Hum Genet (1990) 86:215-218
9 Springer-Verlag1990
Heterozygous expression of X-linked chondrodysplasia punctata Complex chromosome aberration including deletion of MIC2 and STS Doris Wiihrle 1, Gotthold Barbi l, Wolfgang Schulz 2, and Peter Steinbach 1 1Abteilung Klinische Genetik der Universit~it, Frauenstrasse 29, W-7900 Ulm, Federal Republic of Germany eInstitut f/it PhysiologischeChemie I der Universit~t, Moorenstrasse 5, W-4000Dfisseldorf, Federal Republic of Germany Received April 25, 1990/ Revised June 30, 1990 Summary. Two females showing partial expression of Xlinked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2-Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2-Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2-Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.
Introduction Chondrodysplasia punctata (CDP) represents a genetically heterogeneous group of bone dysplasias characterised by punctate epiphyseal calcifications in early infancy (McKusick 1986). Three main types have been reported: (1) a severe autosomal recessive rhizomelic type (Spranger et al. 1971), (2) a Conradi-Htinermann type, presumedly autosomal dominant (Spranger et al. 1970), and (3) an X-linked dominant CDP that is lethal in males (Happle et al. 1977). Additionally, several families with CDP have been reported suggesting an X-linked recessive pattern of inheritance. In some of these cases, bone Offprint requests to: D. Wtihrle
dysplasia was shown to be associated with small deletions at Xp22.3. The clinical features may, therefore, be explained by deletions of contiguous genes located at this band (Curry et al. 1984; Ballabio et al. 1988). The affected males showed disproportionately short statures with shortening of extremities and hypoplasia of distal phalanges. The face was dysmorphic, often with a flat nasal bridge. In the newborns, punctate calcifications were present in the epiphyses of cartilages and bones, including vertebral bodies, long bones, tarsal, wrist and ankle bones. The patients were psychomotorially retarded and later developed mental retardation. In some patients, especially in the mentally retarded, ichthyosis occurred because of steroid sulfatase (STS) deficiency (e.g. Curry et al. 1984). Female carders of X-linked recessive CDP had norreal gonadal function and apparently normal fertility. However, an increased frequency of spontaneous abortion was reported. This increase was associated with an alteration of the sex ratio in favour of girls, suggesting an increase of lethality in male fetuses. The phenotypic differences between carrier females and their non-carrier relatives were only subtle (Curry et al. 1984). The cartiers showed moderate short stature with a mean height of 154 cm (versus 166 cm in the controls) and mild shortening of the tibiae (Curry et al. 1984; Agematsu et al. 1988; Ballabio et al. 1988). Punctate epiphyseal calcifications, disappearing in older male patients, have not been detected among carrier females. We report here on a mother and her daughter who were both short statured and carded the same aberrant X chromosome. The aberration was the result of a complex rearrangement involving a deletion of band Xp22.32 as demonstrated by Southern blot analysis. This finding enabled us to establish the diagnosis of X-linked C D P in the absence of a male index patient in this family.
Case report The proposita is a 32-year-old female. Her puberty had been normal. Menarche was at 11 years followed by irregular menstrual cy-
216 cles. She was disproportionately short of stature suggesting that she could have hypochondrodysplasia. Her length was 148 cm with short limbs and a relatively long trunk. She also had a mild facial dysplasia including hypertelorism and a flat nose bridge. She was first examined at the age of 18 years. Chromosome analysis revealed an aberrant X chromosome with an enlarged short arm, and she was diagnosed as having Turner's syndrome as a result of an isochromosome of the long arm of the X chromosome. Since the proposita gave birth to a daughter, this chromosome was re-interpreted as resulting from a translocation involving the X and another unknown chromosome. The proposita's daughter was born after an uneventful pregnancy. Birth weight was 3280 g, length 49 cm. At the age of 8 4/12 years, she was 128 cm (25%ile) and weighed 37 kg (97%ile). Her arms and legs were relatively short. She had inherited the mother's aberrant X chromosome.
