasscreened for molecular rearrangements bySouthern analysis. The proband in each familywas a male with classical signs of EDA, andthe panel is identical to the one previouslyscreened for deletions at polymorphic lociclosely linked to the EDA locus.4 DNAsamples were extracted, digested with EcoRI,electrophoresed, and transferred to nylonmembranes by previously described tech-niques.9Four separate cosmid contigs from an X

chromosome cosmid library,'0 containing 26individual cosmids, had been previously iden-tified to be either within or contiguous to theEDA locus (fig 1).3 A single anchor cosmidfrom cosmid contigs A, B, and C had beenpreviously hybridised to the patient panel, butno cosmids from the most distal contig groupD had been used.' Cosmid ICRFc104Cl1.138from group D, and ICRFc104A09.80 fromgroup B, were used as radiolabelled probes andhybridised to Southern blots ofDNA from thepatient panel. LX5c4, the distal end clone of aYAC (4757) that was used to identify thecosmid contigs in the EDA region, was alsoused as a probe.' The use of cosmids as probesfor hybridisation to human genomic DNAwas accomplished by previously describedmethods."

ANALYSIS OF FLANKING POLYMORPHICMARKERSFamilies were selected for study of the co-segregation of EDA and flanking polymorphicmarkers, if they contained a mother who waseither an obligate carrier, or a clearly manifest-ing carrier based on the congenital absence of

Department ofMolecular andMedical Genetics,L-103, Oregon HealthSciences University,3181 SW Sam JacksonPark Road, Portland,Oregon 97201, USAJ ZonanaM JonesJ Gault

Institute of MedicalGenetics, Universityof Wales College ofMedicine, Cardiff, UKA ClarkeN S T Thomas

Department ofPaediatric Genetics,University of Munich,GermanyB Muller

Correspondence toDr Zonana.Received 8 September 1993Revised version accepted forpublication 22 November1993

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AnLy

I I | TelDXS732 I LA5c4 DXS453

Cosmid

contigs----

H04 *143 G09-96A09-80

C03184 C11138

DELETIONS

EDA 1015

EDA 1003 | . . . . . . . . .

EDA 1058

EDA 1008

Figure 1 Location ofgenomic clones isolatedfrom the EDA region and microdeletions in four unrelated malepatients with EDA. AnLy represents the X chromosome breakpoint in a female with EDA and a balanced Xautosome translocation.3 LA54 is the distal end clone of a YAC used to identify the cosmid contigs from the EDAregions.3

two or more of her permanent teeth,'2 with atleast one affected child. In addition, her par-ents had to be clinically unaffected with onlythe single affected offspring, have no otherfamily history of the disorder, and be availablefor study. Thus, if the disorder segregatedwith the haplotype of flanking markers on theX chromosome of the maternal grandfather,the mutation would be proven to be de novosince the disorder is completely penetrant inmales.'3 The mutation would most likely haveoccurred during spermatogenesis, althoughpostzygotic mutation in the mother cannot beexcluded. If the disorder segregated with thehaplotype of the maternal grandmother's Xchromosome, the mutation would either haveoccurred de novo during oogenesis, or thegrandmother would be a non-manifesting car-rier. Since the latter two possibilities cannot bereadily distinguished, the ratio of male to fe-male origin of de novo mutations in this studyrepresents a minimal estimate.Blood samples were obtained from the pro-

bands and appropriate family members forDNA preparation and analysis. The segrega-tion of alleles at seven polymorphic loci fromthe Xq 1-q21.1 region were studied to ident-ify informative markers flanking the EDAlocus (table).

