Sperm chromosome analysis to assess potential germ cell mosaicism
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Clinical Genetics 1988: 34: 85-89
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Sperm chromosome analysis to assess potential germ cell mosaicism B. BRAND RIFF^, L. A. GORDON~, B. B. CRAWFORD", s. A. SCHONBERG'. M. GOLAEI',s. CHARZAN', M. s. GOLBUS~ AND A. v. CARRANO' IBiomedical Sciences Division, University of California, Lawrence Livermore National Laboratory. Livermore, Wniversity of California, Berkeley, and 'University of California, San Francisco, Ca.: ' USA Human sperm chromosome complements were examined to assess the possibility that the conceptions of two children with the same chromosomal defect, del( 13)(q2Zq32), from chromosomally normal parents were the result of a paternal germ cell mosaicism. Analysis of 216 complements, both by quinacrine banding and by measuring the relative length of chromosome 13, showed no unusual subpopulation of 13s; this decreased the likelihood of a paternal origin of the deletion. Sperm chromosomal analysis is a useful adjunct to availab!e techniques in clinical genetics. When counseling cases involving either structural or numerical de now chromosome abnormality, it is of importance to discuss the possibility of germ cell line mosaicism as well as to offer prenatal diagnosis for subsequent pregnancies. Received 13 July 1987. accepted for publication 19 March 1988 Key wordr: gonadal mosaicism; human sperm chromosomes; recurrent chromosome aberrations
Fusion of human sperm with eggs from golden hamsters is at present the only noninvasive method available to study the chromosomes of human germ cells. The test has been used to establish baseline frequencies of structural and numerical abnormalities in sperm chromosomes of apparently normal men (Martin et al. 1983, Brandriff et al. 1984, 1985a, 1988, Kamiguchi & Mikamo 1986). Of clinical interest, the system is also being used to study meiotic segregation in reciprocal and Robertsonian translocations, and paracentric inversions (Brandriff et al. 1986, Balkan & Martin 1983a, b, Martin 1984). For the clinical geneticist, this method may prove to be a valuable tool in understanding the etiology of certain abnormalities. Here we report sperm chromosomal analysis in a case in which germ cell
mosaicism was one possible explanation for a recurrence of the identical interstitial deletion in offspring of parents with normal somatic karyotypes. Material and Methods
Ascertainment of Family This family was initially studied following the birth of a male child with multiple congenital abnormalities suggestive. of an unbalanced chromosome constitution; the spectrum of anomalies included growth retardation, hypertonia, microcephaly, and dysmorphic facial features, including epicanthal folds, upslanting palpebral fissures and small cupped ears. He was severely delayed in both mental and motor development. Cytogenetic analysis revealed the
B R A N D R I F F ET A L
karyotype 46,XY,del( 13)(q22q32). Both parents were also studied and a normal chromosomal constitution was confirmed in lymphocytes in each. In view of this finding, the couple was given a low recurrence risk, but offered prenatal diagnosis for future pregnancies. It was the parental preference not to have such studies performed. A subsequent pregnancy, following the birth of a normal child, was terminated when a lobar holoprosencephaly with one large ventricle was diagnosed by ultrasound. An amniotic fluid cell culture revealed a second occurrence of the same interstitial deletion, karyotype 46,XX,del( 13)(q22q32). Post mortem examination showed minor dysmorphic facial features despite the presence of holoprosencephaly documented on autopsy. Cytogenetic findings are summarized in Table 1. A pedigree with the respective chromosomes 13 is presented in Fig. 1. Sperm Chromosome Analysis To test the possibility that a paternal germ cell mosaicism was the origin of the identical deletions, the father (GCl) provided one semen sample and sperm chromosome analysis was performed. The sample was stored 1 day in TEST-yolk buffer at 4°C. Gamete coincubation, egg culture, and fixation were as described previously (Brandriff et al. 1984, 1985a, b). Table 1 Summary of cytogenetic findings in somatic tissues Subject' Tissue
Karyotype (no. of metaphases)
48,XY (52) 4 6 m (50) 46,XX (50) 46,XX (50) 46,XY,del(l3)(q22q32) (20)
1.2 2.1 2.2 2.3
Blood Skin Blood Skin Blood Not studied Amniocytes
Subject designation refers to pedigree position, Fig. 1.
