Hum Genet (199l) 86:297-299

~) Springer-Verlag 1991

Linkage relationship between incontinentia pigmenti (IP2) and nine terminal X long arm markers

A . Sef iani 1' 12, R . M'rad 1 , L. S imard 2, A . V i n c e n t 3, C. Jul ier 4 , L. H o l v o e t - V e r m a u t t, S. H e u e r t z 1, N . D a h l 5, J . F . Sta lder 6 , M. O. Pe ter 7, C. M o r a i n e 8 , J. Malev i l l e 9 , J. B o y e r ~~ , I. Oberl~ 3 , D . L a b u d a 2, and M . C. H o r s - C a y l a 1' xt

1 Unit6 INSERM U. 12, H6pital des Enfants-Malades, 149, Rue de S~vres, F-75743 Paris Cedex 15, France 2H6pital Sainte Justine, 3175, Chemin Sainte Catherine, Montreal H3T IC5, Canada 3LGME, Facult6 de M6decine, 11, Rue Humann, F-67085 Strasbourg, France 4CEPH, 27, Rue Juliette Dodu, F-75010 Paris, France SBiomedical Center, Box 589, S-75123 Uppsala, Sweden 6CHU de Nantes, Place Alexis-Ricordeau, F-44035 Nantes Cedex, France 7CHR de Mulhouse, B.P. 1070, F-68051 Mulhouse Cedex, France 8CHR de Tours, 2, Boulevard Tonnel6, F-37044 Tours Cedex, France 9CHU de Bordeaux, 168, Cours de ]'Argonne, F-33077 Bordeaux Cedex, France a~ de Gu6ret, 39, Avenue de la Sdnatorerie, F-2301l Gu6ret, France 11Universit6 Pierre et Marie Curie, 4, Place Jussieu, F-75252 Paris Cedex 05, France 12Facult6 de M6decine, Rabat, Morocco

Received May 15, 1990 / Revised August 6, 1990

Summary. Linkage data for familial incontinentia pig- menti (IP2) and nine X chromosomal markers are re- ported. Previously found linkage between IP2 and the DXS52 locus is confirmed with the maximum lod score of 6.19 at a recombination fraction of 0.03. Linkage is also established with loci DXS134, DXS15 and DXS33. Multipoint analysis allows us to localize the IP2 locus out- side a block of seven linked markers of the Xq28 region.

Introduct ion

Incontinentia pigmenti is a rare dominant X-linked ge- nodermatosis that is lethal in males. Both familial and sporadic cases have been documented. Some of the sporadic cases are associated with an X/autosome trans- location. We have previously localized the IP2 gene re- sponsible for the familial cases to Xq28 by linkage with DXS52, and have excluded its location in other regions (Sefiani et al. 1988, 1989a; Sinnett et al. 1988). None of the breakpoints for the cases associated with an X/auto- some translocation are in the region Xq28 (Sefiani et al. 1989b). In the present paper, we present additional families and localize the IP2 gene in the region Xq28 more precisely.

Materials and methods

Fami l ies

We investigated 12 families in which 52 mitosis are potentially in- formative. The pedigrees are shown in Fig. 1. In these families, at

Offprint requests to: M.-C. Hors-Cayla, Unit~ INSERM U. 12, HSpital des Enfants-Malades, 149, Rue de S6vres, F-75743 Paris Cedex 15, France

Family 1

Family 3 Family 2

Family 4 Family 5 Family 6

Family 7

Family 10 Family 11 Family 12

Fig.l. Pedigrees of families with familial incontinentia pigmenti (IP2); affected females are indicated by solid circles. Family mem- bers identified by an asterisk were not included in the linkage anal- yses

298

Table 1. Loci and probes used

Probe Locus Localization Enzyme Contact Reference

55.E DXS105 q27 BclI/BglII/MspI J.-L. Mandel Arveiler et al. (1988)

U6.2 DXS304 q28 BclI N. Dahl Dahl et al. (1989)

