Molecular definition of the 11p15.5 region involved in Beckwith-Wiedemann syndrome and probably in predisposition to adrenocortical carcinoma
Isabelle Henry1, Marc Jeanpierre2, Philippe Couillin1, Fernande Barichard1, Jean-Louis Serre1, Hubert Journel3, Annie Lamouroux4, Catherine Turleau5, Jean de Grouchy5, and Claudine Junien1
1INSERM U.73, Château de Longchamp, Bois de Boulogne, F-75016 Paris, France
2INSERM U.129, Hôpital Cochin, F-75014 Paris, France
3 Service de Pédiatrie, Hôpital Ponchaillou, F-35000 Rennes, France
4CNRS, Laboratoire de Neurobiologie, F-91190 Gif sur Yvette, France
5 INSERM U.173, Hôpital Necker-Enfants-Malades, F-75730 Paris, France
Summary. To define more precisely, in molecular terms, the region involved in Beckwith-Wiedemann syndrome (BWS), we have studied patients with BWS and a constitutional dupli- cation of 11p15 using eight 11p15 markers. In the first case with a de novo duplication and extra material on 11p, the region spanning pter to CALCA, excluded, was duplicated. In the second case, the rearrangement was characterized using somatic cell hybrids established with lymphocytes from the father who carried a balanced translocation t(11;18)(p15.4;p11.1). The breakpoint lay exactly in the same region. It could thus be in- ferred that the two sons, who were the first cases reported of BWS with dup11p15 and adrenocortical carcinoma (ADCC), carried a duplication similar to that observed in the first case. Together with evidence for specific somatic chromosomal events leading to loss of 11p15 alleles in familial cases of ADCC, it can be hypothesized that a gene involved in predisposition to ADCC maps to region 11p15.5.
Introduction
Trisomy 11p15 has been described in six patients (Waziri et al. 1983; Turleau et al. 1984; Journel et al. 1985) specifically con- sidered as having a Beckwith-Wiedemann syndrome (BWS) (Beckwith 1963; Wiedemann 1964). Retrospectively, features of the syndrome mainly characterized by exomphalos, macro- glossia, and gigantism were also seen to be present in the rare patients previously reported with trisomy 11p15 (Turleau and de Grouchy 1985). Cytogenetic forms can be distinguished mainly by mental retardation and congenital heart disease, however most cases are sporadic. Rare families with several affected individuals are known. Autosomal dominance with variable expressivity has been demonstrated by segregation analysis (Niikawa et al. 1986).
Special consideration must be given to the increased inci- dence (7.5%) of different types of tumours that were observed in patients with BWS, including nephroblastoma (59% of the tumours), adrenocortical carcinoma (15%), hepatoblastoma and rhabdomyosarcoma (Wiedemann 1983). Although adreno- cortical carcinoma is a rare childhood tumour (0.4% of malig-
nant tumours) it occurs with an increased frequency (40-fold) in patients with BWS. Adrenocortical carcinoma is also more frequently associated (12% versus 1%), in the same individual or in the same family, with tumours of the same type as those observed in BWS (Benaily et al. 1975).
The relationship between specific chromosomal abnor- malities and predisposition to childhood tumours has been ex- tensively demonstrated in retinoblastoma, with deletion of band 13q14, and in Wilms’ tumour with a deletion of band 11p13. In BWS, however, the relationship between the dupli- cation of 11p15 and the increased risk for several types of tu- mours is not as obvious since among the 12 cases of BWS with 11p15 duplication reported, only one had developed a tumour (Journel et al. 1985). The first step in understanding this re- lationship is to characterize, in molecular terms, the region the duplication of which can be responsible for BWS.
In the present report two such cases, one of which was the unique case reported presenting with a rare tumour, adreno- cortical carcinoma, have been studied. Using eight 11p15 markers, we have shown by gene copy number determination and analysis of somatic cell hybrids, that the region involved was similar in both patients.