Cytogenetic analyses were performed with quinacrine rather than Giemsa banding, as the number of complements available to be studied from the single sample required the more consistent although less detailed quinacnne stain. To assure that a subpopulation of shorter chromosomes 13 did not remain undetected, 130 complements were restained with Giemsa, photographed and the lengths of selected chromosomes were measured with a hand-held micrometer. The measured length of chromosome 13 in each spread was normalized to the 4 and 12 chromosomes from the same complement to compensate for variation in chromosome condensation among different sets. The same procedure was conducted on 65 cymplements from a historical control donor (donor A, Brandriff et al. 1984, 1988).
No interstitial deletions in 13q were identified in 2 16 quinacrine-banded sperm chromosome complements. Twenty-one cells (9.7%) contained other types of structural abnormalities (breaks, fragments and exchanges), of which 9 complements (4.2%) contained chromosome exchanges, either dicentrics or translocations. Although the frequency of cells with structural aberrations was within the range for normal men observed in this laboratory (1.9% to 14.5%), the proportion of cells with chromosome exchanges was the highest observed to date among 20 historical control men where the next highest frequency observed was 2.4%. Three of the cells containing exchanges had multiple structural aberrations, including multiple chromosome exchange events, a phenomenon infrequently observed in several other normal men studied (Brandriff et al. 1988). A single hypohaploid cell (0.5%) was within the range ob-
SPERM CHROMOSOMES AND GERM CELL MOSAICISM
Flg. 1. Recurrence of identical del(13) in sibs from normal Darents
served in our historical controls. No hyperhaploid complements were seen. Fig. 2 shows the histograms of relative lengths of chromosome 13/4, 13/12 and 12/ 4 (control) for donor GCl and a historical control donor A. Donor GCI and donor A showed similar distributions in all cases. The variation evident within each distribution was most likely due to unequal chromosome condensation within complements. There was no evidence of a subset of smaller chromosomes 13.
The occurrence of an identical interstitial chromosome deletion in two of three offspring of apparently chromosomally normal parents is unusual. It does not seem likely that these aberrations arose as independent de novo events. A possible cause might be germ cell mosaicism in one of the parents. Banding patterns of chromosomes 13 in the parents compared to those in the child and the fetus were not sufficiently dif-
Fig. 2. Distribution of relative lengths of chromosome 13 in sperm chromosomes.
BRANDRIFF ET A L
ferent to confirm the parental origin of the deleted 13's. We investigated the father's sperm chromosomes since these cells are relatively easy to obtain and artificial insemination with donor semen would have been a reasonable alternative to offer this couple in the case of a positive finding. However, results from 216 analyzed sperm chromosome complements produced no evidence in support of a paternal germ cell mosaicism and suggests this was probably not responsible for the recurrence of the deletion. In a similar case, an identical de now interstitial deletion in the long arm of chromosome 16 in two sisters has been reported by Hoo et al. (1985). The parents of these sisters had normal chromosomes, and no mosaicism could be detccted in lymphocytes or skin fibroblasts. The deleted chromosomes 16 could be established by heterochromatic polymorphisms to have been derived from the mother. These authors speculated that a paracentric inversion, undetectable by available cytogenetic techniques, might account for the recurrence of the same deletion. In fact, a maternal paracentric inversion in 13q was reported to be associated