Clinical Genetics 1986: 30: 7 6 7 9

Paternal non-disjunction in a 46,XYl 47,XXY individual with a fragile 1 7 ~ 1 2 in

the mother N. TOMMERUP~. T. TONNESEN1 AND K. -H. GUSTAVSON*

'Department of Medical Genetics, The John F. Kennedy Institute, Glostrup, Denmark and 'Department of Clinical Genetics, Akademiska sjukhuset, Uppsala, Sweden

In a family where the mother carried a fragile site at 17~12, RFLP-analysis with the X-specific probe L1.28 showed that the 46,XY/47,XXY mosaicism detected in her Klinefelter son was due to a non-disjunctional event in paternal meiosis I, followed by a secondary loss of an X- chromosome by a mitotic non-disjunction. Thus, an association between the primary meiotic non-disjunction and the presence of the fragile site could be excluded.

Received 1 February, accepted for publication 15 April 1986

Key words: Fragile site 17p 12; 46,XY/47,XXY mosaic; RFLP-analysis.

The simultaneous observation of aneuplo- idy and heritable fragile sites in several cases has lead to the speculation whether heri- table fragile sites may predispose to chro- mosomal non-disjunction (Sutherland & Hecht 1985). Recently we have found a pos- sible association between heritable fragile sites and 2nd meiotic or early mitotic non- disjunction of chromosome 21 (Tom- merup & Mikkelsen, submitted). In that study banding heteromorphisms on chro- mosome 21 were used to clarify the type of non-disjunction responsible for the trisomic state. However, this is not feasible when the numerical aberration involves chromo- somes with less conspicious banding hetero- morphisms ~ like the X-chromosome.

A number of restriction-fragment-length- polymorphisms (RFLPs) have been ident- ified on the human X-chromosome (see HGM 8, 1985) and RFLP-analysis has re- cently been used for non-disjunction studies in abortuses with X-chromosome monoso-

my (Hassold et al. 1985). In this paper we describe the successful use of RFLP-analy- sis for non-disjunction study in a family with a mosaic Klinefelter individual whose mother carries a heritable fragile site at 17~12.

Material and Methods

Peripheral blood was obtained from a 16- year-old 46,XY/47,XXY Klinefelter indivi- dual, his mother who carries a fragile site at 1 7 ~ 1 2 and his father with a normal karyotype. The family has previously been described in detail (Gustavson & Kjessler 1978).

PHA-stimulated lymphocytes were grown in medium 199 + 5% newborn calf serum with and without the addition of Di- stamycin A (DA) (100 pg/m1/24 h) (Suther- land et al. 1984). Following routine chromo- some preparation 25 Q-banded metaphases were analysed from each culture for the

N O N - D I S J U N C T I O N ; 4 6 , X Y / 4 7 , X X Y & F R A ( 1 7 ) 77

presence of sex chromosomes and fra (17)

DNA was extracted from peripheral EDTA-blood and digested with a 4-fold ex- cess of Taq I under conditions recommend- ed by the supplier (Boehringer). Samples (10 pg) were subjected to electrophoresis on a 0.7% agarose gel and blotted onto a nitrocellulose-filter with 20X SSC (3.OM NaCl; 0.3M Na-citrate). The DNA probe L1.28 is a 1.35 kb EcoRI genomic DNA fragment cloned in bacteriophage PAT1 53 (Davies et al. 1983). The plasmid bound probe was nicktranslated to a specific ac- tivity of 6 x lo8 cpm/pg. Prehybridization was performed at 42°C for 6h in 45% for- mamide, 4X SSC, 0.1M Na-phosphate-buf- fer (pH 6.5), 5X Denhardt, 0.1% Na-pyro- phosphate, 0.1% SDS and 250 pg/ml de- natured salmon sperm DNA. Hybridization was performed for 16h at 42°C in 45% for- mamide, 4X SSC, 0.1M Na-phosphate-buf- fer (pH 6.5), lx Denhardt, 0.1% Na-pyro- phosphate, 0.1 YO SDS, 100 pg/ml denatured salmon sperm DNA and 10% Dextran sul- phate. After hybridization filters were washed for 10 min at 25°C in 2X SSC/0.5% SDS, then for 60 min at 25°C in 2X SSC/ 0.5% SDS and finally for 3h at 68°C in 1X SSC/O.5% SDS. Autoradiography was for 1-4 days.