Materials and methods
Individuals studied
With no associated tumour. Patient SAU has been previously described in detail (Turleau et al. 1984). Main features were: prematurity, macrosomia, macroglossia, abdominal hypotonia with ombilical hernia, and neonatal hypoglycaemia, all per- taining to BWS. Other features evocative of a chromosomal disorder were a malformative uropathy, a small atrial defect, microcephaly, and moderate mental retardation.
With associated tumour. Individual PEL carried a balanced re- ciprocal translocation, 46,XY,t(11;18)(p15.4;p11.1). His two sons, presenting with BWS, were carriers of a duplication for the distal part of chromosome 11 (Journel et al. 1985). Both were severely affected and died soon after birth. Adrenocorti- cal carcinoma was revealed at autopsy in the second child.
t (11;18) (p15.4;11.1)
dup(11 p15) BWS
dup{ 11p15) BWS ADCC
Since the child with adrenocortical carcinoma died shortly after birth, we could only examine DNA from the father to determine the region involved (Fig. 1).
Somatic cell hybrids
Somatic cell hybrids (PEL16, PEL40) were established by fus- ing a lymphoblastoid cell line from individual PEL and a mouse cell line Sp2/0-Ag14, using polyethylene glycol as a fusing agent. Hybrid clones were selected by indirect immunofluores- cence using antibodies recognizing antigens specific for 11p13 (MIC4 and MIC11) and for 11q (MDU1) (Van Heyningen et al. 1985; Goodfellow et al. 1984; Couillin et al. 1989). Karyo- typic analysis of PEL16 and PEL40 failed to reveal any sec- ondary rearrangement of chromosomes 11.
Cytogenetic analysis
Prometaphase karyotypes were obtained after thymidine syn- chronization followed by R-banding (RTHG), 5-bromodeo- xyuridine incorporation and fluorescence-photolysis-Giemsa staining (RTBG), or G-banding (Viegas-Péquignot and Dut- rillaux 1978).
Probes
Southern blots were hybridized to the following 11p probes: HRAS1 (c-Ha-ras-1), a 2.9-kb SacI genomic fragment (Chang et al. 1982); INS (insulin), a 9.0-kb HindIII genomic fragment (Bell et al. 1984); IGF2 (insulin-like growth factor II), a 1.1- kb EcoRI cDNA fragment (Dull et al. 1984); TH (tyrosine hydroxylase), a 0.8-kb PstI cDNA fragment (Craig et al. 1985); D11S12 (pADJ 762), a 5.5-kb EcoRI ramdon genomic fragment (Barker et al. 1984); HBB (ß globin), a 4.4-kb PstI genomic fragment (Lawn et al. 1978); PTH (parathyroid hor- mone), a 2.5-kb PstI-EcoRI cDNA fragment (Vasicek et al. 1983); CALCA (calcitonin), a 0.58-kb PstI cDNA fragment (Craig et al. 1982). MYC (c-myc), a 3.0-kb SacI genomic frag- ment (Dalla Favera et al. 1982) was used as a non-chromo- some 11 internal control in dosage analysis.
Southern blot experiments
DNA was prepared from lymphocytes, fibroblasts, hybrid cells or lymphoblastoid cell lines as described by Sanders- Haigh et al. (1980). Aliquots of 10-30 µg DNA were digested with restriction endonucleases according to the manufacturer’s recommendations. Restriction endonucleases were purchased from Promega Biotech, [32P]dCTP and the nick-translation kit from Amersham. DNA was electrophoresed and then trans- ferred to nitrocellulose or nylon membrane as described by Southern (1975). Hybridization with the 32P-labelled probes (specific activity, 2-6 × 108 cpm/ug) was carried out for 16h in
A
B
buffers containing 10% dextran sulphate as described by Wahl et al. 1979. For gene copy number determination the filters were freed of probe in alkali and rehybridized with a non- chromosome 11 probe, MYC, used as an internal control. The intensity of the hybridization signals was measured with a SEBIA densitometer. The values for normal control subjects and for patients were estimated through 3-7 independent de- terminations and through 3-8 independent determinations, respectively. The value of the ratio 11p probe versus non-11p internal control probe was calculated for each independent determination. Statistical analysis (Student’s t test) was per- formed using independent values obtained for normal indi- viduals and for the patients.