ST35-691 DXS305 q28 TaqI/Pstl J.-L. Mandel Vincent et al. (1989)

F814 DXS52, F8C q28 BclI J.-L. Mandel Heilig et al. (1988)

MN12 DXS33 q28 BglI K. Davies Patterson et al. (1987)

cpX67 DXS134 q28 MspI P. Pearson HoNer et al. (1987)

DX13-7 DXS15 q28 BglfI K. Davies Patterson et al. (1987)

767 DXS115 q28 PstI P. Pearson Arveiler et al. (1988)

least two females were affected. The families were ascertained, by the authors, from different pediatric and genetic centers. Women were scored as affected if they exhibited early and late cutaneous manifestations accompanied or not by dental ocular or neurologi- cal anomalies. There were a few exceptions as follows. In family 6, patient II 1 does not know if she exhibited early cutaneous man- ifestions; she was considered as affected because of the observa- tion of late cutaneous manifestations and five miscarriages. Patient 12 in family 8 and patient 1 1 in family 10 have only dental manifes- tations; they were scored as obligate carriers because of the occur- rence of two affected daughters. The same diagnosis was reached for patient I 1 in family 9, for patient 1 2 in family 11 and for pa- tient II 2 in family 12 for whom we had no precise information. The clinical description of family 4 has been previously reported (Iancu et al. 1975).

DNAstudies

D N A extraction from peripheral lymphocytes, Southern blotting, hybridization to radioactively labeled whole plasmids, and auto- radiographs were carried out using standard methods as previously described (Sefiani et al. 1988). Restriction enzyme digestions were performed as indicated by the manufacturers. Restriction frag- ment length polymorphism loci are given in Table 1.

Linkage analysis

Analysis of data was performed with version 3.5 of L INKAGE (Lathrop et al. 1984); the analysis was conducted assuming a gene frequency of 2.5 x 10 -5, and a dominant inheritance with 100% lethality in carrier males and 95% penetrance in carrier females. Confidence intervals for the recombination fractions were calcu- lated as described by Conneally et al. (1985).

Table 2. Pairwise linkage analysis of IP2 and markers from the Xq27-Xq28 region. The estimated recombination rates (8) corre- sponding to the maximum lod scores (z max) are presented. Confi- dence intervals are indicated in parentheses. The number of infor- mative meioses with and without phase ranged from l0 to 43

Probe ~) z max

DXS105 0.12 0.66

DXS304 0.16 1.24

DXS305 0.17 0.95

DXS52 0.03 (0-0.13) 6.19

DXS33 0 (0-0.18) 2.46

DXS134 0 (0-0.12) 3.76

DXS15 0 (0-0.14) 3.66

F8C 0.07 1.64

DXS115 0 1.72

Muhipoint analysis The LINKMAP program was used to determine the location scores corresponding to the location of IP2 in the different inter- vals of the following genetic map: DXS105 - (0.20) - DXS304 - (0.084) - DXS305 - (0.016) - DXS52, DXS33, DXS134, DXS15, - (0.013) - F8C, DXSl l5 . Recombination fractions between ad- jacent loci are indicated in parentheses. The recombination frac- tion does not exceed 0.005 between the extreme markers of the block DXS52, DXS33, DXS134 and DXS15 (I. Oberl6 et al., in preparation).

Results and discussion

Two point linkage data between the IP2 locus and mark- ers from the Xq28 region are presented in Table 2. Sig- nificant lod scores were obtained with DXS52 (z max =