with a case of 13q- deletion in a child (Sparkes et al. 1979). However, as the authors pointed out, a deletion would be an unusual consequence of a paracentric inversion, because meiotic crossing over in these inversions leads to dicentric and acentric chromosomes. At least nine families have been reported in which there has been recurrence of de ~ O Y Otranslocation (21q;21q) Down syndrome. In three of the families there is direct evidence for the existence of parental somatic cell mosaicism in addition to the presumptive evidence for germ cell line mosaicism (Steinberg et al. 1984). Sperm chromosomal analysis can be used, as demonstrated here, as an adjunct to other tools currently available in clinical
cytogenetics. Even though our results were negative, they were useful in reducing the number of options to present to the parents, who were anxious to have another child. We also wish to point outthat even though the recurrence of apparently de now arising aberrations is extremely rare, such cases do exist, and should be taken into consideration when counseling clients. We believe that prenatal diagnosis should be offered for future pregnancies in all such cases, regardless of whether ascertainment was through a balanced or unbalanced karyotype. Acknowledgements
Work performed in part,,by the Lawrence Livermore National Laboratory under the auspices of the U.S.Department of Energy under contract number W-7405-ENG-48. References
Balkan, W. H. & R. H. Martin (1983a). Chromosome segregation into the spermatozoa of two men heterozygous for different rdprocaf translocations. Hum. Genet. 63, 345-348. Balkan, W. H. & R. H. Martin (1983b). Segregation of chromosomes into the spermatozoa of a man heterozygous for a 14;21 Robertsonian translocation. Am. J. Med. Genet. 16, 169-172. Brandriff, B., L. Gordon, L. Ashworth, G. Watchmaker, A. Carrano & A. Wyrobek (1984). Chromosomal abnormalities in human sperm: comparisons among four healthy men. Hum.Genet. 66, 193-210. Brandriff, B., L. Gordon, L. Ashworth, G. Watchmaker, D. H. Moore XI & A. V. Carrano (1985a). Chromosomes of human sperm: variability among normal individuals. Hum. Genet. 70, 18-24. Brandriff, B., L. Gordon & G. Watchmaker (198Sb). Human sperm chromosomes obtained ' from hamster eggs after capacitation in TESTyolk buffer. Gum. Res. 11. 253-259. Brandriff, B.. L. Gordon, L. K. Ashworth. V. Littman, G. Watchmaker & A. V. Carrano (1986). Cytogenetics of human sperm: meiotic
SPERM CHROMOSOMES A N D GERM CELL MOSAICISM segregation of two translocation camers. Am. J. Hum.Genet. 38, 197-208. Brandriff, B. F., L. A. Gordon, D. Moore I1 & A. Y Carrano (1988). An analysis of structural aberrations in human sperm chromosomes. Cytogenet. Cell Genet 47, 29-36. Hoo, 1. J., R. B. Lowry, C.C. Lin & R. H.A. Haslam (1985). Recurrent de novo interstitial deletion of 16q in two mentally retarded sisters. C h . Genet. 27, 420-425. Kamiguchi, Y. & K. Mikamo (1986). An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova. Am. 3. Hum. Genet. 38, 724-740. Martin, R. H.,W.Balkan, K. Bums, A. W.Rademaker, C. C. Lin & N. L. Rudd (1983). The chromosome constitution of 1000 human spermatozoa. Hum. Genet. 63, 305-309. Martin, R. H.(1984). Analysis of human sperm chromosome complements from a male hetero-
zygous for a reciprocal translocation t(l1;22)(q23;ql I). Clin. Genet. 25, 357-361. Sparkes, R. S., H. Muller & I. Klisak (1979). Retinoblastoma with 13q - chromosomal deletion associated with maternal paracentric inversion of I3q. Science 203, 1027-1 029. Steinberg, C., E. H. Zackai, D. L. Eunpu, M. T. Mennuti & B. S. Emanuel (1984). Recurrence rate for de now 21q21q translocation Down syndrome: a study of 1 12 families. Am. J. Med. Genet. 17, 523-530. Address: Brigitte I.: Brandriff. Ph.D. Biomedical Sciences Division, L-452 Lawrence Livermore Nationui Laboratory RO.Box 808 Livermore, CA 94SSO USA