J . ment. Defic. Res. (1980) 24, 159 159

ANEUPLOIDY IN CULTURE

ARABELLA SMITH* and GAE ELLIOTT

Cytogenetics Unit, Oliver Latham Laboratory, Health Commission of New South Wales,

Australia

INTRODUCTIONOf three major studies of aneuploidy in cuUuie, two were based on peripheral

blood cultures of normal populations (Jacobs, Court-Brown and Doll, 1961 ;Fitzgerald and McEwan, 1977) and one was based on bone marrow studies of abnor-mal as well as normal individuals (Court-Brown, Jacobs and Doll, 1960). In theformer two studie.s, the major finding was an age-related efTet t of sex chromosomeaneuploidy in females. There have been surprisingly few further reports givingstandard data on large populations. Such information would be useful in the inter-pretation of chromosome counts in clini< al cytogenetics, particularly in delineatinglow-grade constitutional mosaicism.

To help clarify this situation in our hands, particularly with respect to pre-parative faults, we analysed data from chromosome cultures performed in tlie usualway, assessing the frequency and type of random gain and random loss of chromo-somes, and compared the results for age, sex and different referral groups.

METHODSChromosome counts

The dala analysed consisted of cells from a hundred consecutive clinicallyrtfcned patients, excluding only those wilh poor growth—less tlian twenty-fiveanalysable cells. The parameters of culture and analysis were the same for eachpatient.

Chromosome analysis was performed after three-day cultures by standard pro-cedures (Moorhcad, Nowell, Mellman, Battips and Hungerford, 1960), using cultineMedium Hams FIO (Flow Laboratories) in each case. G banding (Seabright, 1971)and C banding (Arrighi and Hsu, 1971) were carried out routinely and representa-tive \ hotographs and karyotypes ^vere made in each case.

Analysable cells were those that were well spread, bul nevertheless a compactunit. The slides were iirst scanned, and the aim was to choose optimal cells forcounting. Cells which appeared incomplete were not counted, and areas whereseveral cells were close together were avoided (figure 1). The choice of cells examinedwas somewhat subjective, which was an unavoidable limitation of the ; tudy, but oncea cell was chosen and counted it was not discarded.

For each patient a minimum of twenty conventional, three G banded and fourC banded cells was examined and in most cases thirty cells were analysed. The

*Rcprint requests to: Dr A. Smith, Cytogenetics Unit, Oliver Lathum LaboratoryHealth Commission of New South Wales, P.O. Box 53. North Ryde, NSW 2113, Australia

Received 4th March, 1980

160 ANEUPLOIDY IN CULTURE

' ^'M ^ '

Fig. I. Two G banded ct̂ Us. The one on ihc left was analysed, bciiis well spreadbut nevertheless a compact unit. The chromosomes in the cell on the right weretoo contracted, and this cell was not analysed. AUhough these two cells wereclose together, they were clearly distinct units, but on the whole, cells as close

together as this were avoided.

microscopic indices were recorded for all cells examined, and for those cells with anon-modal chromosome number, the extra or missing chromosome(s) or group(s)was noted. A break in any chromosome, if present, was also noted, as was the findingof fragments or rearrangements {other than a constitutional translocation).

Patient referrals

The clinical referrals were divided into five convenient groups—mental retarda-tion, male and female infertility, habitual abortion, Down syndrome, and a mis-

ARABELLA SMITH and GAE ELLIOTT 161

cellaneous group consisting mainly of relatives of abnormal patients, individualsseeking genetic counselling, and cases referred with abnormal physical development.

RESULTSOf the hundred patients, sixty-nine were males and thirty-one were females.

The age range of ihe patients varied from two weeks to sixty years, the majority^68 per cent—were in the twenty-forty age bracket.

A total of 3,175 cells was examined from these himdred patients, and of these,258 cells gave hypomodal counts (8.1 per cent) and sixty-two cells demonstratedhypermodal counts (1.9 per cent) giving a total of 320 non-modal cells (10 per cent).Of the hypomodal cells, 213 had foi ty-five chromosomes and forty-five cells had forty-four chromosomes. Of the sixty-two hypermodal cells, sixty had forty-seven chromo-somes, two cells had forty-eight chromosomes.

These findings were evaluated separately for sex of the patient, age of the patientand reason for referral. Analysis of counts according to age (Table I) showed thatthere was no significant difference between the number of hypomodal cells in anyage group, compared with the expected 8.1 per cent, and similarly the hypermodalcells did not vary significantly in any age group from the expected 1.9 per cent ofcells counted. Analysis of counts according to sex (Table 2) did not show any sig-nificant differences from the expected number of non-modal cells for either sex.Analysis of results according to clinit al referral (Table 3) did not show any significantdifferences in any referral group from the total number of hypo- and hypermodalcells from the group as a whole.

