Disappearing Trisomy 8 Mosaicism

Disappearing Trisomy 8 Mosaicism* HON FONG LOUIE MARK, Ph.D.t and JO-ANN BLAYMORE BIER, M.D.$ fLaboratory of Cytogenetics, FISH, and Genotoxicology and fChild Development Center, Rhode Island Hospital and Brown University School of Medicine, Providence, RI 02903 ABSTRACT A case is presented of a patient with disappearing trisomy 8 mosaicism initially thought to have stigmata of the fragile X syndrome. This case is interesting for two reasons. First, it demonstrates the occurrence of “disappearing mosaicism,” a phe nomenon first described by LaMarche et al,1 in 1967. Our patient, initially studied in 1991 by two laboratories and found to be mosaic for chromosome 8 trisomy, was apparently normal by both GTG-banding and fluorescent in situ hybridization (FISH) when studied in 1996. Second, this case further underscores the fact that except under special circumstances, it is important that GTG-banding analysis be performed so that the entire human genome be examined in addition to scoring for the fragile X mutation on Xq27.3. In a recent review of the existing database at Rhode Island Hospital on chromosomal abnormalities found in patients referred because of a question of the fragile X syndrome during the period from January 1, 1990 to June 30, 1995, it was found that the frequency of other chromosomal abnormalities in patients referred because of a question of fragile X syndrome equaled or exceeded that of patients found to be positive for fragile X. Our figures, consistent with those reported in the literature, underscore the value of routine karyotyping in this population of patients. Introduction was reviewed to establish the true frequency of chromosomal abnormalities found in patients referred for fragile X syndrome at our center.2 Much emphasis in the recent literature has In this report, a representative case of a patient focused on the occasional findings of fragile X from this population is presented in detail. syndrome in the pediatric population with This patient is also interesting from another delayed development, while other clinical perspective, in that he provides an example of findings in this same patient population have an under-recognized phenomenon, disappear been somewhat neglected. In a previous study, ing mosaicism, first described in 1967. the existing database at Rhode Island Hospital The patient, referred to us initially to rule out fragile X, was found to have a low percent age mosaicism for trisomy 8. Features of the * Send reprint requests to: Hon Fong Louie Mark, trisomy 8 syndrome include long facies, promi Ph.D., Director, Laboratory of Cytogenetics, FISH, and Genotoxicology, Rhode Island Hospital, 593 Eddy Street, nent forehead, large ears, transverse palmar Providence, RI 02903. crease, and mental retardation, which overlap 293 0091-7370/97/0700-0293 $01.50 © Institute for Clinical Science, Inc. 294 MARK AND BIER those of the fragile X phenotype.3 Other fea The global developmental delay, combination of an unusual cognitive profile, and many of the behaviors dem tures of the trisomy 8 syndrome, such as crypt onstrated during his evaluation, were consistent with the orchidism, skeletal malformations and micro diagnosis of pervasive developmental disorder (PDD). gnathia, are not generally present in the fragile Given his relative macrocephaly, his minor auricle anoma lies, his low muscle tone and ligamentous laxity, as well as X syndrome. Sometimes the only significant his unusual behavioral characteristics, developmental findings in trisomy 8 mosaicism are mental delay and diagnosis of PDD, chromosome studies were retardation or psychological problems.4 requested in order to rule out the fragile X syndrome. Chromosomal analysis revealed mosaic trisomy 8 at this age. Case Report A repeat physical examination was done in October of 1995 when the patient was six-and-three-quarter-years old. His behavior during this evaluation was significant for The patient (ME, figure 1) a two-and-one-half-year-old occasional mouthing of objects, fair eye contact, perse- male, was referred to the Rhode Island Hospital Child verative behaviors, communication and socialization diffi Development Center in June of 1991 because of develop culties, all consistent with the diagnosis of autism. Non mental delay. He was bom prematurely to a 28 year old verbal cognitive testing results showed skills at the 4 years, gravida 1, para 0 female by cesarian section followed by a 9 months level. His mother reported ongoing language, 33-week pregnancy, which was complicated by cigarette social interaction, and behavioral concerns. Physical smoking, hypertension and pre-eclampsia. Birthweight examination revealed that his face was mildly narrow, but was 1900 grams. After a relatively benign course in the overall there were no significant dysmorphic features. A intensive care nursery, he was discharged. repeat peripheral blood specimen was obtained in August When seen at two-and-one-half-years, the patient’s of 1996 for the purpose of assessing the status of his tri mother reported that he had been in good health gener somy 8 mosaicism. A buccal smear was used to substitute ally, with the exception