Family 7

1

, I) 2 1 1 2 1 2 2 1 2 1

2 2 2

2

1 (1) 2 1 1 2 1 1

OXS 105 DXS 304 OXS 305 OXS 15 OXS 134 OXS 52 FSC DXS 115

2 2 2 1 1 1 2 2

. (

1 2 1 1 2 1 1 2 1 2 2 3 1 1 1 1

2 1 1 1 1 2 2 1 2 1 3 2 1 1 1 1

i nd nd 2 1 1 1 2 2

[] 4 2

1 2

Family 10 OXS 305 1 ,2 i ,: l oxss2 t (1) F8C

I

1 2

1 1 nd nd 2 2 1 1 1 1 1 2 2 1 2 1

( 5

Fig. 2. Two recombinant families. Family 7: phase is deduced from other meioses in generation II. The recombinant allele for DXS105 is indicated by an uppercase figure in the genotype of individual II,1. Family 10: the genotype of the father, in brackets, is deduced from the genotypes of his daughters, nd Not done; allele in brackets allele deduced

299

-25 Centromere

20

._~

i

�9 ' ' 0

Map dtstances IMorgans I 2100 20 630 1 Odds

450

Fig. 3. Support for placements of the IP2 locus with respect to the map of the Xq27-Xq28 loci: A DXS105; B DXS304; C DXS305; D, E, F, G DXS52, DXS33, DXS134, DXS15; H, IF8C, DXSll5. Locus A (DXS105) was arbitrarily assigned a map position of 0. Distances between loci were calculated using the mapping function of Haldane. The location score value is defined as twice the natural logarithm of the odd ratio (Lathrop et al. 1984). Odds against the placement of IP2 in each interval are indicated

6.19 at 0 = 0.03), DX S 134 (z max = 3.76 at 0 = 0) DXS15 z max = 3 . 6 6 at 0 = 0 ) and DXS33 (z max = 2 . 4 6 at = 0). Lod scores are positive with all the o ther markers

tested in this region: DXS105, DXS304, DXS305, FSC and DXS115.

Observat ion of key recombinants and multipoint anal- ysis have been used to investigate the most likely place- men t of the IP2 locus relative to the Xq28 markers . Accord ing to Mandel et al. (1989), the mos t probable o rder of the markers is f rom cen t romere to te lomere: DXS105 - DXS304 - DXS305 - (DXS52, DXS33 , DXS134, DXS15) - FSC - DXS115. Figure 2 shows the segregat ion of informative markers in 2 recombinants . In family 10, it is possible to deduce the genotype of the fa ther f rom the genotypes of the two daughters ; it ap- pears that the two affected daughters received a differ- ent X - c h r o m o s o m e f ragment in the region carrying the markers DXS305, D X S 5 2 and F8C. If we assume only one recombina t ion event per meiosis, this observat ion means that the IP2 locus is outside the group of markers const i tuted by DXS305, DXS52 and F8C. In family 7, the phase is inferred f rom the four o ther non- recombin- ant incloses. This r ecombinan t shows that DXS105 is outside a block constituted by DXS304, DXS305, DXS52, DXS134, F8C, DXS115 and the IP2 locus. Taking into account the informat ion that the IP2 locus is l inked to the block (DXS52, DXS33 , DXS134, DXS15) , that it shows a positive lod score with F 8 C and DXS115, and that it is less tightly l inked to DXS304 than to F 8 C , we suggest that the IP2 locus mos t likely maps distal to F8C.

Results f rom the mult ipoint analysis are shown in Fig. 3; a l though the data allow us to conf i rm linkage to the Xq28 markers ( location score o f 25.4 equivalent to a lod score of 5.5), they are insufficient to locate IP2 in a precise interval of the map. The intervals ( D X S 3 0 4 - DXS305) and (F8C, DXS115 - tel) are the most likely, all o ther intervals having odds of less than 1 : 240 against their containing the IP2 locus.

The above results conf i rm the localization o f the fa- milial form of incontinentia pigmenti (IP2) to the Xq28 region. However , as the families studied are very small, we cannot exclude possible genetic he terogenei ty ; analy- ses of addit ional families and probes are current ly being pursued to ascertain the precise location of IP2 and the genetic homogene i ty o f the familial fo rm of the disease.