Table IAnalysis of chromosome counts according to age

Total numberAge group Number of of cells Hypomodal Hypermodal

(years) subjects counted cells % cells "/„

0-4 8 262 20 7.6 9 3.4**5-14 !3 406 35 8.6 5 1-2

15-24 11 365 42 11.5* 8 2.225-34 51 1554 117 7.5 30 1.935-44 13 439 34 7.7 7 1.645-64 4 149 10 6.7 3 2.0

100 3175 258 8.1 62 1.9

"Difference between the observed 11.5 and the expected 8.1 was not significant (P = 0.5).**Difference between the observed 3.4 and the expected 1.9 was not significant (P — 0.5).

(Using X* test; see footnote. Table 5.)

The finding of breaks, fragments, or rearrangements (other than a consti-tutional transiocation) was a rare occurrence, occurring in seventeen of the 3,175cells examined (0.5 per cent). These results are shown in Table 4, related to thereason for referral.

162 ANEUPLOIDY IN CULTURE

Table 2Analysis of chromosome counts according to sex

Sex

MaleFemaleTotal

Number ofsubjects

6931

100

Total numberof celts

counted214810273175

Hyf>omodalcells164!)4

258

/o7.69.18.1

Hypermodalcells372562

/ o1.72.4L9

Table 3Analysis of chromosome counts according to referral

ReferralMental

retardationInfertilityHabitual

abortionDown

syndromeMiscellaneous

Number afsubjects

1038

14

1325

Total numberof cellscounted

3301184

443

436782

Hypomodalcells

20103

32

3766

/a

6.0fi.7

7.2

8.48.4

Hypermodalcells

1015

5

1616

/a

3.01.2

1.1

3.62.0

100 3175 258 8.1 C2 1.9

Table 4

Number of cells with breaks, fragments or rearrangements by clinical referral

Referral Breaks Fragments Rearrangements*

Mental retardationInferliiity [all males)Habitual abortionDown syndromeMiscellaneous ; * *

*Other than cons'itional transiocation.**The one eell with a rearrangement shrfwcd -16, XX. t (1 ; 11) ip21 ; q 14) in a fi male

relative of a Down syndrome proband.

An analysis of ihe chromosomes "missing" or "extra" in the hypo and hyp'.r-modal cells was undertaken and these results are shown in Table 5. To simplify theanalysis, the data froni G banded cells (which precisely identified the chromosome)were pooled with the data from the standard cells aud C banded cells (which largelyclassified the chromosome according to group). The chromosomes involved in thelosses were non-randomly distributed, with an excess of the small chromosomes from

ARABELLA SMITH and GAE ELLIOTT 163

Table 5

Distribution of chromosomes "lost" from the hypo/nodal cells and ^^gained" in the hypermodalcells examined, with the respective statistical significance when compared to random loss and gain

for each group

ChromosomeGroup

ABCDEFGY

""Statistical analysis

X'' was calctUatcd for Ios

Number"lost"

79

53426934800

303

Statistical*Signijicance

P < 0.001P < 0.001P < 0.001

NS**P < 0.001

NSP < 0.001

NS

was performrd u^ing the X^(observed

.acs and gams as — —

Number"gained"

23

3454952

64

StatisticalSignijicance

NSNS

P < 0.001NSNSNSNSNS

tesl for goodness ol fit. I'o— expected)'

1

**Not sii^nificant.Group K and G bring lost, and a deficit of the large chromosomes lVoin CIroups A,B and C being lost. The gains were truly random except for Group C, which wasgained more often than expected by random gain, but the numbers here were small.

DISCUSSIONDue to difficulties in some cases in the clinical cytogenetic situation of deter-

mining mosaicism, we felt the need for basic data with regard to aneuploidy inculture. Tables such as those of Hook (1977) are useful, but precisely defining cultureconditions for one's own laboratory, and comparing a given result with knownbaseline data, would be more helpful in assessing a mosaic. We have analy.sed onehundred consecutive clinical referrals where good growth occtirred using Hams FIOculture medium on .seventy-two-hour cullures, and found overall 8.1 per cent of cellsto be hypodiploid and 1.9 per cent to be hyperdiploid.

Gourt-Brown et al. (1960) di.scussed very well the problem of cultural artefactsand in iheir study they found more ancuploid cells with forty-live chromosomes thanforty-seven chromosomes or any other number. We also found this, but the "randotnloss" of a chromosome was not in fact random; it seems that the small chromosomesare more frequently lost than the larger ones. This did not, however, apply to theY chromosome, which was also found by Fitzgerald and McEwan (1977).

We did not find any age difference in our study, but there were only fourpatients over forty-five years of age. Jacobs et al. (1961), studying a comparablenumber of patients, found an increasing number of cells with forly-fi\e chromosomesin women over sixty years. A sex difference was also found by Fitzgerald and McEwan

164 ANEUPLOIDY IN CULTURE

(1977) which applied to all age groups, though more significant in the older women.The total number of cells examined (from normal volunteers) in his study was similarto ours.

