Hum Genet (1992) 90:181-183

human .. genet, cs 9 Springer-Verlag 1992

Clinical case report

Multiple minute marker chromosomes derived from Y identified by FISH in an intersexual infant L. Diekmann 1, K. Palm 1, R. A. Pfeiffer 2, U. Trautmann 2, W. Scholz 3, E. Schroers 3, P. Vogt 4, and M. K/ihler 4 1St~idtische Kinderklinik, Dortmund, Federal Republic of Germany 2Institut for Humangenetik der Friedrich-Alexander Universitfit, Erlangen-Ntirnberg, Federal Republic of Germany 3Institut fiir Medizinische Genetik der Ruhr-Universitgt, Bochum, Federal Republic of Germany 4Institut far Humangenetik der Ruprecht-Karls-Universit~it, Heidelberg, Federal Republic of Germany Received January 28, 1992 / Revised April 24, 1992

Summary. C h r o m o s o m a l analysis in a child with ambiguous sex showed mosaicism of at least two cell lines with one or m o r e m a r k e r chromosomes or none at all. They were shown to be derived from the Y c h r o m o s o m e by fluorescent in situ hybridisation (FISH) using different D N A probes that cover parts of the long and the short arm.

Introduction Small m a r k e r c h r o m o s o m e s in cell lines with one X, and even, albeit exceptionally, two X chromosomes (Miro et al. 1982) in Turner-like and in intersexual individuals have been suspected of being structurally abnormal Y chromosomes, which m a y contain specific sex determining functions. Identification with the G i e m s a - l l technique (Kosztolanyi 1988) has only been partially conclusive. Real evidence, however, has finally become attainable by molecular (Disteche et al. 1984; Beverstock et al. 1989) and/or in situ hybridisation of c h r o m o s o m e specific D N A probes (Kozma et al. 1988; Crolla et al. 1989). Rearranged Y chromsomes are likely to be lost during mitosis, hence leaving 45,X/46,XY mosaicism. During morphogenesis unequal regional distribution of Y specific capacity is expected to cause mixed gonadal dysgenesis, which may be asymmetric, and subsequently abnormal genital differentiation. There is a considerable risk of gonadal malignancy. We report on the unusual finding of mosaicism of cells with 45,X and cells with one or more additional Y derived minute chromosomes [f(Y)] in an infant with ambiguous sex.

Correspondence to: R. A. Pfeiffer

Case report SP 910701 is the first child of healthy parents. Maternal and paternal age was 23 and 24 years respectively. The first pregnancy was aborted. Delivery at 38 weeks and pregancy were uneventful. Weight was 3.140g, length was 51 cm, HC was 33 cm. The child was referred because of ambiguous genitalia. No other malformations or abnormalities were found. There was scrotal hypospadias (Prader III) with sinus urogenitalis visualized by genitography. NMR tomography demonstrated a rudimentary uterus. Bilateral herniotomia revealed an ovarian like gonad and tube on the left side while no gonad was found on the opposite side. Histology showed prepuberal testis organization. Serum testosterone at 3 months of age was 22.0 ng/dl, 17-beta estradiol was less than i0 pg/ml. LH was increased from 7.0 up to 22 ng and FSH from 2.6 ng/ml up to 5.0 ng/dl after adequate stimulation.

Results

Cytogenetic and fluorescent in situ hybridisation (FISH) studies In metaphase spreads from phytohaemagglutinin ( P H A ) activated lymphocytes 45 chromosomes with only one X, or 46 to 49 chromosomes were counted a m o n g which one or several m a r k e r chromosomes were noticed (Table 1). One m a r k e r chromosome, permanently present, ex-

Table 1. Distribution of cell lines with various numbers of f(Y) Karyotype

Mitoses (n = 400)

