Am J Hum Genet 28:62-68, 1976

Incidence of Aneuploidy in a Human Population WALTER B.

GOAD,'

ARTHUR ROBINSON,2 AND THEODORE T. PUCK3

It has recently proved feasible to examine large numbers of human newborns for aneuploidy of the sex chromosomes and of chromosome 21 [ 1, 2 ]. We have screened 40,371 newborns delivered at two Denver hospitals for these conditions since 1964. The overall frequency of these particular chromosomal anomalies is about 0.3 % per live birth, which represents a high incidence of medically significant, human genetic abnormality. The screening is highly effective and accurate; it therefore comprises a sensitive and reliable monitor of an important class of genetic events in the population studied. Events that occur completely at random will appear with constant probability per birth, that is, with an incidence uniform in time. This component of the total incidence of aneuploidy might be called "endemic." Our results indicate, with high statistical confidence, that in addition there is a component of comparable intensity that by contrast is "epidemic." MATERIALS AND METHODS

Screening for sex-chromosomal anomalies was accomplished by determining the Barrand Y-body status of cells from the amniotic membrane. If the observed number of Barr bodies per cell disagreed with that expected on the basis of the phenotypic sex of the baby, or if the structure or percentage of Barr bodies appeared aberrant, a buccal smear and a complete chromosomal analysis were carried out. In 0.08% of all cases studied, the amniotic preparation suggested a sex chromatin positive condition which was not confirmed either by the buccal smear or the karyotype analysis. It was hypothesized that these cases were X-chromosomal mosaics, and that by chance the cells with the abnormal karyotype were localized in the cells of the extraembryonic tissues early in embryogenesis, when nondisjunction had occurred. These cases were not included among the newborns with abnormal karyotypes. Since the beginning of 1971 we have also screened 15,641 newborns for Y-chromosome Received June 4, 1975; revised August 14, 1975. This work was performed under the auspices of the U.S. Energy Research Development Administration and supported in part by the U.S. Public Health Service grant 5R01-HD00622 from the National Institutes of Health. This paper is contribution no. 628 from the Department of Biophysics and Genetics and contribution no. 199 from the Eleanor Roosevelt Institute for Cancer Research, University of Colorado Medical Center. 1 Los Alamos Scientific Laboratory, Los Alamos, New Mexico. 2 National Jewish Hospital and Research Center, Denver, Colorado, and Department of Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado. Reprint requests should be sent to this address. 3 Eleanor Roosevelt Institute for Cancer Research, University of Colorado Medical Center, Denver, Colorado. © 1976 by the American Society of Human Genetics. All rights reserved.

62

INCIDENCE OF ANEUPLOIDY

63

constitution (8,011 phenotypic males), utilizing fluorescence microscopy of touch preparations from the transected umbilical cord [3], and again subjecting every detected anomaly to complete chromosomal analysis. Using this method it is possible occasionally to miss the presence of a Y-body. However, if such an error occurred, an apparent discrepancy from the sexual phenotype would be signaled in the initial screening so that a complete chromosomal analysis would be called for. In eight such cases, the fluorescent karyotype revealed a small, nonfluorescent or slightly fluorescent, long arm of the Ychromosome. From this we estimate that about 1/1000 males has a Y-chromosome with markedly diminished fluorescence. In a 47,XYY karyotype, only the conjunction of one fluorescent and one nonfluorescent Y-body could escape detection, which is very unlikely. Therefore, as in the Barr body analysis, Y-chromosomal fluorescent screening, as utilized in the total procedure described, provides an accurate epidemiological methodology. Finally, screening for trisomy of chromosome 21 was accomplished by a preliminary physical examination of each baby, followed by complete chromosomal analysis in every case where there was the slightest suspicion of the presence of Down syndrome. RESULTS

The overall results, classified by aberrant karyotype, are presented in table 1. Our results on the incidence of trisomy 21 are in agreement with a number of other TABLE 1 CHROMOSOMAL ABNORMALITIES-DENVER CHROMOSOMAL STUDY-1964-1974 Aneuploidy

No.

Incidence

(%) Sex chromosomal abnormalities: Phenotypic males: .................... 47,XXY ............... ..................... 47,XYY ............... Mosaics ............... ..................... Phenotypic females: ..................... 47,XXX .............. 45,X .................. .................... Mosaics ............... ..................... Total ............... ..................... Trisomy 21: To mothers < 35 ........... ................ To mothers > 35 .......... ................