ResultsIDENTIFICATION OF FAMILIES WITH DE NOVOMOLECULAR DELETIONSFamily EDA 1003Subject III1 (fig 2A) had in a previous studybeen shown to be deleted for cosmidsICRFc104G09.96 and ICRFc104C03.184

Informative markers flanking the EDA locus

Locus Location Omax (Zmax) Reference

AR Xql 1.2-q12 - 14PGKlPl Xqll.2-ql2 0-046 (13-04) 2DXS339 Xqll.2-ql3 0-000 (28-19) 2DXS732 Xql2-ql3.1 0 000 (21 20) 2DXS453 Xql2-ql3.1 0-009 (24-33) 2DXS441 Xql3.2-ql3.3 - 15DXS72 Xq21.1 0.033 (9-77) 2

from cosmid contigs B and C respectively, butthe end points of the deletion were not identi-fied and other family members were notstudied.3 Additional probes were used and theproband was shown to be deleted (no hybridis-ation signal) when L,5c4 and cosmidICRFc104C1 1.138 were used as probes (fig 1).Hybridisation with cosmid ICRFc1O4AO9.80from contig group B identified the proximalend point of the deletion. There was absence ofall seven normal sized EcoRI fragments but a

unique 5-6 kb junctional fragment was present(fig 3). DNA digested separately with HindlIland BglII, blotted and hybridised to cosmidICRFc1O4AO9.80, showed absence of all nor-

mal sized fragments and the presence ofunique junctional fragments of 16 and 5 9 kbrespectively. These unique sized fragmentswere not observed upon hybridisation of cos-

mid ICRFc1O4AO9.80 to genomic DNA fromover 100 unrelated X chromosomes. SubjectIII-3, his affected brother, also has a moleculardeletion, and their obligate carrier mother(II-2) displayed the expected junctional frag-ment. However, neither of the maternal grand-parents (I 1 and 1-2) nor the proband's sister(III2) were deleted.The proband (III 1), a 10 year old male, and

his 4 year old brother, have classical findings ofEDA, including marked hypodontia, hypotri-chosis, and significant hypohidrosis. Both boyshad normal growth and development and no

signs of any other significant disability or dis-order. Their mother (II-2), an obligate carrierof EDA, has obvious physical manifestationsof the disorder, with congenital absence of 13permanent teeth, a patchy distribution ofsweating over her body, and noticeable hypo-trichosis of her scalp hair. No other familymember has signs or symptoms of EDA.

Analysis of the segregation of alleles at thepolymorphic loci flanking the EDA locusshowed the mother (II-2) to be informative forthe flanking loci DXS339 and DXS441. Thehaplotype of flanking markers segregating withthe disorder is that of the maternal grandfather(fig 2A). Therefore, the mutation probably

Cen

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Detection of de novo mutations and analysis of their origin in families with X linked hypohidrotic ectodermal dysplasia

A EDA 1003

3 S3392 FS441

kb

11

IIIAl.

I

-5 6^o -5 1

-42- 3.7--3-2

* -2 9- 28

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11

111

-2.1

S339S453

*26

C EDA 1008

11

>3E:-- 1 4

(1) (2)

(1) (2) 3

III

(5)

Figure 2A-C Pedigrees offamilies with de novo molecular deletions with hap,of linked polymorphic loci. Black square denotes affected males; dot in circle deobligate carrier female; asterisk in circle denotes manifesting carrier female.

occurred during spermatogenesis, alth4postzygotic mutation in the mother canexcluded. As expected, the proband'swho did not display the junctional fralinherited the haplotype of the maternalmother. Barring gonadal mosaicismgrandfather, the findings eliminate pcarrier status for six female relativesfamily.

Family EDA 1058Except for a single normal sized fragmehybridisation signal was seen when cICRFc104C11.138 from contig D was ua probe on hybridisation with genomicrestricted with EcoRI and HindIII in sIII-1 from family EDA 1058 (figs 2B,

Figure 3 Southern blot of EcoRI digested genomicDNA from EDA family 1003, probed with cosmidICRFclO4AO9.80. Lanes: (1) Ii1, (2) 12, (3) II2,(4) III1I, (5) III3, (6) III2, (7) II1. Note the5-6 kb junctionalfragment in the affected sons and theirmother in lanes 3, 4, and 6.

was not deleted for any of the cosmids fromcontig groups A-C, but showed no signalwhen LX5c4 was used as a probe. The proband(III 1), a 6 year old male, had only two perma-nent teeth, inability to sweat, fine white scalphair, and bilateral absent nipples. His mother(II2) had a normal physical examination, andno family history suggestive of EDA. She had