Our study did not detect any significant differences in hypomodal or hyper-modal counts with different diagnostir groups and the bone marrow studies ofCourt-Brown el al. (1960J also did not show significant differences for those patientswith abnormal karyotypes. In the study of Jacobs el al. (1961) there was no significantdifference found for Klinefelter's syndrome or Down syndrome in young patients,but the increase of hypomodai cells with age was more pronounced in those withabnormal karyotypes.

Reports of mosaic individuals vary widely. Baikie, Darlnall and Lickiss (1972)cautiously described a "possible" mosaic XX/XXX female with XXX present ineight of seventy-eight cells (10.25 per cent), while Nielsen and Freidrich (1971)confidently outlined an individual mosaic for a pericentric inversion of chromosomeone found in two of five hundred cells examined (0.4 per cent). Under the conditionsspecified in this report, to classify a Down syndrome mosaic with a small normal cellline we would need to find significantly more than 8.1 per cent of cells to have acount of forty-six, particularly as G chromosomes are more frequently "randomly"lost than some of the other groups. Similar calculation can be applied to otherconditions where mosaicism can be encountered.

In this analysis, we found very few breaks, fragments or rearrangements (otherthan a constitutional translocation). These occurred in seventeen of 3,175 cells(0.5 per cent). Such breaks are considered to be less common in .seventy-two-hourcultures thati forly-eight-hour cultures (Hatcher and Hook, 1976). Kim, Hsu,Paciuc, Cristian, Quintana and Hirschhorn (1975) gave the normal value of chromo-some breakage in iheir laboratory a.s between 0-4 per cent, and described four adultswith a history of foetal wastage who showed chromosome breakage significantlyabove this level. Our data in this respect are not numerous, but there appears tobe an excess of cells with fragments among infertile males.

SUMMARY

On one hundred consecutive clinically referred patients, a total of 3,175 cellswas fully analysed. The parameters of culture and analysis were the same for allpatients, and a breakdown of cells showed 258 cells (8.1 per cent) with hypomodalcounts and sixty-two cells (1.9 per cent) with hypermodal counts. The great majorityof the hypomodal cells contained forty-five chromosomes and the distribution ofchromosome loss from these cells was non-random, with fewer than expected of thelarge chromosomes (groups A, B and C) being lost, and more than expected of thesmall chromosomes (group E and G) being lost. The distribution of the chromosomesgained in the hypermodal cells was random (except for the C group where the num-bers were small). In this data, there was no significant difference of aneuploidy inculture noted for sex, age, or patient referral group.

ARABELLA SMITH and GAE ELLIOTT 165

REFERENCESARRIGIIT, F . E . and Hsu, T. G. (1971) Localisation of heterochromatin in htiman chromo-

somes. Cytogenetks 10, 81.B.\IK:E, A. G. , b.\RTNALL, N. A. and LICKISS, .J. N. (1972) XXY son of a possibly XX/XXX

mother. Lancet i, 697.COURT-BROWN, W . M . , JACOBS, P. A. and Doi.i., R. (1960) Interpretation of chromosome

counts made on bone marrow cells. Lancet i, 160.FITZGERALD, P. H. and MCEWAN, C . M . (1977) Total aneuploidy and age-related sex chromo-

some aneuploidy in cultured lymphocytes of normal men and women. Hum. Genet. 39,329.

HATCHER, N . H . and HOOK, E . B. (1976) Early in-vitro division ol PHA-stimulated cord bloodlymphocytes: implications for study of chromosome breakage. Am. J. hum. Genet. 28, 290.

HOOK, E . B. (1977) Exclusion of chromosomal mosaicism: Tables of 90%, 95%, and 99%.Confidence litnits and comments on use. Am. J. hum. Genet. 29, 94.

JACOBS, P. A., COURT-BROWN, \V . M . and DOI-L, R . (1961) Distribution of human chromo-some counts in relation to age. Nature 191, 1178.

KIM, H . J . , HSU, L. Y . F., PACIUC, S., CRISTIAN, S., QUINTANA, A. and HIRSCHHORN, K .(1975) Cytogenetics of fetal wastage. New Engl. J. Med. 293, 844.

MooRHEAD, P. S., NowELL, P. C , MEIXMAN, W . J., BATTIPS, D . M . and HUNGERFORD, D . A.(1960) Chromosome preparations of leucocytes cultured from human peripheral blood.Exp. Cell Res. 20,613.

NIELSEN, J. and FREIDBICH, U . (1971) Pure gonadal dysgcnesis. Ciin. Genet. 3, 52.SEABRIGHT, M . (1971) A rapid banding technique ibr human chromosomes. Lancet ii, 971.