Acknowledgements. We thank Drs. K. Davies and P. Pearson for providing probes. We are also indebted to IP families and the clini- cians who referred them to us: Dr.A. David (C.H.U., Nantes), Dr.C.Foldes (C.H.G., Saint Germain en Laye), Professor B. LeMarec (C. H. R. Rennes).

R e f e r e n c e s

Arveiler B, Oberl6 I, Vincent A, HoNer MH, Pearson PL, Man- del JL (1988) Genetic mapping of the Xq27-q28 region: new RFLP markers useful for diagnostic applications in fragile X and hemophilia B families. Am J Hum Genet 42 : 380-389

Conneally PM, Edwards JH, Kidd KK, Lalouel JM, Morton NE, Ott J, White R (1985) Report of the committee on methods of linkage analysis and reporting. (8th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 40: 356-359

Dahl N, HammarstrOm-Heeroma K, Ommen GB van, Pettersson U (1989) A polymorphic locus at Xq27-q28 detected by the probe U6.2 (DXS304). Nucleic Acids Res 17:2884

Heilig R, Oberl6 I, Arveiler B, Hanauer A, Vidaud M, Mandel JL (1988) Improved DNA markers for efficient analysis of fragile X families. Am J Med Genet 30 : 543-550

HoNer MH, Bergen AAB, Skraastad MI, Carpenter N J, Veenema H, Connor JM, Bakker E, Ommen GJB van, Pearson PL (1987) Efficient isolation of X chromosome specific single- copy probes from a cosmid library of a human X/hamster hy- brid-cell line: mapping of new probes close to the locus for X- linked mental retardation. Am J Hum Genet 40:312-328

Iancu T, Komlos L, Shabtay F, Elian E, Halbrecht I, B06k JA (1975) Incontinentia pigmenti. Clin Genet 7 : 103-110

Lathrop GM, Lalouel JM, Julier C, Ott J (1984) Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 81 : 3443-3446

Mandel JL, Willard HF, Nussbaum G, Romeo G, Puck JM, Davies KE (1989) Report of the committee on the genetic con- stitution of the X chromosome. Cytogenet Cell Genet 51 : 384- 437

Patterson M, Kenwrick S, Thibodeau S, Faulk K, Mattei MG, Mattei JF, Davies KE (1987) Mapping of DNA markers close to the fragile site on the human X chromosome at Xq27.3. Nucleic Acids Res 15 : 2639-2651

Sefiani A, Sinnett D, Abel L, Szpiro-Tapia S, Heuertz S, Craig I, Fraser N, Kruse TA, Frydman M, Peter MO, Schmutz JL, Gilgenkrantz S, Mitchell G, Frdzal J, M61anqon S, Lavergne L, Labuda D, Hors-Cayla MC (1988) Linkage studies do not confirm the cytogenetic location of incontinentia pigmenti on Xpll. Hum Genet 80: 282-286

Sefiani A, Abel L, Heuertz S, Sinnett D, Lavergne L, Labuda D, Hors-Cayla MC (1989a) The gene for incontinentia pigmenti is assigned to Xq28. Genomics 4: 427-429

Sefiani A, Heuertz S, Turleau C, Thibaud D, Grouchy J de, Hors- Cayla MC (1989b) Incontinentia pigmenti: Xq breakpoint is not the same in a case of r(x) and in X/autosome transloca- tions. Ann G6nEt (Paris) 32 : 149-151

Sinnett D, Sefiani A, Lavergne L, Mitchell G, M6lanqon S, Abel L, Gilgenkrantz S, Frdzal J, Hors-Cayla MC, Labuda D (1988) Linkage exclusion of incontinentia pigmenti (1P) locus from the short arm of X chromosome by RFLP markers. Genome 30 [Suppl] 1 : 233

Vincent A, Kretz C, Oberl6 I, Mandel JL (1989) A new poly- morphic marker very closely linked to DXS52 in the q28 region of the human X chromosome. Hum Genet 82 : 85-86