Percentage

49,X,+4f(Y) 48,X, + 3f(Y) 47,X,+2f(Y) 46,X,+ f(Y) 45,X

288 54 10 2 46

72 14 2.5 0.5 11

182

Fig.1. Metaphase showing four Y-derived minute chromosomes and a nucleus wilh four specific signals after fluorescent in situ hybridisation (FISH) with the biotin-labeled Y library probe LLOYNSO1

hibited a tiny G-positive band and was definitely larger than the other minute ones, which were similar in size. In some metaphases the larger marker appeared as a ring. Occasionally the minute marker chromosomes were clustered or aligned. Association with acrocentric chromosomes was not seen. All marker chromosomes were Q-negative and C-negative. In fibroblasts cultured from various biopsies (fascia, both gonads, left tube) a similar mosaicism was noted. However the cell line with 45,X was proportionately increased in all samples. In cells containing one or more markers the large marker was invariably present. This cell line predominated in cultures from the left side (testicular gonad, tube) compared with the opposite side and with fascia (37.5% vs 20%). The paternal karyotype was 46,XY with a morphologically normal Y chromosome. In situ hybridisation of Y specific probes was performed using the Y library probe LLOYNSO1 (ATCC), the centromeric probe DYZ3, the probe DYZ5 (labelling Ypl 1.2) and cos Y6.65 (labelling Yq11.22-23). FISH was carried out using the protocol published by Lichter et al. (1988) with minor modifications. All marker chromosome were labelled with the library (Fig. 1), p-arm and centromere probes. As the q-arm probe did not give any signal the marker chromosomes should consist of pericentric chromatin with loss of the Yql 1.22-23 region (beside Yql2).

Discussion

The morphological abnormality of genitalia fits into the clinical manifestations of X O / X Y mosaicism (Knudtzon et al. 1987: Rosenberg et al. 1987). Differentiation of gonads is possibly asymmetric ("ovarian-testicular dys-

genesis") and caused either by regional diminution and possibly unequal distribution of sex determining factors. A similar case has been reported by Taillemite et al. (1978). From the configuration of the extra chromosome, but limited to conventional staining, r(Y) was concluded. Numerous observations of minute extra chromosomes considered to be structurally abnormal Y chromosomes (Surana et al. 1971; summarized by Fryns et al. 1985) have been published; however multiple such markers are rare. Btihler (1980) reported a girl with Turner features in which mosaicism of 45,X and cell lines with one, two and three small marker chromosomes were seen in lymphocytes and also in fibroblasts. The author was unable to identify the minute chromosomes. Since both gonads were streaks without evidence of dysgenetic testicular tissue the origin of the minute chromosomes could be X chromosomal but also autosomal as well. Fryns et al. (1978) reported on a male infant in which seven different cell lines were noted. No normal Y was found. In the majority of the lymphocytes a large and almost metacentric Y chromosome and in 23% a deleted nonfluorescent ~'fragment" were present. Moreover in a few cells two metacentric Ys were noticed. "Spot like chromatin material" in a few cells and one acentric fragment in single cells were found in a normal male infant. The extra chromsomes were thought to have been derived from an r(Y) (Ruthner and Golob 1974), Cells with one or even two r(Y) were observed in a cryptorchid boy while the 45,X cell line was present in only 3% (Mattei et al. 1979). A presumptive r(Y) chromosome, which was noted in all analysed metaphases from lymphocytes was found in an 18-year-old male with a normal male habitus. From molecular studies deletions of the long arm in interval 5 and of the short arm and in the pseudoautosomal region distal to MIC2 were suggested (Pohlschmidt et al. 1991). Multiple occurrence of minute marker chromosomes probably of various origin seems to be unusual. Indeed, Mascarello et al. (1987) who reported on a dysmorphic girl with 46,XX with variable numbers of small marker chromosomes did not refer to similar observations. The origin of the markers was not identified. Because of weak D A - D A P I fluorescence of some they might have been autosomal. If we assume that in the present case the large marker is a ring chromosome, an additional minute marker might have originated by abnormal mitotic segregation or after preceding sisterchromatid exchange (SCE) (Geard 1974). This marker should then have been doubled. Variable numbers could be explained by mitotic loss, but artefacts have also to be accounted for. Nevertheless the stability of a cell line with two or three minute markers is noteworthy. Selection against these cell lines seems to be minimal. However the distribution of different cell lines seems to differ in various tissues. The proportion of cells containing the marker lends support to the correlation of regional Y potential and testicular differentiation of the primary gonad. Whether the deletion of the normal paternal Y chromosome and secondary minute markers occurred in a

183 p r e m e i o t i c or in a postzygotic cell m i g h t be c o n c l u d e d from a study of s p e r m cells with Y specific D N A p r o b e s which, h o w e v e r , was n o t g r a n t e d .