Results
Cytogenetic analysis
Patient SAU had extra material on the tip of one 11p. The banding pattern of this chromosome corresponds to a normal 11p as far as band p15.5 (Turleau et al. 1984) (Fig. 2A).
Individual PEL had a reciprocal balanced translocation t(11;18)(p15.4;p11.1). His two sons had inherited the der(18) and were therefore trisomic for part of 11p15 (Journel et al. 1985) (Fig. 2B).
Dosage of 11p markers in patient SAU
The DNA from patient SAU was analysed for gene copy number determination using eight 11p15 markers. The con- sensus gene order is the following: tel-HRAS1-INS/IGF2-TH- D11S12-HBB-CALCA/PTH (Kazazian and Junien 1987). Densitometer scanning profiles of the autoradiograms were performed and the computed peak area of the probe tested was compared with the computed peak area of the internal control. The values obtained for patient SAU were statisti- cally different from those obtained for normal individuals (t test). The ratios showed that patient SAU was trisomie for the distal region of 11p encompassing the linkage group HRAS1- INS/IGF2-TH-D11S12-HBB (Table 1, Fig.3) but had only two copies of the closely linked markers PTH and CALCA. Using a Student’s t-test with the values obtained for HRAS1 and HBB, we could infer that a duplication occurred for the proximal part of the segment (P<0.025). We could not, how- ever, exclude a triplication for the distal part (HRAS1-INS). We thus demonstrated that the region involved in BWS lies distal to the closely linked markers CALCA and PTH.
| HRAS1 | INS | IGF2 | TH | D11S12 | HBB | CALCA | PTH | |
|---|---|---|---|---|---|---|---|---|
| Mean ratios | 2.04 | 2.04 | 1.64 | 1.85 | 2.25 | 1.54 | 1.12 | 1.08 |
| N/C | (8/7) | (5/3) | (7/3) | (5/3) | (3/3) | (5/6) | (3/3) | (3/3) |
a
b
c
A 1 2 3B1 2 3 C 1
2
Parental origin of the de novo duplicated region
To determine the parental origin of the extra 11p15.5 material in patient SAU we compared the genomic pattern of patient SAU and her parents by analysis of ß globin restriction frag- ment length polymorphisms RFLPs revealed by AvalI. As shown in Fig. 3B the signal for allele 1, inherited from the father, is two times more intense than that for allele 2 inher- ited from the mother. As shown by densitometric tracings the patient inherited two identical alleles from her father (Fig. 3C).
Molecular characterization of somatic cells hybrids with t(11;18) (p15.4;p11.1)
Of 12 cases of BWS with dup11p15 reported, only one devel- oped a tumour. In this case (the son of case PEL reported here), adrenocortical carcinoma was revealed by autopsy (Journal et al. 1985). To characterize the 11p region involved in BWS with genetic predisposition to adrenocortical car-
16
40
SP2
PEL
40 16 SP2 PEL
4.4
2.9
2
6.7
2
2.0
HBB / Ava II
CALCA/ TaqI
cinoma, we established somatic cell hybrids with a lympho- blastoid cell line from the father who carried the balanced re- ciprocal translocation. As shown in Fig. 4, hybrid PEL16, which has retained the 11p- chromosome, is negative for HBB, positive for CALCA (allele 2 of 6.7kb) and positive for PTH (data not shown), whereas hybrid PEL40 which has re- tained the 18p+ derivative chromosome is positive for HBB (allele 2 of 2.0kb, and constant bands of 2.9 and 4.4 kb) and negative for PTH and CALCA. These concordant data preclude secondary rearrangements in hybrid PEL40 and PEL16 and indicate that the breakpoint in individual PEL unambiguously lay between HBB and CALCA/PTH, thus showing that the duplicated region in the sons of individual PEL involved exactly the same markers as in patient SAU.