S339 been evaluated after the birth of the proband,and a sweat pore count of her palms wassuggested to be "moderately reduced" at 12 to14/cm. During a subsequent pregnancy, shewas studied and found to be informative forthe DXS339 and DXS453 loci closely flankingthe EDA locus. Her male fetus (III2) wasshown to have the maternal grandmother'shaplotype, rather than the maternal grand-father's haplotype carried by the affected pro-band (fig 2B) and, as expected, proved to beunaffected. During her most recent pregnancy,another male fetus was detected with the samehaplotype as the proband. She was counselled

lotPes that he would be affected if she was a carrier,but this could not be determined. She con-tinued the pregnancy and shortly after delivery,of this infant the molecular deletion was

ough a detected in the proband. Neither brothermot be (III2 and III-3) was deleted upon hybridisa-sister, tion with cosmid ICRFc104C1 1.138, indicat-gment, ing that the proband's deletion arose eithergrand- during oogenesis in subject II2, or she was ain the gonadal mosaic for the mutation (fig 4).ossiblein the

Family EDA 1008The proband (III1) is an 11 year old malewith six secondary teeth, sparse, fine scalphair, and marked hypohidrosis (fig 2G). His

tnt, no mother (II-2) is a manifesting carrier withvosmid conical shaped teeth, absent bilateral maxillaryised as incisors, and patchy sweating. The maternalDNA grandparents are clinically unaffected, and;ubject there is no other family history of EDA. Ana-4). He lysis of the proband's DNA shows him to be

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r ---

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- 1-85 Figure 5 Southern blot of HindIII, EcoRI, and BglIIdigested genomic DNA from the proband (III2) fromEDA family 1008 (lanes 1, 3, 5) and a control (lanes2, 4, 6), probed with cosmid ICRFc1O4C11.138. Notethe absence of multiple normal sizedfragments with eachdigest in lanes 1, 3, and 5.

- 1 3 hybridisation with cosmid ICRFc104C1 1.138(data not shown).

Figure 4 Southern blot and HindIII digested genomicDNA from EDA family 1058, probed with cosmidICRFcI04C11.138. Lanes: (1) IIlI, (2) III I, (3)III2, (4) III3, (5) II2, (6) I l, (7) 12. Note thedeletion of all but one normal fragment in subject III Iin lane 2, but normal pattern in subject III3 in lane 3.

missing multiple fragments on hybridisation ofcosmid ICRFc1O4C11.138 to DNA digestedseparately with several restriction enzymes (fig5). He was not deleted for any of the cosmidsused as probes from contig groups A-C, nor

for the YAC end clone LX5c4. Analysis ofpolymorphic loci flanking the EDA locusshowed that the proband had inherited hisallele at the DXS339 locus from the maternalgrandfather. Since the proband's mother isclearly affected, and no recombinants havebeen observed to date between the DSX339and EDA loci, the mutation probably arose

during spermatogenesis in the maternal grand-father. Neither he nor the maternal grand-mother showed evidence of the deletion upon

IDENTIFICATION OF DE NOVO MUTATIONS BYHAPLOTYPE ANALYSISNine families with unidentified mutations hada pedigree structure potentially informative foranalysis of possible de novo mutations withrelevant samples available (fig 6). Informativeflanking markers were present in all nine fami-lies. In seven of the families studied, the haplo-type segregating with the disorder was inher-ited from the maternal grandfather, and in twocases from the maternal grandmother.