Materials and methods To elucidate the chromosome aberration, the proposita's X chromosomes were first re-examined after Q-, G-, and R-banding. Late replicating was analysed after BrdU-labelling in the late Sphase (Vogel et al. 1978). Metaphases were prepared from peripheral lymphocytes and cultured skin fibroblasts using standard methods. As the cytogenetic results suggested that the proposita could have a partially duplicated X chromosome, a molecular study with X-specific DNA probes was also performed. Genomic DNA was extracted from cultured fibroblasts of the proposita essentially following the protocol of Davies et al. (1986). Samples of DNA (10gg) were digested with restriction endonucleases, electrophoresed through 1% agarose gels, and blotted upon nitrocellulose membranes (Southern 1975). The membranes were hybridised with DNA probes radiolabelled by nick translation at specific activities of 100-150 laCi/lag. The following single copy anonymous DNA fragments, mapping to the short arm of the X chromosome, were used: pl9B (MIC2, Goodfellow et al. 1986), pGMGX9 (DXS237, GiUard et al. 1987), pD2 (DXS43, Aldridge et al. 1984), p99-6.1 (DXS41, Aldridge et al. 1984), pC7 (DXS28, Dorkins et al. 1985), pERT87-15 (DXS164, Kunkel et al. 1985), p754 (DXS84, HoNer et al. 1985), and pL1.28 (DXS7, Davies et al. 1983). Autoradiographic signals were quantified with an LKB laser densitometer (Ultroscan XL). To distinguish between the autoradiographic signals derived from the normal and the aberrant X chromosomes, DNA was also analysed from somatic cell hybrid clones that had lost the aberrant X but retained the normal X chromosome of the proposita. The cell hybrids were constructed by fusion of the proposita's fibroblasts with HPRT deficient mouse cells (RAG). Hybrid clones were isolated in hypoxanthine/aminopterin/thymidine (HAT) medium, which selects for the presence of the active human X chromosome.
Results and discussion In the cytogenetic analysis o f lymphocytes and fibroblasts f r o m the proposita, all cells e x a m i n e d s h o w e d an X chrom o s o m e with several additional bands on an abnormally large short a r m (Fig. 1). T h e a b e r r a n t X c h r o m o s o m e was late replicating. W h e r e a s the G - b a n d i n g p a t t e r n gave no evidence of the possible origin of the additional c h r o m o s o m e material, s o m e Q - b a n d e d c h r o m o s o m e s suggested that the a b e r r a n t X c h r o m o s o m e could have originated f r o m an inverse duplication. Structural Rb a n d i n g p a t t e r n s of sufficient resolution clearly d e m o n strated that the brightly fluorescing b a n d Xp22.3 was not detectable o n the a b e r r a n t X c h r o m o s o m e .
Fig.1. The proposita's X chromosomes stained by GAG-banding (G), QFQ-banding (Q) and RFA-banding (R). The aberrant X chromosome is shown on the right of each pair. Note that the brightly fluorescing terminal R band Xp22.3 (arrow) is missing on the aberrant X chromosome
Fig. 2. Examination of RFLPs at DNA marker loci on the short arm of the X chromosome (Xp). Genotypes of the proposita are ~ven in the right column. The physical locahsafion of the probes on Xp is also shown
Fig, 3, Southern blot analysis of genomic DNA from the proposita using various probes specific for Xp. Each probe gave two hybfidisation signals indicating that the proposita was heterozygous at these loci. In each lane, the upper bands correspond to allele 1, the lower bands to allele 2 of the respective marker. With the exception of L1.28, each probe gave bands of significantly different intensities