of frequent episodes of otitis for a more invasive means of obtaining another tissue, as media. Developmental milestones were delayed, as the study of other tissues is the recommended approach for patient crawled at 1 year, did not stand or walk indepen cases of mosaicism. dently until 2 years, and had only recently begun to use single words. He was only beginning to spoon feed himself at the time of the evaluation. The patient’s physical exami nation at two-and-one-half-years was remarkable for a Materials and Methods large head size in proportion to his body, an unusual hair whorl pattern, ears of normal size, but with mildly under C onventional C y to g en etic T ec h n iq u e s developed antehelices, mildly low overall muscle tone, and ligamentous laxity. The patient had an unusual cognitive profile with overall functioning at approximately the 19 The peripheral blood specimens were col months level, but with visual motor skills at the 30 month lected in heparinized tubes. Lymphocytes level. He had poor eye contact and some self-stimulatory, stereotypic behaviors, and showed limited interest in social were cultured for 72 hours with phytohemag contact with others. He showed a lack of communicative glutinin using standard techniques.5 6-7 Fragile intent, and expressive and receptive language skills were X site expression in the 1991 specimen was tested to be at the 12 to 15 month level and 18 to 20 month induced by one or more standard treatment level, respectively. protocols upon request from the referring phy sician. Scoring of other sites besides the fragile X site at Xq27.3 was performed to provide an internal control. Briefly, fluorodeoxyuridine (FUdR, 10 |xl of 10' ' M final concentration),* methotrexate (MTX, 0.1 ml of 10~5 M final concentration)* and Trimethoprim (TMP, 0.1 ml of 13 mg^mL final concentration)* were added to each 5 ml of culture 72 hours after the initiation of the culture. Twenty-one hours after the addition of the induction agents, 32 |j l 1 of stock Colcemid (10 |xg/mL)t were added. Three hours later (or 24 hours after the

* Sigma Chemical Co., St. Louis, MO. F igure 1. Recent photograph of the patient, M.E. t Gibco Life Technologies, Inc., Grand Island, NY. DISAPPEARING TRISOMY 8 MOSAICISM 295 addition of additives), cells were harvested specific human chromosomes and are par according to a modification of the methods of ticularly suited for the purpose of chromo Moorhead et al.5 The GTG-banding analyses some enumeration. were simultaneously performed to rule out Briefly, slides containing metaphase chro chromosome rearrangements.8,9 mosome spreads were pre-treated using When fragile X cytogenetic testing was per RNAse and 2XSSC, dehydrated in a cold- formed, at least 50 percent of the cells scored graded ethanol series, denatured in formamide for fragile Xq27.3 were derived from cultures at 70° to 71°C and hybridized overnight at in which fragile site expression was induced by 37°C in a humid chamber. On the following anti-metabolites (eg, 5-fluorodeoxyuridine and day, the coverslips were removed, and the methotrexate) in accordance with various slides were post-washed under stringent con guidelines, such as those from the Pacific ditions using 65% formamide and 2XSSC Northwest Regional Genetics Group. The at 43°C. presence of the fragile site in putative cases For detection, slides were treated with 50 (jd was confirmed by GTG-banding. When a frag of fluorescein-labeled avidin at 37°C for 20 ile X study was performed, it was considered minutes. The interphase nuclei were counter positive when at least 4 percent of metaphases stained with 20 |xl of a 1:1 dilution of pro- in the cytogenetic analysis contained a fragile pidium iodide/antifade for a final concentra X chromosome. Borderline cases were usually tion of 0.3 |jLg/ml per slide. The slides were repeated and/or confirmed by molecular test covered with glass coverslips and examined ing. In a cytogenetic study, at least 100 cells under a Zeiss epifluorescence photomicro were scored. Scoring of other breaks and gaps scope using an FITC exciter filter set. Suitable was used as a means of internal control. Where fields were photographed using Ektachrome indicated, DNA testing was performed by ref ASA 400 color film. erence laboratories. In general, when DNA testing was ordered, a routine three-day stimu Results and Discussion lated culture of peripheral blood was also per The fragile X syndrome is considered to be formed to rule out the presence of constitu one of the major inherited causes of mental tional chromosomal abnormalities present retardation in males. Clinical features usually elsewhere in the genome. associated with the fragile X syndrome include The designation of chromosomal abnormali developmental delay, learning disabilities, ties were in accordance with An International mental retardation, autism, avoidance behavior System for Human Cytogenetic Nomenclature.10 as well as hyperactivity and attention deficit, speech and language delay, unusual hand man nerisms, long and narrow faces with moder Fluorescent in sit u Hybridization (FISH) ately increased head circumference (>50th percentile), prominence of the jaw and fore For