Acknowledgements. We are obliged to Mrs. S. Reichardt for skilful assistance in FISH. Part of this work was supported by a grant from the Deutsche Forschungsgemeinschaft to P.V. (Vo403/1-3).

References Beverstock GC, Macfarlane JD, Veenema H, Hoekman H, Goodfellow PJ (1989) Y chromosome specific probes identify breakpoint in a 45/46,X,del(Y) (pter-q11.1) karyotype of an infertile male. J Med Genet 26 : 330-342 Bfihler E (1980) A synopsis of the human Y chromosome. Hum Genet 55 : 145-175 Crolla JA, Smith M, Docherty Z (1989) Identification and characterisation of a small marker chromosome using non-isotopic in situ hybridisation with X and Y specific probes. J Med Genet 26: 192-194 Dist6che C Luthy D, Haslam DB, Hoar D (1984) Prenatal identification of a deleted Y chromosome by cytogenetics and a Y-specific repetitive DNA probe. Hum Genet 67: 222-224 Fryns JP, Cassiman JJ, van den Berghe H (1978) Unusual in vivo rearrangements of the Y chromosome with mitotic instability in vitro. Hum Genet 44: 349-355 Fryns JP, Kleczkowska A, van den Berghe H (1985) Clinical manifestations of the Y chromosome deletions in man. In: The Y chromosome, part B. AR Liss, New York, pp 151-179 Geard CR (1974) Ring chromosomes and sister chromatid exchange. In: Tice RR, Hollaender (eds) Sister chromatid exchanges, Plenum Press, New York, London, pp 91-101

Knudtzon J, Aarskog D (1987) 45,X/46,XY mosaicism. A clinical review and report of ten cases. Eur J Pediatr 146 : 266-271 Kosztol~nyi G (1988) Giemsa-11 technique elucidating three structurally altered nonfluorescent Y chromosomes: r(Y), idic(Yp), dir tan dup (Yp) Ann G6n6t 31:235-240 Kozma R, Fear C, Adinolfi M (1988) Fluorescence in situ hybridization and Y ring chromosome. Hum Genet 80: 95-96 Lichter P, Cremer T, Borden J, Manuelidis L, Ward DC (1988) Delineation of individual human chromosomes in metaphase or interphase cells by in situ suppression hybridisation using recombinant DNA libraries. Hum Genet 80 : 224-234 Mascarello JTM, Jones MC, Chambers SR (1987) A patient with extreme variation in number and size of small marker chromosomes. Hum Genet 75 : 191-194 Mattei JF, Mattei MG, Lucas C, Giraud F (1979) Les anomalies de structure du chromosome Y, ~ propos de 10 observations. J G6not Hum 27 : 53-55 Miro R, Caballin MR, Coll MD, Marina S, Egozcue J (1982) An XX male with a 46,XX/47,XX,+Y(q12-qter)karyotype. Hum Genet 60: 82-84 Pohlschmidt M, Rappold G, Krause M, Ahlert D, Hosenfeld D, Weissenbach J, Gal A (1991) Ring Y chromosome: molecular characterization by DNA probes. Cytogenet Cell Genet 56: 65-68 Rosenberg C, Frota-Pessoa O, Vianna-Morgante AM, Chu TH (1987) Phenotypic spectrum of 45,X/46,XY individuals. Am J Med Genet 27: 553-559 Ruthner U, Golob E (1974) 45,X/45,X,ace(?Yp)/46,X,r(Y) in a phenotypically normal newborn male. Humangenetik 22 : 177180 Surana RB, Forbath P, Cohen PE (1971) Minute Y chromosome. Ann Genet 14:145-148 Taillemite JL, van den Akker J, Portmo MF, Le Porrier N, Roux Ch (1978) Mosaique 45,X/46,XY avec Y petit et non fluorescent. Ann Gdn~t 21 : 116-119

Multiple minute marker chromosomes derived from Y identified by FISH in an intersexual infant.

Chromosomal analysis in a child with ambiguous sex showed mosaicism of at least two cell lines with one or more marker chromosomes or none at all. The...
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