T o determine the extent of the distal deletion and to elucidate the origin o f the additional b a n d s in the aberrant X c h r o m o s o m e , the p r o p o s i t a ' s D N A was analysed on S o u t h e r n blots hybfidised with single c o p y p r o b e s m a p p i n g to p o l y m o r p h i c loci on the short a r m o f the X c h r o m o s o m e (Fig. 2). Surprisingly, all p r o b e s m a p p i n g to X p 2 2 . 2 - X p 1 1 . 3 were informative, the proposita being
217 Table 1. Densitometric quantitation of hybridisation signalsa of loci on segment Xpll.3-p22.2. The values obtained for alleles 1 and 2 of probes D2,754 and L1.28 were compared. The alleles 1 of the D2 (Xp22.2) and 754 loci (Xp21.1) were present in two copies, respectively (see ratio) Probe
Allele 1
Allele 2
Ratio
D2 754 L1.28
1.3 1.9 0.9
0.6 0.9 0.9
2.2 2.1 1.0
a Average of 3 measurements
heterozygous at all loci tested. With the exception of L1.28, all probes gave hybridisation signals of significantly different intensities. Examples are shown in Fig. 3. This was also demonstrated by densitometry. The more intense bands were apparently of double dosages, indicating that the sequences detected by probes D2 and 754 (Table 1) were present in three copies in the proposita's D N A and that the short arm of her aberrant X chromosome was partially duplicated. Since the duplication involved the loci DXS43 to DXS84, but not DXS7, it extended from Xp22.2 to Xp21.2. This duplication was, however, probably not responsible for the clinical features of the proposita since this part of the X chromosome largely undergoes inactivation. However, there could be a deletion involving band Xp22.3. Therefore, we tested the two loci MIC2 and STS located on Xp22.3-pter, a segment that usually escapes X chromosome inactivation. MIC2 encodes a cell-surface antigen that is recognised by the monoclonal antibody 12E7. The gene is located in the pseudoautosomal region of the sex chromosomes (Xp22.32-pter, Y p l l - p t e r ) (Darling et al. 1986). Both chromosomes carry homologous alleles, being exchanged in male meiosis. Probe 19B (MIC2) is a genomic fragment of the MIC2 gene detecting a TaqI polymorphism with two alleles. In the proposita's D N A , only a single hybridisation band of 3.2 kb was obtained corresponding to allele 2 of this marker. STS catalyses the hydrolysis of sulfate ester bonds in a number of substrates. The gene was mapped at Xp22.32 proximal to the MIC2 locus (Human Gene Mapping 8 1985). There is no functional STS homologue on the human Y chromosome. Probe GMGX9 (DXS237) is closely linked to STS (lod score 16.46 at Omax = 0.00; Wirth et al. 1988). This probe defines a high frequency biallelic H i n d I I I polymorphism (Gillard et al. 1987). The proposita was typed A1 at the GMGX9 locus. To allow for a densitometric detection of a deletion at the two loci tested, the 19B and GMGX9 membranes, which also contained D N A from appropriate controls, were simultaneously hybridised with the autosomal probe XV2c (D7S23, Estivill et al. 1987) to obtain a reference signal (Fig. 4). The results of the densitometric evaluation are summarised in Table 2. The hybridisation bands obtained with both probes in the proposita's D N A gave signal densities corresponding to 50% (19B) and 57% (GMGX9) of the values obtained from control females homozygous for the corresponding allele. It was also
Fig.4. Southern blot analysis using probe GMGX9 (DXS237) closely linked to STS at Xp22.3. Lanes 1, 2 proposita; lanes 3, 4 female control homozygous for allele 1; lanes 5, 6 heterozygous control. A1 allele 1, A2 allele 2 of the GMGX9 marker. R Reference signal of probe XV2c (D7S23) used for densitometric quantitation Table 2. Densitometric quanfitafion of hybridisafion signals of loci on Xp22.3. R, Reference signalobtained from probe XV2c (D7S23). The mean ratios A1/R and A2/R and the resulting copy numbers of loci are also given 3.2-kb band (A2) of probe 19B (MIC2) Proposita A2 hl-
.
A2 R A2/R Copies (A2)
1.4 1.5 0.9 0.8 0.7 1
.
Control A2-A2 .
.