20 4 3

.050 .026 (=4/15,641) .0074

12 11 16

.030 .027 .040

66

.180

28 15

1.000*

.072

NOTE.-40,371 newborns were screened: 20,666 males; 19,705 females. Incidence was calculated by assuming that the observed rate of XYY (1971-1974) applied over the entire period 1964-1974. * Approximately 3.7% of all newborns in the study had mothers > 35.

studies [4] in demonstrating an increased frequency for older mothers. Therefore, we have further classified cases of Down syndrome into those whose mothers were under 35 and those whose mothers were 35 or older at the time of birth. In figure 1 the birth dates of the aneuploid babies are displayed. It is obvious that they are not uniformly distributed. Yet the total number of births per month is approximately uniform, fluctuating by about 10%o in each direction from the

0

0 0

No

-

Cl)

0

U>

0

a

0 .0 0

t3

o

v

*

0 0

Iu. >)~.0.

0)

E_

oO

0=

0

0 o

0

N 0Cd

_i .0

a 0

Cd

0

.0.0 0

_

O. 0

I

.0 0

.0.O -

.0

Al > 0

a

, C

0

64

0

65

INCIDENCE OF ANEUPLOIDY

average. We first observed, and remarked [ 1], that if the year is divided into two equal periods, May-October and November-April, there is a large disparity in aneuploid births (table 2). For any other division into two consecutive periods the TABLE 2

CONTRAST BETWEEN SEASONS OF HIGH AND Low INCIDENCE OF CHROMOSOMAL ABNORMALITIES NOVEMBER-APRIL

MAY-OCTOBER

NEWBORNS SCREENED

(19,318)

(21,053)

Puniform incidence*

Sex chromosomal abnormalities ..... ..... Trisomy 21 to mothers < 35 ...... ...... Total ............................. Trisomy 21 to mothers > 35 ...... ......

23 6 29 8

43 22 65 7

6 X 10-3 1 X 10-3

*

2 X 10-4

~.5

Probability of a difference as large as (or larger than) that observed, given incidence constant in time.

disparity is smaller. There is no suggestion of a seasonal variation in trisomy 21 babies born to older mothers. For all other babies, we asked the question: had the incidence been uniform in time, what is the probability that so large a disparity would occur in this particular sample? The answer is approximately 10-4 for the combined sex chromosomal and chromosome 21 aneuploidies (table 2). This amounts, statistically, to a direct assay of correlation with time. We might instead base a hypothesis on data from the first year or even the first two years which strongly indicate a seasonal effect (fig. 1). We then could test this hypothesis on the remainder of the data. If one omits the first year only, the probability of so large a disparity had the incidence been uniform rises to about 10-3, and if the first two years are omitted, to 10-2. In any case, a true annual variation in incidence is strongly indicated. The counts in table 2 indicate that the ratio of abnormality between the seasonal high and low incidences is approximately two. We can-be 95% confident* that the increase is at least 1.5-fold. Another view of the seasonal incidence is obtained by plotting monthly incidences (fig. 2). It seems sensible to construct these with some allowance for dispersion in the time elapsed between the significant event (nondisjunction), whose correlation with time we are seeking to discover, and birth. There is at least the dispersion of gestation times of about 15 days. We have included its effect as follows: the estimates of incidence at each midmonth are weighted averages of cases occurring before or after that date, weighted according to distance (in time) from midmonth by a linear function of half-width 15 days. The error estimates are cal*All of the numbers given here that relate to statistical significance are calculated by the method previously described [2]. Dr. Nathan Mantel has called our attention to an alternative method of calculation [5]. When the data are recalculated by the latter method, virtually the same numerical results are obtained. Hence the same conclusions are reached from two different statistical approaches.

GOAD ET AL.

66 OV ). .O_

.401.30_ a)

(10a)

