Hum Genet (1989) 83 : 83-87

© Springer-Verlag 1989

Detection of an unbalanced translocation (4;14) in a mildly retarded father and son by flow cytometry

Alexander Cooke, John L. Tolmie, James M. Colgan, Caroline M. Greig, and J. Michael Connor

Duncan Guthrie Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ, UK

Summary. A child with impaired intelligence, minor dys- morphisms, obesity and genital hypoplasia was found to have an apparently balanced translocation, 46,XY,t(4;14)(q12;q13), following cytogenetic analysis. The same rearrangement was also detected in the child's father, who had similar phenotypic abnormalities to his son. Detailed study of flow karyotypes produced from lymphoblastoid cell lines established that in both patients the translocation was in fact unbalanced with approximately 11 million base pairs of D N A (corresponding to about 6.0% of chromosome 4 or 11.0% of chromosome 14) being lost.

Table 1. Clinical findings in the proband and his father

Feature Proband Father of proband

Height 90th centile 10th centile Weight > 97th centile > 97th centile OFC > 75th eentile > 95th centile Mild mental impairment + + Late puberty + ? Small penis + + Undescended testis Bilateral Right Inguinal hernia - + Minor retinal pigment

abnormalities + -

Introduction

The precise delineation of the chromosomal breakpoints of a translocation can frequently prove difficult in cytogenetic analysis (Raimondi et al. 1983). In cases where appropriate high-resolution banding has not been achieved, or when there is clinical evidence suggesting the possibility of chromosome imbalance, flow cytometry can provide an alternative measure of the change in D N A content of the chromosomes involved (Martin et al. 1988).

We have previously demonstrated that ethidium bromide flow karyotypes provide an accurate method of defining chro- mosome D N A content (Harris et al. 1986), confirming the presence of suspected cytogenetic deletions (Cooke et al. 1987), or detecting deletions that were not resolved by micro- scopy (Cooke et al. 1988). We now describe the results of flow karyotype analysis on a father and son with similar phenotypic abnormalities in whom conventional cytogenetic analysis re- vealed the presence of an apparently balanced reciprocal translocation involving chromosomes 4 and 14.

Case report

The proband was referred to the genetics clinic because of genital hypoplasia, minor retinal pigmentary abnormalities and mild mental retardation at age 11 years. At this time, chromosome analysis of peripheral lymphocytes revealed the presence of an apparently balanced reciprocal translocation 46,XY,t(4;14)(q12;q13), but as the same translocation was also present in his father, it was thought unlikely to be of sig- nificance.

Offprint requests to: A. Cooke

Review of the family, with close attention to the father's past medical history and clinical features, showed that he had minor dysmorphisms in common with his son (Table 1), as well as a striking facial resemblance (Fig. 1) Detailed flow karyotype analysis was therefore undertaken to provide an in- dependent check on the cytogenetic assessment regarding the apparently balanced translocation.

Materials and methods

Cytogenetic analysis

PHA-stimulated lymphocyte cultures were used to yield meta- phase and prometaphase chromosome preparations, which were stained by standard Giemsa banding techniques.

Sample preparations for flow cytometry

Chromosome samples for flow cytometry were initially pre- pared from PHA-stimulated lymphocyte cultures (Young et al. 1981). Later, lymphoblastoid cell lines were established from the child and his parents, and chromosome samples pre- pared as previously described (Harris et al. 1986). Flow karyo- types for each family member were produced as before (Cooke et al. 1988) and analysed with the aid of a microcom- puter program so that Gaussian distributions could be fitted to the peaks (Harris et al. 1986).

Results

Flow karyotypes were produced from the father, mother and child; examples of these are shown in Fig. 2. Relative fluores-

84

INg.IA, B. The proband (A) and his father (B)

cence values for the peaks in each flow karyotype were ob- tained as previously described (Cooke et al. 1987), and the final results were expressed as the mean of seven separate flow karyotypes produced for each individual (Table 2). The allocation of chromosomes to particular peaks in the flow karyotypes was aided by the calculation of the area encompas- sed by each peak (minus background). An example of peak area estimation using Gaussian distributions for chromosomes 1-15 in the father is shown in Fig. 3 and Table 3.

Flow karyotypes were produced from both lymphocyte cultures and lymphoblastoid cell lines. No significant varia- tions (other than quality of resolution) were noted between different samples for the same individual.

As might have been predicted from the cytogenetic results, both translocation carriers show only one chromosome pre- sent in the normal 4 peak, and a deficiency of one chromo- some in the 13-15 region of the flow karyotype. In the area of the flow karyotype corresponding to chromosomes 7 and X, all three individuals show a similar pattern with four chromo- somes being present. Thus, in the two males, it can be assumed that one of the chromosomes present in the X peak represents one of the translocation products, which from cytogenetic analysis is the chromosome with the 14 short arm and centro- mere and most of the long arm of chromosome 4, i.e. t(14-4) (Fig. 4). The relative fluorescence value for this peak in the father is 639.5 (SD 1.5), whereas in the child the value is 642.5 (SD 2.1).

18 19 17

16 If' 15 13

z2,v 21 ~2'19 18 I]

9i12 MOTHER

t X7 2

] -12 CHILD

111 ,l'II~ ,6 ~ 5 3 2 1 I

21

1 16 i[5 t~ "!