2.4 2.4 0.7 0.7 1.4 2
4.0-kb (A1) and 2.0-kb + 1.5-kb bands (A2) of probe GMGX9 Proposita A1 A1 A2 R AI~ Copies(A1)
2.6 2.9 . . . 1.0 0.7 3.0 1
Control A1-A1
Control A1-A2
3.1
2.5 2.4 1.7 1.2 0.9 0.8 2.9 1
2.2
. 0.6 0.4 5.3 2
shown that the proposita's GMGX9 peak was very similar to the corresponding peak in the D N A of a heterozygous control female. It was therefore concluded that the proposita was hemizygous for the loci MIC2 (19B) and DXS237 (GMGX9, closely linked to STS) mapping on Xp22.3-pter. This is in accordance with the cytogenetic finding that band Xp22.3 is not detectable on the aberrant X chromosome. The aberrant X chromosome is probably deleted for the pseudoautosomal segment carrying the MIC2 gene and also a distal part of the heterologous region including the STS gene; it is interpreted as Xp21.1-Xp22.2::Xp22.2-Xqter. To elucidate how this complex chromosome aberration was generated, mouse human cell hybrids were constructed that retained the normal X chromosome of the proposita. Analysis of these hybrid clones for the markers from Xp21.1-Xp22.2 revealed that all those alleles that were present in a single dosage were carried on the normal X chromosome of the proposita. The duplicated X chromosome, therefore, had two identical alleles indicating that the aberration resulted from an intrachromo-
218 somal rearrangement. This rearrangement, however, was not a simple duplication. It involved a deletion of gene loci on the segment X p 2 2 . 3 - p t e r escaping X inactivation.
Conclusion Our cytogenetic and molecular genetic results confirm that the phenotype of the proposita and her daughter represent heterozygous expressions of the Curry-type of X-linked chondrodysplasia punctata probably as a result of a deletion of contiguous genes located on X p 2 2 . 3 pter. This type of chondrodysplasia punctata has usually shown a recessive pattern of inheritance. In the family reported here, the trait may, however, be classified as Xlinked dominant with male lethality because of a non-viable imbalance involving functional disomy of X p 2 1 . 3 p22.2 combined with nullisomy of X p 2 2 . 3 - p t e r .
Acknowledgements. We gratefully acknowledge X. Estivill, M.A. Ferguson-Smith, P.N. Goodfellow, L.M. Kunkel, and J.L. Mandel for their probes. We also thank ATCC for providing probes. This work was supported by the Deutsche Forschungsgemeinschaft
(DFG). References Agematsu K, Kenichi K, Hironori M, Yutaka N, Yasuo N, Taro A (1988) Chondrodysplasia punctata with X;Y translocation. Hum Genet 80:105-107 Aldridge J, Kunkel L, Bruns G, Tantravahi U, LaLande M, Brewster T, Moreau E, Wilson M, Bromley W, Roderick T, Latt SA (1984) A strategy to reveal high frequency RFLPs along the human X chromosome. Am 2 Hum Genet 36:546564 Ballabio A, Parenti G, Carrozzo R, Coppa G, Felici L, Migliori V, Silengo M, Franceschini P, Andria G (1988) X/Y translocation in a family with X-linked ichthyosis, chondrodysplasia punctara, and mental retardation: DNA analysis reveals deletion of the steroid sulphatase gene and translocation of its Y pseudogene. Clin Genet 34: 31-37 Curry C, Magenis RE, Brown M, Lanman JT Jr, Tsai J, O'Lague P, Goodfellow P, Mohandas T, Bergner EA, Shapiro IA (1984) Inherited chondrodysplasia punctata due to a deletion of the terminal short arm of an X chromosome. N Engl J Med 311: 1010-1015 Darling SM, Banting GS, Pyre B, Wolfe J, Goodfellow PN (1986) Cloning an expressed gene shared by the human sex chromosomes. Proc Natl Acad Sci USA 83:135-139 Davies KE, Pearson PL, Harper PS, Murray JM, O'Brien T, Sarfarazi M, Wflliamson R (1983) Linkage analysis of two cloned
DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the human X-chromosome. Nucleic Acids Res 8: 2303-2312 Davies LG, Dibner MD, Battey JF (1986) Basic methods in molecular biology. Elsevier, New York Amsterdam London Dorkins H, Junien C, Mandel JL, Wrogemann K, Moison JP, Martinez M, Old JM, Bundey S, Schwartz M, Carpenter N, Hill D, Lindlof M, Chapelle A de la, Pearson PL, Davies KE (1985) Segregation analysis of a marker localized Xp21.2Xp21.3 in Duchenne and Becker muscular dystrophy families. Hum Genet 71 : 103-107 Estivill X, Farrall M, Scambler PJ, Bell GM, Hawley K.MF, Lench NJ, Bates GP, Kruyer HC, Frederick PA, Stanier P, Watson EK, Williamson R, Wainwright BJ (1987) A candidate for the cystic fibrosis locus isolated by selection of methylation Bee islands. Nature 326: 840--845 Gillard EF, Affara NA, Yates JRW, Goudie DR, Lambert J, Aitken DA, Ferguson-Smith MA (1987) Deletion of a DNA sequence in eight of nine families with X-linked ichtliyosis (steroid sulphatase deficiency). Nucleic Acids Res 15 : 3977-3985 Goodfellow PJ, Darling SM, Thomas NS, Goodfellow PN (1986) A pseudoautosomal gene in man. Science 234: 740-743 Happle R, Matthass H-H, Macher E (1977) Sex-linked chondrodysplasia punctata? Clin Genet 11:73-76 Hofker MH, Wapenaar MC, Goor N, Bakker E, Ommen GJB van, Pearson PL (1985) Isolation of probes detecting restriction fragment length polymorphisms from X-chromosome specific libraries: potential use for diagnosis of Duchenne muscular dystrophy. Hum Genet 70:148-156 Human Gene Mapping 8 (1985) 8th International Workshop on Human Gene Mapping. Cytogenet Cell Genet 40, nos 1-4 Kunkel LM, Monaco AP, Middlesworth W, Ochs HID, Latt SA (1985) Specific cloning of DNA fragments absent from the DNA of a male patient with an X-chromosome deletion. Proc Natl Acad Sci USA 82: 4778-4782 McKusick VA (1986) Mendelian inheritance in man. Catalogs of autosomal dominant, autosomal recessive and X-linked phenotypes, 7th edn. Johns Hopkins University Press, Baltimore London Southern E (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503-517 Spranger JW, Bidder U, Voelz C (1970) Chondrodysplasia punctata (Chondrodysplasia calcificans) Typ Conradi-Htinerman. Fortschr Geb Rtntgenstr Nuklearmed 113: 717-727 Spranger JW, Opitz JM, Bibber U (1971) Heterogeneity of chondrodysplasia punctata. Humangenetik 11 : 190-212 Vogel W, Schempp W, Sigwarth I (1978) Comparison of thymidine, fluorodeoxyuridine, hydroxyurea, and methotrexate blocking at the G1/S phase transition of the cell cycle, studied by replication patterns. Hum Genet 45 : 183-198 Wirth B, Herrmann FH, Neugebauer M, Gillard EF, Wulff K, Stein C, Figura K yon, Ferguson-Smith MA, Gal A (1988) Linkage analysis in X-linked ichthyosis (steroid sulfatase deficiency). Hum Genet 80:191-192
9 Springer-Verlag1990
Heterozygous expression of X-linked chondrodysplasia punctata Complex chromosome aberration including deletion of MIC2 and STS Doris Wiihrle 1, Gotthold Barbi l, Wolfgang Schulz 2, and Peter Steinbach 1 1Abteilung Klinische Genetik der Universit~it, Frauenstrasse 29, W-7900 Ulm, Federal Republic of Germany eInstitut f/it PhysiologischeChemie I der Universit~t, Moorenstrasse 5, W-4000Dfisseldorf, Federal Republic of Germany Received April 25, 1990/ Revised June 30, 1990 Summary. Two females showing partial expression of Xlinked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2-Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2-Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2-Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.