FISH, modifications of the procedures head with particularly large and mildly dys of Pinkel et al.,11 Mark et al.,12 Mark,13 Mark morphic ears, hyperextensible joints, high et al,14'15 Miranda, Mark and Medeiros,16 and arched palate, pes planus, pectus excavatum, Afify, Bland and Mark17 as well as manufac mitral valve prolapse and macroorchidism.2 turer’s instructions^ were followed. A biotin- Some clinical features of the syndrome overlap labeled, chromosome-specific a-satellite those associated with low level mosaicism for probe was used. Alpha-satellite DNA probes trisomy 8, such as the long face, prominent hybridize to highly repeated alphoid forehead, large ears, transverse palmar crease sequences at the peri-centromeric regions of and mental retardation.18 It is probably for this reason that this patient with the mosaic tri somy 8 syndrome was referred for fragile t Oncor, Gaithersburg, MD. X analysis. 296 MARK AND BIER Our patient was initially referred in 1991 for banding patterns in all the cells examined. No the purpose of ruling out fragile X syndrome. abnormal cells were detected in this meta Chromosomal analysis revealed a modal chro phase study. mosome number of 46 per cell with a male sex Furthermore, fluorescent in situ hybridiza constitution. A single cell with trisomy 8 (5 tion using a chromosome 8-specific a-satellite percent) was found in the first 20 cells ana probe (D8Z2)$ revealed 19 interphase nuclei lyzed. An additional 100 cells were therefore out of a total of 3160 nuclei (0.6 percent) analyzed. Results of this and subsequent analy examined to be trisomic. This is compared sis yielded no additional cells with fragile X or with a frequency of 0.2 percent positivity for 3 trisomy 8, while autosomal fragile sites were signals in normal controls. induced at 8 percent. Fluorescent in situ When a blood smear slide was analyzed hybridization by a reference laboratory using using FISH and the same probe, only one probe pJM128 was performed because FISH interphase out of 512 cells (0.2 percent) was permits the examination of a large number of found to be trisomic. cells. It revealed the presence of 22 out of A buccal smear specimen was also obtained 1000 nuclei (2.2 percent) with three positive in an attempt to score cells from another tis signals. Similar results were obtained in our sue, as the optimal approach in cases of laboratory using a commercial chromosome 8 mosaicism is the analysis of more than one tis specific «-satellite probe (D8Z1)4 sue. Out of 224 buccal mucosal cells, only 1 After an exhaustive search of slides prepared interphase nucleus, representing 0.4 percent from a second specimen, metaphases showing of the total cell population, was trisomic. The hybridization of D8Z1 to three chromosomes quality of buccal smear preparations, however, (figure 2) were found among numerous meta is in general not comparable to blood and skin. phases and interphase nuclei showing only two The possibility of a skin biopsy to obtain cells signals. The findings of three positive signals for a fibroblast cuture, although previously for the chromosome 8 probe were consistent explored, was ultimately ruled out because the with the findings of three of the chromosome patient’s mother was not enthusiastic about 8 in a GTG-banded cell originally detected by this option. the reference laboratory in the metaphase Taken together, the results of the study con study (data not shown). These results, from ducted five years after the initial study are two independent laboratories, were inter interpreted as apparently normal. preted as low level mosaicism for trisomy 8. It LaMarche et al1 first described the phe was further noted that this level of mosaicism nomenon of disappearing mosaicism in a could have been easily missed in a standard 20 female with mosaic trisomy 18 syndrome at cell GTG-banded analysis. birth. At 10 months of age, chromosome analy Because of the observation that trisomy 8 sis revealed only normal metaphases. A growth mosaicism declines with age, a finding consis advantage of the normal cell line over the tent with the hypothesis of “disappearing abnormal population of cells with time was mosaicism”, another sample of blood was suggested by these authors for this particular taken at the age of approximately 7 years and 8 mosaicism. Since the original findings by these months, in August of 1996.119 Results of chro authors were first described, we have not mosomal analysis of a routine three-day cul encountered other follow-up reports on this ture of 120 peripheral blood leukocytes hypothesis. However, it has been known for derived from such a sample revealed the num some time that trisomy 8 mosaicism declines ber of chromosomes to be 46 per cell with a with age leading one to question if there is a male sex constitution and normal appearing steady decline in trisomic cells so that eventu ally these cells disappear completely.19 This t Oncor, Gaithersburg, MD. hypothesis is indeed intriguing. However, to DISAPPEARING TRISOMY 8 MOSAICISM 297

Figure 2. Fluorescent in situ hybridization (FISH) using a chromo some 8 specific a-satellite probe illustrating 3 signals in metaphase (top) and interphase (bottom) cells.