Discussion
We have therefore further defined, with molecular probes, the region the duplication of which can be responsible for BWS. The region involved in the 11p rearrangements in our two patients extends from pter/HRAS1 to HBB inclusive. In the case of patient SAU, we could further identify the pater- nal origin of the duplication of 11p+. Since she inherited two identical alleles from her father it can be concluded that this duplication (either in tandem or in mirror) did not result from an unequal meiotic cross-over, but most likely from an un- equal mitotic recombination involving sister chromatids dur- ing mitosis in a germ cell line.
Constitutional chromosomal abnormalities in patients with different types of malignancy represent a breakthrough in localizing the genes for susceptibility to cancer. There are now several examples in which the localization proposed on the basis of cytogenetic findings could be further substantiated.
This was achieved either by family studies in retinoblastoma (Sparkes et al. 1983) and in familial adenomatous polyposis (Herrera et al. 1986; Bodmer et al. 1987), or by demonstrating a specific loss of alleles mapping to the same region in sporadic tumours of the same type, in nephroblastoma (Koufos et al. 1984), meningioma (Seizinger et al. 1987), and retinoblastoma (Cavenee et al. 1983).
Using DNA markers and somatic cell hybrids we have characterized the region, flanked by HRAS1 and HBB, that is duplicated in a BWS patient with adrenocortical carcinoma. The present report is the first to describe such a triple associa- tion: 11p15.5 duplication/BWS/adrenocortical carcinoma. Several observations may support this tentative assignment of a gene involved in predisposition to adrenocortical carcinoma to region 11p15: (1) We have recently observed a specific loss of heterozygosity limited to the same region in tumour cells from two unrelated familial cases of adrenocortical carcinoma (Henry et al. 1987). Other types of tumours were observed in these families, including breast cancer and rhabdomyosarcoma. Interestingly, as recently shown by mitotic recombination mapping, the same region is also involved in rhabdomyosar- coma and breast cancer (Scrable et al. 1987; Ali et al. 1987). (2) A loss of heterozygosity for HRAS1 was demonstrated in an adrenal adenoma from an adult with BWS (Hayward et al. 1987). (3) This tentative assignment of a gene involved in pre- disposition to adrenocortical carcinoma is also in agreement with the increased frequency of this tumour in BWS. In this respect, since a loss of heterozygosity for chromosome 11 has been found in the Wilms’ tumour cells from a BWS patient with a normal karyotype (Mannens et al. 1987), it would be important to demonstrate whether BWS patients with a termi- nal duplication of 11p15.5 also exhibit loss of heterozygosity for this region in their tumour cells. This would address the question of the possible significance of the causal relationship between a constitutional duplication of 11p15 and a somatic deletion of the same region in the tumour cells. Obviously, more cases of adrenocortical carcinoma or families will have to be studied to confirm the existence of a gene involved in ge- netic predisposition to adrenocortical carcinoma.
Acknowledgements. We thank Henriette Nicolas, Marie-Claude Grisard and Nicole Ravisé for technical assistance. Probes used in this study were kindly provided by A. Bank, R. White, H. M. Kronenberg, E.C. Hang, R. Pictet, A. Ullrich, I. McIntyre, and R. Gallo. This work was supported by grants from INSERM (Institut national de la santé et de la recherche médicale), CNRS (Centre national de la re- cherche scientifique ATP 955194), ARC (Association pour la Re- cherche sur le Cancer), Ligue Nationale Française contre le cancer, and Réseau de recherche clinique INSERM (J.de G.).