Introduction Chondrodysplasia punctata (CDP) represents a genetically heterogeneous group of bone dysplasias characterised by punctate epiphyseal calcifications in early infancy (McKusick 1986). Three main types have been reported: (1) a severe autosomal recessive rhizomelic type (Spranger et al. 1971), (2) a Conradi-Htinermann type, presumedly autosomal dominant (Spranger et al. 1970), and (3) an X-linked dominant CDP that is lethal in males (Happle et al. 1977). Additionally, several families with CDP have been reported suggesting an X-linked recessive pattern of inheritance. In some of these cases, bone Offprint requests to: D. Wtihrle
dysplasia was shown to be associated with small deletions at Xp22.3. The clinical features may, therefore, be explained by deletions of contiguous genes located at this band (Curry et al. 1984; Ballabio et al. 1988). The affected males showed disproportionately short statures with shortening of extremities and hypoplasia of distal phalanges. The face was dysmorphic, often with a flat nasal bridge. In the newborns, punctate calcifications were present in the epiphyses of cartilages and bones, including vertebral bodies, long bones, tarsal, wrist and ankle bones. The patients were psychomotorially retarded and later developed mental retardation. In some patients, especially in the mentally retarded, ichthyosis occurred because of steroid sulfatase (STS) deficiency (e.g. Curry et al. 1984). Female carders of X-linked recessive CDP had norreal gonadal function and apparently normal fertility. However, an increased frequency of spontaneous abortion was reported. This increase was associated with an alteration of the sex ratio in favour of girls, suggesting an increase of lethality in male fetuses. The phenotypic differences between carrier females and their non-carrier relatives were only subtle (Curry et al. 1984). The cartiers showed moderate short stature with a mean height of 154 cm (versus 166 cm in the controls) and mild shortening of the tibiae (Curry et al. 1984; Agematsu et al. 1988; Ballabio et al. 1988). Punctate epiphyseal calcifications, disappearing in older male patients, have not been detected among carrier females. We report here on a mother and her daughter who were both short statured and carded the same aberrant X chromosome. The aberration was the result of a complex rearrangement involving a deletion of band Xp22.32 as demonstrated by Southern blot analysis. This finding enabled us to establish the diagnosis of X-linked C D P in the absence of a male index patient in this family.
Case report The proposita is a 32-year-old female. Her puberty had been normal. Menarche was at 11 years followed by irregular menstrual cy-
216 cles. She was disproportionately short of stature suggesting that she could have hypochondrodysplasia. Her length was 148 cm with short limbs and a relatively long trunk. She also had a mild facial dysplasia including hypertelorism and a flat nose bridge. She was first examined at the age of 18 years. Chromosome analysis revealed an aberrant X chromosome with an enlarged short arm, and she was diagnosed as having Turner's syndrome as a result of an isochromosome of the long arm of the X chromosome. Since the proposita gave birth to a daughter, this chromosome was re-interpreted as resulting from a translocation involving the X and another unknown chromosome. The proposita's daughter was born after an uneventful pregnancy. Birth weight was 3280 g, length 49 cm. At the age of 8 4/12 years, she was 128 cm (25%ile) and weighed 37 kg (97%ile). Her arms and legs were relatively short. She had inherited the mother's aberrant X chromosome.
Materials and methods To elucidate the chromosome aberration, the proposita's X chromosomes were first re-examined after Q-, G-, and R-banding. Late replicating was analysed after BrdU-labelling in the late Sphase (Vogel et al. 1978). Metaphases were prepared from peripheral lymphocytes and cultured skin fibroblasts using standard methods. As the cytogenetic results suggested that the proposita could have a partially duplicated X chromosome, a molecular study with X-specific DNA probes was also performed. Genomic DNA was extracted from cultured fibroblasts of the proposita essentially following the protocol of Davies et al. (1986). Samples of DNA (10gg) were digested with restriction endonucleases, electrophoresed through 1% agarose gels, and blotted upon nitrocellulose membranes (Southern 1975). The membranes were hybridised with DNA probes radiolabelled by nick translation at specific activities of 100-150 laCi/lag. The following single copy anonymous DNA fragments, mapping to the short arm of the X chromosome, were used: pl9B (MIC2, Goodfellow et al. 1986), pGMGX9 (DXS237, GiUard et al. 1987), pD2 (DXS43, Aldridge et al. 1984), p99-6.1 (DXS41, Aldridge et al. 1984), pC7 (DXS28, Dorkins et al. 1985), pERT87-15 (DXS164, Kunkel et al. 1985), p754 (DXS84, HoNer et al. 1985), and pL1.28 (DXS7, Davies et al. 1983). Autoradiographic signals were quantified with an LKB laser densitometer (Ultroscan XL). To distinguish between the autoradiographic signals derived from the normal and the aberrant X chromosomes, DNA was also analysed from somatic cell hybrid clones that had lost the aberrant X but retained the normal X chromosome of the proposita. The cell hybrids were constructed by fusion of the proposita's fibroblasts with HPRT deficient mouse cells (RAG). Hybrid clones were isolated in hypoxanthine/aminopterin/thymidine (HAT) medium, which selects for the presence of the active human X chromosome.