( P W

Results

Cytogenetics: In all 50 cells analysed, two X-chromosomes were found in the proband along with a Y-chromosome. The fragile site at 1 7 ~ 1 2 was not seen even in the DA- treated culture. In the mother, the fragile site at 1 7 ~ 1 2 was found in 19/25 cells in the normal culture, and in 22/25 cells in the DA-treated one. The father had normal chromosomes.

DNA: Hybridization with the X-chromo- some specific sequence L1. 28, which detects

Taq Z RFLP alleles of 10.2 and 9.0 kb, im- mediately turned out to be informative. The mother was homozygous for the 10.2 kb allele, the father carried the 9.0 kb allele, whereas both alleles were present in the pro- band (Fig. 1). The proband has therefore inherited both a maternal and a paternal X- chromosome, and since he also carries a Y-chromosome, non-disjunction leading to the 47,XXY complement has occurred in paternal meiosis I. This, in turn, indicates that the 46,XY cell line detected shortly after birth must have arisen by a secondary mitotic non-disjunction.

We could not detect any reproducible dosage difference between the 10.2 and 9.0 kb allele in the proband which might have indicated that a significant proportion of DNA-containing cells in his peripheral blood still had a 46,XY karyotype. So, at the age of 16 years, both the cytogenetic

Fig. 1. Identification of a paternal X-chromosome (9.0 kb allele) (arrow) in a 46,XY/47,XXY Klinefelter mosaic by hybridization with the X-specific probe LI. 28 to Ta9 I digested DNA.

78 T O M M E R U P E T A L

and the hybridization data indicates that the 46,XY cell line has disappeared from his peripheral blood. Thus, we cannot tell which of the two X-chromosomes were lost by the mitotic non-disjunction.

Discussion

Since non-disjunction of the sex-chromo- somes has occurred in the paternal meiosis I , and since it is the mother who carries the fragile site at 17~12, we must conclude that there is no connection between the fragile site and the aneuploid state in her offspring. Still, a connection between the fragile site and the subsequent mitotic non-disjunction cannot be disproved in the present case. In this context it may be important that many of the reported cases with simultaneous ob- servation of aneuploidy and heritable fragi- le sites are mosaics (Tommerup, in prep- aration). Furthermore, an increased tend- ency for mitotic non-disjunction of the X- chromosome has recently been reported in peripheral lymphocytes from obligate car- riers of the fragile X chromosome (Brsn- dum Nielsen 1986).

While it is frequently assumed that the extra chromosome in mosaics arises from early mitotic non-disjunction, several data indicates that this is not so. Evidence from Xg blood groups in XYjXXY individuals (Race & Sanger 1975), advanced maternal age in mosaic trisomy 21 (Richards 1974) and analysis of chromosome heteromor- phisms (Hassold et al. 1984) suggests that the abnormal cell line in most mosaics are meiotic of origin; with respect to autosomal mosaics, the data also suggests that the vast majority are due to non-disjunction at ma- ternal meiosis I. Thus, the finding that the 47,XXY cell line in the present case is of meiotic origin is not unexpected.

Regarding the suggested association be- tween heritable fragile sites and aneuploid conditions more cases should be studied to

prove or disprove that this association is real. Since RFLPs have been detected on all human chromosomes (HGM 8 1985), the successful use of RFLP-analysis for assess- ment of X-chromosomal non-disjunction (Hassold et al. 1985, this paper) promise that this strategy will be possible in any aneuploid condition.

Acknowledgments

We thank Professor P. L. Pearson, Leiden for providing the L1. 28 probe. The skilled technical assistance of Marianne Askvold, Else Rsngaard Clausen and Connie Lund is greatly appreciated. This study was sup- ported by the Danish Medical Research Council (Grant 12-3808), by Danmark’s Sundhedsfond and by the Swedish Medical Research Council (Grant 5445).

References

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Davies, K. E., P. L. Pearson, P. S. Harper, J. M. Murray, T. O’Brien, M. Sarfarazi & R. Williamson (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.

Gustavson, K.-H. & B. Kjessler (1978). A Vari- ant Chromosome 17 in a Mother with Re- peated Abortions and a 46,XY/ 47,XXY Kli- nefelter Son. Ups. J . Med. Sci. 83, 119-122.

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Richards, B. W. (1974). Investigation of 142 mo- saic mongols and mosaic parents of mongols; cytogenetic analysis and maternal age at birth. J . Ment. Defic. Res. 18, 199-204.

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Address: Niels Tommerup The John E: Kennedy Institute GI. Landevej 7 DK-2600 Glostrup Denmark