References
Ali IU, Lidereau R, Theillet C, Callahan R (1987) Reduction to homo- zygoty of genes on chromosome 11 in human breast neoplasia. Science 238: 185-188
Barker D, Holm T, White R (1984) A locus on chromosome 11p with multiple restriction site polymorphisms. Nature 303:72-73
Beckwith JB (1963) Extreme cytomegaly of the adrenal fetal cortex, omphalocele, hyperplasia of kidneys and pancreas, and leydig-cell hyperplasia: another syndrome? Western Society for Pediatric Research, Los Angeles, Calif, 1963
Bell GI, Merryweather JP, Sanchez-Pescador R, Stempien MM, Priestley L, Scott J, Rall LB (1984) Sequence of a cDNA clone en- coding human preproinsulin-like growth factor II. Nature 310: 775-777
Benaily M, Schweisguth O, Job JC (1975) Les tumeurs corticosur- rénales de l’enfant. Arch Fr Pediatr 23: 441-454
Bodmer WF, Bailey CJ, Bodmer J, Bussey HJR, Ellis A, Gorman P, Lucibello FC, Murday VA, Rider SH, Scambler P, Sheer D, Solomon E, Spurr NK (1987) Localization of the gene for famil- ial adenomatous polyposis on chromosome 5. Nature 328:614- 616
Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL (1983) Expression of re- cessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305 :779-784
Chang EH, Gonda MA, Ellis RW, Scolnick EM, Lowy DR (1982) Human genome contains four genes homologous to transforming genes of Harvey and Kirsten murine sarcoma viruses. Proc Natl Acad Sci USA 79:4848-4852
Couillin P, Azoulay M, Henry I, Ravisé N, Grisard MC, Jeanpierre C, Barichard F, Metezeau P, Candelier JJ, Lewis W, Van Hey- ningen V, Junien C (1989) Characterization of a panel of somatic cell hybrids for subregional mapping along 11p and within band 11p13. Subdivision of the WAGR complex region. Hum Genet 82 (in press)
Craig RK, Hall L, Edbrooke MR, Allison J, McIntyre I (1982) Partial nucleotide sequence of human calcitonin precursor mRNA nuc- leotide sequence of human calcitonin. Nature 295:345-347
Craig SP, Buckle VS, Craig IW, Lamouroux A, Mallet J (1985) Localisation of the human tyrosine hydroxylase gene to chromo- some 11p15. Cytogenet Cell Genet 40:610 (abstr)
Dalla Favera R, Gelmann EP, Martinotti S, Franckini G, Papas TS, Gallo RC, Wong Staal F (1982) Cloning and characterization of different human sequences related to the onc gene(v-myc) of avian myelocytomatosis virus (MC 29). Proc Natl Acad Sci USA 79:6497-6501
Dull JT, Gray A, Hayflick JS, Ullrich A (1984) Insulin-like growth factor II precursor gene organization in relation to insulin gene family. Nature 310:777-781
Goodfellow P, Tunnacliffe S, Pymb B, Solomon E, Walsh F, Knowles B, Andrews P, Parkar M, Povey S (1984) Multiple loci on chro- mosome 11 control expression of cell surface antigens. Cytogenet Cell Genet 37:481 (abstr)
Hayward NK, Little MH, Mortimer RH, Clouston WM, Smith PJ (1987) Generation of homozygosity at the c-Ha-ras-1 locus on chro- mosome 11p in an adrenal adenoma from an adult with Wiedemann- Beckwith syndrome. Cancer Genet Cytogenet 30: 127-132
Henry J, Huerre-Jeanpierre C, Azoulay M, Chaussain JL, Junien C (1987) A recessive oncogene for familial adrenocortical carcinoma (ADCC) maps to 11p. (9th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 46:629 (abstr)
Herrera L, Kakati S, Gibas L, Pietrzak E, Sandberg AA (1986) Brief clinical report: Gardner syndrome in a man with an interstitial de- letion of 5q. Am J Med Genet 25: 473-476