Results and discussion In the cytogenetic analysis o f lymphocytes and fibroblasts f r o m the proposita, all cells e x a m i n e d s h o w e d an X chrom o s o m e with several additional bands on an abnormally large short a r m (Fig. 1). T h e a b e r r a n t X c h r o m o s o m e was late replicating. W h e r e a s the G - b a n d i n g p a t t e r n gave no evidence of the possible origin of the additional c h r o m o s o m e material, s o m e Q - b a n d e d c h r o m o s o m e s suggested that the a b e r r a n t X c h r o m o s o m e could have originated f r o m an inverse duplication. Structural Rb a n d i n g p a t t e r n s of sufficient resolution clearly d e m o n strated that the brightly fluorescing b a n d Xp22.3 was not detectable o n the a b e r r a n t X c h r o m o s o m e .
Fig.1. The proposita's X chromosomes stained by GAG-banding (G), QFQ-banding (Q) and RFA-banding (R). The aberrant X chromosome is shown on the right of each pair. Note that the brightly fluorescing terminal R band Xp22.3 (arrow) is missing on the aberrant X chromosome
Fig. 2. Examination of RFLPs at DNA marker loci on the short arm of the X chromosome (Xp). Genotypes of the proposita are ~ven in the right column. The physical locahsafion of the probes on Xp is also shown
Fig, 3, Southern blot analysis of genomic DNA from the proposita using various probes specific for Xp. Each probe gave two hybfidisation signals indicating that the proposita was heterozygous at these loci. In each lane, the upper bands correspond to allele 1, the lower bands to allele 2 of the respective marker. With the exception of L1.28, each probe gave bands of significantly different intensities
T o determine the extent of the distal deletion and to elucidate the origin o f the additional b a n d s in the aberrant X c h r o m o s o m e , the p r o p o s i t a ' s D N A was analysed on S o u t h e r n blots hybfidised with single c o p y p r o b e s m a p p i n g to p o l y m o r p h i c loci on the short a r m o f the X c h r o m o s o m e (Fig. 2). Surprisingly, all p r o b e s m a p p i n g to X p 2 2 . 2 - X p 1 1 . 3 were informative, the proposita being
217 Table 1. Densitometric quantitation of hybridisation signalsa of loci on segment Xpll.3-p22.2. The values obtained for alleles 1 and 2 of probes D2,754 and L1.28 were compared. The alleles 1 of the D2 (Xp22.2) and 754 loci (Xp21.1) were present in two copies, respectively (see ratio) Probe
Allele 1
Allele 2
Ratio
D2 754 L1.28
1.3 1.9 0.9
0.6 0.9 0.9
2.2 2.1 1.0
a Average of 3 measurements
heterozygous at all loci tested. With the exception of L1.28, all probes gave hybridisation signals of significantly different intensities. Examples are shown in Fig. 3. This was also demonstrated by densitometry. The more intense bands were apparently of double dosages, indicating that the sequences detected by probes D2 and 754 (Table 1) were present in three copies in the proposita's D N A and that the short arm of her aberrant X chromosome was partially duplicated. Since the duplication involved the loci DXS43 to DXS84, but not DXS7, it extended from Xp22.2 to Xp21.2. This duplication was, however, probably not responsible for the clinical features of the proposita since this part of the X chromosome largely undergoes inactivation. However, there could be a deletion involving band Xp22.3. Therefore, we tested the two loci MIC2 and STS located on Xp22.3-pter, a segment that usually escapes X chromosome inactivation. MIC2 encodes a cell-surface antigen that is recognised by the monoclonal antibody 12E7. The gene is located in the pseudoautosomal region of the sex chromosomes (Xp22.32-pter, Y p l l - p t e r ) (Darling et al. 1986). Both chromosomes carry homologous alleles, being exchanged in male meiosis. Probe 19B (MIC2) is a genomic fragment of the MIC2 gene detecting a TaqI polymorphism with two alleles. In the proposita's D N A , only a single hybridisation band of 3.2 kb was obtained corresponding to allele 2 of this marker. STS catalyses the hydrolysis of sulfate ester bonds in a number of substrates. The