the best of our knowledge, very little pertain vorosa for helpful comments. The continued support of Dr. Roger Mark and the staff of the Laboratory of Cyto ing to this topic has appeared in the cytoge genetics, FISH and Genotoxicology at Rhode Island Hos netic literature. pital is acknowledged.

Acknowledgments References Thanks are extended to Dr. John F. Stone for stimulat 1. LaMarche PH, Heisler AB, Kronemer NS. Disap ing discussions, personnel from our laboratory as well as pearing mosaicism: Suggested mechanism is growth the reference laboratory (Genetrix, Phoenix, AR) for tech advantage of normal over abnormal cell population. nical help, and Drs. Paul H. LaMarche and Erlinda Pol- RI Med J 1967;50:184-9. 298 MARK AND BIER 2. Mark HFL, Bier JB, Scola P. The frequency of chro comparison of approaches. Applied Cytogenetics mosomal abnormalities in patients referred for fragile 1992;18:149-56. X analysis. Ann Clin Lab Sci 1996;26:323-8. 3. Hagerman R, Amiri J, Cronister A. Fragile X check 13. Mark HFL. Fluorescent in situ hybridization as an list. Am J Med Genet 1991;38:283-7. adjunct to conventional cytogenetics. Ann Clin Lab 4. Caspersson TJ, Lindsten J, Zech L et al. Four patients Sci 1994;24:153-63. with trisomy 8 identified by the fluorescence and 14. Mark HFL, Sikov W, Safran H, King T, Griffith RC. Giemsa banding techniques. J Med Genet 1972;9:1-7. Fluorescent in situ hybridization for assessing the pro 5. Moorhead PS, Nowell PC, Mellman WJ et al. Chro portion of cells with trisomy 4 in a patient with acute mosome preparations of leukocytes cultures from non-lymphoblastic leukemia. Ann Clin Lab Sci 1995; human peripheral blood. Exp Cell Res 1960;20:613-6. 25:330-5. 6 . Yunis JJ. High resolution of human chromosomes. Sci 15. Mark HFL, Grollino MG, Sulaiman RA, Lathrop JC. ence 1976;191:1268-70. Fluorescent in situ hybridization (FISH) assessment 7. Yunis JJ, Soveng AL, Bowe AE. Fragile sites are tar of chromosome copy number in gestational tropho gets of diverse mutagens and carcinogens. Oncogene blastic disease. Ann Clin Lab Sci 1995;25:291-6. 1987;1:59-60. 8 . Sumner AT, Evans HJ, Buckland RA. New technique 16. Miranda RN, Mark HFL, Medeiros LJ. Fluorescent for distinguishing between human chromosomes. in situ hybridization in routinely processed bone mar Nature New Biol 1971;232:31-2. row aspirate clot and core biopsy sections. Am J Pathol 9. Seabright M. A rapid banding technique for human 1995;145:1-6. chromosomes. Lancet 1971;2:971-2. 17. Afify A, Bland KI, Mark HFL. Fluorescent in situ 10. Mitelman F (Ed.): An International System for hybridization assessment of chromosome 8 copy num Human Cytogenetic Nomenclature. Basel, Switzer ber in breast cancer. Breast Cancer Res Treatment land: S. Karger, 1995. 1996;38:201-8. 11. Pinkel D, Strawne R, Gray J. Cytogenetic analysis using quantitative high sensitivity, fluorescence 18. deGrouchy J, Turleau C. Clinical Atlas of Human hybridization. Proc Nat Acad Sci USA 1986;83: Chromosomes. New York: John Wiley and Sons, 2934-8. 1984:126-33. 12. Mark HFL, Santoro K, Jackson CL. Cytogenetic char 19. Berry AC, Mutton DE, Lewis DGM. Mosaicism and acterization of somatic cell and radiation hybrids: a the trisomy 8 syndrome. Clin Genet 1978;14:105-14.