Journel H, Lucas J, Allaire C, Le Mée F, Defauve G, Lecornu G, Jouan H, Roussey M, Le Marec B (1985) Trisomy 11p15 and Beckwith-Wiedemann syndrome: report of two new cases. Ann Génét (Paris) 28:97-101
Kazazian HH, Junien C (1987) Report of the committee on the genetic constitution of chromosome 10, 11, 12. (9th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 46: 188-212
Koufos A, Hansen MF, Lampkin BC, Workman ML, Copeland NG, Jenkins NA, Cavenee WK (1984) Loss of alleles at loci on human chromosome 11 during genesis of Wilms’ tumour. Nature 309: 170-172
Lawn RM, Fritsch EF, Parker RC, Blacke G, Maniatis T (1978) The isolation and characterization of linked-ß-globin genes from a cloned library of human DNA. Cell 15: 1157-1174
Mannens M, Slater RM, Heyting C, Bliek J, Hoovens J, Bleeken- Wagemakers EM, Voûte PA, Coad N, Franks RR, Pearson PL (1987) Chromosome 11, Wilms’ tumour and associated congenital diseases. Cytogenet Cell Genet 46:655 (abstr)
Niikawa N, Ishikiriyama S, Takahashi S, Inagawa A, Tonoki H, Ohta Y, Hase N, Kamei T, Kajii T (1986) The Wiedemann-Beckwith syndrome: pedigree studies on five families with evidence for au- tosomal dominant inheritance with variable expression. Am J Med Genet 24: 41-55
Sanders-Haigh L, Anderson F, Francke U (1980) The ß globin gene is on the short arm of human chromosome 11. Nature 283: 397 — 400
Scrable HJ, Witte DP, Lampkin BC, Cavenee WK (1987) Chromo- somal localization of the human rhabdomyosarcoma locus by mitotic recombination mapping. Nature 329: 645-647
Seizinger BR, Monte S de la, Atkins L, Gusella JF, Martuza RL (1987) Molecular genetic approach to human meningioma: loss of genes on chromosome 22. Proc Natl Acad Sci USA 84: 5419-5423 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503- 517
Sparkes RS, Murphree AL, Lingua RW, Sparkes MC, Field LL, Funderburk SJ, Benedict WF (1983) Gene for hereditary retino- blastoma assigned to human chromosome 13 by linkage to esterase D. Science 219:971-973
Turleau C, Grouchy J de (1985) Beckwith-Wiedemann syndrome. Clinical comparison between patients with and without 11p15 trisomy. Ann Génét (Paris) 28:93-96
Turleau C, Grouchy J de, Chavin-Colin F, Martelli H, Voyer M, Charlas R (1984) Trisomy 11p15 and Beckwith-Wiedemann syn- drome. A report of two cases. Hum Genet 67:219-221
Van Heyningen V, Boyd PA, Seawright A, Fletcher JM, Fantes JA, Buckton KE, Spowart G, Porteous ND, Hill RE, Newton MS,
Hastie ND (1985) Molecular analysis of chromosome 11 deletions in aniridia-Wilms tumor syndrome. Proc Natl Acad Sci USA 82: 8592-8596
Vasicek TJ, McDeviti BE, Freeman MW, Fennick BJ, Hendy GN, Potts JT, Rich A, Kronenberg HM (1983) Nucleotide sequence of the human parathyroid hormone gene. Proc Natl Acad Sci USA 80:2127-2131
Viegas-Péquignot E, Dutrillaux B (1978) Une méthode simple pour obtenir des prométaphases. Ann Génét (Paris) 21: 121-125
Wahl GM, Stern M, Stark GR (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76:3683-3687
Waziri M, Patil SR, Hanson JW, Bartley JA (1983) Abnormality of chromosome 11 in patients with features of Beckwith-Wiedemann syndrome. J Pediatr 102: 873-876
Wiedemann HR (1964) Complexe malformatif familial avec hernie ombilicale et macroglossie. Un “syndrome nouveau”. J Génét Hum 13:223-232
Wiedemann HR (1983) Tumours and hemihypertrophy associated with Wiedemann-Beckwith syndrome. Eur J Pediatr 141: 129
Received July 12, 1988