In the seven families where the mutationwas present on the maternal grandfather'shaplotype, the mutation had to have ariseneither during spermatogenesis or postzygoti-cally in the mother, since the disorder showscomplete penetrance in males. In either case,the carrier risk of the maternal grandmotherand great aunts of the proband is eliminated.The risk to the maternal aunts and their femaleoffspring is also eliminated, barring gonadalmosaicism in the maternal grandfather. In thetwo families where the disorder segregatedwith the haplotype of the maternal grand-mother (EDA 1010 and 1108), it cannot be

9 0

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Detection of de novo mutations and analysis of their origin in families with X linked hypohidrotic ectodermal dysplasia

EDA 5[l22E t--3 t23

11 r 2[1--i22t1 2

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EDA 1020

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III

17 ~7 S339F2 F2 S72

III t:4

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111

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339 ° 36 tS453453 011lI

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III111 "~~~~~~t2

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III t2

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111 b -61 14r4 [7 [7e2 1 F1

EDA 1108

11 t2 EC

III

(I 13 4S732S441

1

13

EDA 1030

11 tl t2[l b b oIII *11 I2Lat1 1 t1

Figure 6 Pedigrees ofEDA families with undefined mutations and possible de novo mutations, with haplotypes at polymorphic marker lociflanking the EDA locus.

ruled out that the mutations were not de novo,but inherited from a non-manifesting grand-mother (fig 6). If DNA samples were availablefor analysis from the unaffected male offspringof these women, one might be able to distin-guish between these two possibilities.

DiscussionWe have been able to identify de novo muta-tions in 10 families with EDA, with three ofthem having detectable de novo deletions. Oneother family (EDA 1015) with a moleculardeletion had been previously detected amongthe families in our panel, but the origin of themutation could not be determined since thedeletion was present in all three generationsstudied.4 The proximal breakpoint of this dele-tion was localised within cosmid contig A(ICRFc1O4HO41.43), with a distal breakpointlocated proximal to cosmid ICRFc104C1 1.138from contig group D (fig 1). The clinicalphenotype of the affected males in all fourfamilies with molecular deletions was no dif-ferent from other males with EDA. Identifica-tion of the de novo nature of the moleculardeletions was extremely helpful in counsellingthese families, and detection of four separatemolecular deletions should aid in the posi-tional cloning of the EDA locus. To date, 5%of the 80 families studied have detectable mo-lecular deletions using cosmids localised to theEDA region as probes. Further deletions maybe identified in the remaining families, includ-ing the seven with apparent de novo muta-tions, as additional genomic clones and cDNAfrom the EDA region are isolated.Although the present sample size is small,

the 3-5:1 ratio (95% CI of 0.8-14.8 to 1) ofmale to female origin of mutations in thefamilies with yet to be defined mutations isconsistent with the results of studies of otherX

linked loci, such as factors VIII and IX, andZFX.'6'9 Sex differences in the origin ofmuta-tion appears to depend on the nature of themutation, with single base substitutions ingeneral, and transitions at the CpG dinucleo-tide in particular, having a male preponder-ance, while deletion cases display a sex ratiocloser to unity.'4 This indicates that the major-ity of the unidentified mutations in the EDAgene may be the result of single base pairsubstitutions. The male to female ratio oforigin of mutation in this study is a minimalestimate since in the cases where the disordersegregated with the X chromosome of thematernal grandmother, the mutation may notbe de novo. Gonadal and somatic mosaicism inmales and females can complicate both originofmutation studies and genetic counselling,202'but such mosaicism in EDA has yet to bedocumented.The ability to prove that a mutation was de

novo eliminated the risk of being a carrier formost female relatives in the 10 families. Ouranalyses indicate the need for caution in theinterpretation of carrier status based on subcli-nical tests such as sweat pore counts.78 Severalof the females suspected of being carriersbased on such tests were found not to be at riskafter molecular analysis. The apparent excessof mutations during spermatogenesis ratherthan oogenesis, if confirmed by additionalfamily studies, would significantly increase therisk of mothers of sporadic cases being car-riers. Hopefully, direct detection of both dele-tions and single base pair substitutions will bepossible in most families once the EDA gene isidentified.The authors would like to thank P Zimmerman and T K Hyattfor their assistance in preparation of the illustrations and JChelly and T Monaco for the X chromosome cosmids used asprobes. This work was supported by the following organisa-tions: PHS RO1 AR40741 from the National Institute ofArthritis, Musculoskeletal and Skin Diseases (JZ), NationalFoundation for Ectodermal Dysplasias (JZ), and The Well-come Trust (AC,NSTT).

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