I

-$

1 1

1

1

I

{

~~~~~~Ti_ ~~~~~~~i

ii ii

.10 L

2

-

I

7 6 5 10 11 12 8 9 Months Of The Year FIG. 2.-Combined monthly incidence of aneuploidy-sex chromosomal and trisomy 21-born to mothers < 35. Shaded circles = direct estimates from this study, allowing for 15-day dispersion in gestation times (see text p. 65-66); dotted line = two season model, 95% confidence level; dashed line = two season model, 99% confidence level (see text p. 65-66). t N

U

3

4

culated from Poisson distributions whose means are these weighted averages; the error bars delineate a range such that the probability is I/e (37%) that the incidence lies outside it. Thus, in speaking of high and low seasons we have fitted this result by a two-season model for the variation in incidence. The fit is constructed using the method of Robinson et al. [2]. In figure 2 we have sketched as a dotted line the two-season model for which we are 95% confident that the seasonal disparity was at least as great as shown (1.6-fold) and as a dashed line the comparable result with 99%o confidence (at least 1.3-fold). An interesting, if barely significant, departure from the overall trend is seen in August and January. Another striking feature of the sex chromosomal data (fig. 1) is the disparity in cases between the years 1969 and 1970 and the remainder of the period (table 3). TABLE 3 CONTRAST OF "EPIDEMIC" YEARS WITH 1968-1970

NEWBORNS SCREENED

Sex chromosomal abnormalities Trisomy 21 to mothers 35 ......

.......... ...... ......

1968-1970

1964-67 AND 1971-74

(12,072)

(28,299)

Puntifhrm incidence*

8 6 5

54* 22 10

4 X 10-4

.15

~.5

* Since Y aneuploidy was not screened for during 1968-1970, four individuals with a 47,XYY karyotype, identified during 1971-1974, were omitted from this calculation.

INCIDENCE OF ANEUPLOIDY

67

Again it is very unlikely (P 4 X 10-4) that the average incidence was the same over the two periods. We conclude that it is very likely that the epidemic component of the incidence of sex chromosome aneuploidy varies both on a scale of seasons and on a longer scale of several years. Thus, it seems that we can identify, with high statistical confidence, three distinct components of the incidence of aneuploidy of the sex chromosomes and chromosome 21: (1) a steady, endemic component of 0.10%-0.15% per birth for sex chromosomal aneuploidy, and around 0.05% for trisomy 21 for mothers under age 35; (2) an epidemic component for both of these groups which approximately doubles the incidence during May-October; and (3) a steady, about 10-fold larger component for trisomy 21 for mothers over 35. In Denver the epidemic component for sex chromosome aneuploidy was almost certainly absent for several years in a row. Owing to its lower absolute incidence, no significant conclusion on this point is possible for trisomy 21. ,

DISCUSSION

Our results continue to confirm the initial tentative conclusion of nonrandomness which we drew for the occurrence of nondisjunction of the sex chromosomes and at least one autosome, number 21. The fact that our studies have thus far revealed significant seasonal variations in occurrence in a reasonably stable population gives weight to the hypothesis that environmental factors are active in the etiology of aneuploidy. The data for trisomy 21 suggest parallel occurrence with the sex chromosome abnormalities. These data are consistent with the possibility that there are etiological factors which predispose to nondisjunction of both the relatively large X chromosome and the very small number 21 chromosome. Therefore, it seems possible that all of the human chromosomes may be susceptible to the same environmental factors. This would make the elucidation of predisposing causes even more urgent from a public health standpoint. In a previous paper [2] we hypothesized on the basis of studies on only 17,000 newborns that viral infections might be implicated in the etiology of these conditions. We have been unable in our current, more extensive studies to confirm or rule out the viral hypothesis. The frequent association of sex chromosome abnormalities with important medical and psychosocial problems has been much discussed in the literature. For this reason we have been studying the natural history and prognosis of these conditions in newborns identified by the study who presumably represent a high-risk group. One fact from this study seems particularly important: abnormalities thus far found in children are relatively minor and hence raise the definite possibility that they may be susceptible to remedial therapy before becoming major and irreversible. The previous hint [2] of socioeconomic correlation has not been confirmed in our overall results, but the absence of any correlation with racial background has continued. The simplicity and accuracy of the screening methods employed commend this

68

GOAD ET AL.

approach for the study of human populations which may be at risk for increased chromosomal aneuploidy due to environmental conditions. SUMMARY

A population of 40,371 individuals consisting of every baby delivered at two Denver hospitals from 1964 to 1974 has been screened for aneuploidy of the sex chromosomes and chromosome 21. The pattern in time with which aneuploidy occurs suggests an epidemic component of the incidence superimposed on an approximately equal constant frequency. The epidemic incidence is most likely to be high for births from May to October, to persist for several consecutive years, and then to be absent for several consecutive years. REFERENCES 1. ROBINSON A, PUCK TT: Studies on chromosomal nondisjunction in man. II. Am J Hum Genet 19:112-129, 1967 2. ROBINSON A, GOAD WB, PUCK TT, HARRIS J: Studies on chromosomal nondisjunction in man. III. Am J Hum Genet 21 :466-485, 1969 3. GREENSHER A, GERSH R, PEAKMAN D, ROBINSON A: Screening of newborn infants for abnormalities of the Y chromosome. J Pediatr 79:305-306, 1971 4. CARTER C, MACCARTHY D: Incidence of mongolism and its diagnosis in the newborn. Brit J Soc Med 5:83-90, 1951 5. MANTEL N, PATWARY KM: Interval estimation of single parametric functions. Bull Int Stat Inst 38:227-240, Part IV, 1961