gene was mapped at Xp22.32 proximal to the MIC2 locus (Human Gene Mapping 8 1985). There is no functional STS homologue on the human Y chromosome. Probe GMGX9 (DXS237) is closely linked to STS (lod score 16.46 at Omax = 0.00; Wirth et al. 1988). This probe defines a high frequency biallelic H i n d I I I polymorphism (Gillard et al. 1987). The proposita was typed A1 at the GMGX9 locus. To allow for a densitometric detection of a deletion at the two loci tested, the 19B and GMGX9 membranes, which also contained D N A from appropriate controls, were simultaneously hybridised with the autosomal probe XV2c (D7S23, Estivill et al. 1987) to obtain a reference signal (Fig. 4). The results of the densitometric evaluation are summarised in Table 2. The hybridisation bands obtained with both probes in the proposita's D N A gave signal densities corresponding to 50% (19B) and 57% (GMGX9) of the values obtained from control females homozygous for the corresponding allele. It was also
Fig.4. Southern blot analysis using probe GMGX9 (DXS237) closely linked to STS at Xp22.3. Lanes 1, 2 proposita; lanes 3, 4 female control homozygous for allele 1; lanes 5, 6 heterozygous control. A1 allele 1, A2 allele 2 of the GMGX9 marker. R Reference signal of probe XV2c (D7S23) used for densitometric quantitation Table 2. Densitometric quanfitafion of hybridisafion signals of loci on Xp22.3. R, Reference signalobtained from probe XV2c (D7S23). The mean ratios A1/R and A2/R and the resulting copy numbers of loci are also given 3.2-kb band (A2) of probe 19B (MIC2) Proposita A2 hl-
.
A2 R A2/R Copies (A2)
1.4 1.5 0.9 0.8 0.7 1
.
Control A2-A2 .
.
2.4 2.4 0.7 0.7 1.4 2
4.0-kb (A1) and 2.0-kb + 1.5-kb bands (A2) of probe GMGX9 Proposita A1 A1 A2 R AI~ Copies(A1)
2.6 2.9 . . . 1.0 0.7 3.0 1
Control A1-A1
Control A1-A2
3.1
2.5 2.4 1.7 1.2 0.9 0.8 2.9 1
2.2
. 0.6 0.4 5.3 2
shown that the proposita's GMGX9 peak was very similar to the corresponding peak in the D N A of a heterozygous control female. It was therefore concluded that the proposita was hemizygous for the loci MIC2 (19B) and DXS237 (GMGX9, closely linked to STS) mapping on Xp22.3-pter. This is in accordance with the cytogenetic finding that band Xp22.3 is not detectable on the aberrant X chromosome. The aberrant X chromosome is probably deleted for the pseudoautosomal segment carrying the MIC2 gene and also a distal part of the heterologous region including the STS gene; it is interpreted as Xp21.1-Xp22.2::Xp22.2-Xqter. To elucidate how this complex chromosome aberration was generated, mouse human cell hybrids were constructed that retained the normal X chromosome of the proposita. Analysis of these hybrid clones for the markers from Xp21.1-Xp22.2 revealed that all those alleles that were present in a single dosage were carried on the normal X chromosome of the proposita. The duplicated X chromosome, therefore, had two identical alleles indicating that the aberration resulted from an intrachromo-
218 somal rearrangement. This rearrangement, however, was not a simple duplication. It involved a deletion of gene loci on the segment X p 2 2 . 3 - p t e r escaping X inactivation.
Conclusion Our cytogenetic and molecular genetic results confirm that the phenotype of the proposita and her daughter represent heterozygous expressions of the Curry-type of X-linked chondrodysplasia punctata probably as a result of a deletion of contiguous genes located on X p 2 2 . 3 pter. This type of chondrodysplasia punctata has usually shown a recessive pattern of inheritance. In the family reported here, the trait may, however, be classified as Xlinked dominant with male lethality because of a non-viable imbalance involving functional disomy of X p 2 1 . 3 p22.2 combined with nullisomy of X p 2 2 . 3 - p t e r .
Acknowledgements. We gratefully acknowledge X. Estivill, M.A. Ferguson-Smith, P.N. Goodfellow, L.M. Kunkel, and J.L. Mandel for their probes. We also thank ATCC for providing probes. This work was supported by the Deutsche Forschungsgemeinschaft
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