12 FATHER

~,~7, o 543, 2 ,

FLUORESCENCE

Fig. 2. Flow karyotypes from the mother (top), child (middle), and father (bottom)

The second product of the translocation, namely the short arm and centromere of chromosome 4 plus most of the long arm of 14, i.e. t(4-14) (Fig. 4), is found in the flow karyotypes of the father and son as a shoulder on the immediate left of the main central peak. The only chromosome found in this posi- tion in flow karyotypes from normal individuals is a chromo- some 9 exhibiting a significant deficiency in heterochromatin (9qh- ) . This unusual polymorphism was not observed cyto- genetically in either patient (Fig. 4), and furthermore it can be seen from the flow karyotypes that whereas both father and child have three chromosomes present in the chromosome 8 peak (presumably repesenting the two homologues of 8 and a 9qh+), the mother has both chromosome 9's present in the main central peak. Therefore, in the child, the 9qh+ must be of paternal origin, and the single chromosome found to the immediate left of the main central peak must be the t(4-14).

In the father, the t(4-14) chromosome had a mean relative fluorescence value of 529.7 (SD 1.0), and an identical mean value was found in the child (SD 1.7).

As ethidium bromide flow karyotypes provide an accurate measure of chromosome DNA content (Harris et al. 1986), it could be predicted that in a balanced translocation, the shift in fluorescence from the normal chromosome homologues to their respective translocation products should be exactly

85

Table 2. Relative fluorescence values for the three family members, expressed as the means obtained from seven flow karyotypes for each indi- vidual. C, Homologue inherited by child; P, paternal; M, maternal

Father Child Mother

Relative Chromosome no. Relative Chromosome no. Relative Chromosome no. fluorescence fluorescence fluorescence

1027.4 1 1024.5 1 1009.9 1 (xl) C 978.8 2 977.1 2 973.2 1 (×1), 2 820.2 3 821.5 3 817.8 3 786.4 4 (xl) 782.4 4 (×1) 782.0 4 749.5 5 747.6 5 749.3 5 704.3 6 703.2 6 703.3 6 662.7 7 664.8 7 659.9 7 639.5 X,t(14-4) 642.5 X,t(14-4) 636.5 X 600.3 8, 9 (xl) C 601.7 8, 9 (xl) P 599.6 8 560 9 (×1), 10-12 560 9 (xl) M, 10-12 560 9-12 529.7 t(4-14) 529.7 t(4-14) 460.4 13 (×1) 460.4 13 (xl) 455.4 13 (×1) 448.1 13 (×1) 446.8 13 (xl) 437.5 13 (×1), 14 (xl) 436.5 14 (× 1) 420.1 15 419.4 14 (×1) M, 15 420.8 14 (xl) C, 15 (xl) C 378.6 t6 379.1 16 382.4 15 (xl), 16 357.8 17 354.5 17, 18 (xl) M 347.8 17, 18 334.7 18 339.3 18 (×i) P 285.9 20 285.9 20 281.8 20 260.0 19 260.6 19 258.7 19 219.9 Y, 22 (xl) 222.5 Y, 22 (x 1) M 215.5 22 195.3 22 (×1) C, 21 201.2 22 (xl) P, 21 (×1) P 202.3 21 (xl)

189.6 21 (×1) M 187.7 21 (xl) C

9-12

15 14 t~4-~

326 FLUORESCENCE

3 2 5

FATHER

~97

Fig. 3. Part of a flow karyotype from the father showing Gaussian distributions fitted to the peaks

equal. This conclusion, however, presupposes that both homologues of a particular chromosome have identical fluorescence values. In reality, this is frequently not the case, as a large number of chromosomes, e.g. 13-15, exhibit a sig- nificant degree of normal size variation (heteromorphism).

Using the relative fluorescence values observed in the father (Table 2), the shift from the normal 4 homologue to the t(4-14) chromosome involves a decrease of 256.7, whereas the increase in fluorescence from the normal 14 to t(14-4) is 203.0. The difference between these values (53.7) represents 6.8% of the size of the normal homologue of chromosome 4.

When the results obtained in the child are used, the shift from 4 to t(4-14) gives a value of 252.7, similar to the equiva-

lent shift in the father. However, the shift from 14 to t(14-4) is significantly greater than that found in the father giving a value of 223.1. In the child's case, the difference in values is 29.6, which corresponds to only about 3.8% of chromosome 4. This apparent disparity can principally be accounted for by the difference in relative fluorescence observed between the normal 14 homologues in the two patients. The child has clearly inherited the smaller 14 homologue found in his mother; this is approximately 4% smaller than the normal 14 present in the father.

It would have been possible to produce a more accurate measure of the shifts involved in the translocation if the rela- tive fluorescence values of the chromosomes concerned could

86

Table 3. Area analysis of part of a flow karyotype from the father using computer-fitted Gaussian distributions

Peak C.V. Area Chromosome Chromosome(s) equivalent present

1 2.22 1275 2.8 14 (xl), 15 2 2.18 862 1.9 13 3 1.85 499 1.1 t(4-14) 4 1.97 3104 6.8 9 (xl), 10, 11, 12 5 2.17 1324 2.9 8, 9 (x 1) 6 1.71 924 2.0 X,t(14-4) 7 1.80 955 2.1 7 8 2.01 894 2.0 6 9 1.69 936 2.1 5

10 1.66 471 1.0 4 11 1.69 978 2.1 3 12 1.65 986 2.2 2 13 1.87 911 2.0 1

Pig. 4A-C. G-banded chromosomes from the father: A 4 and t(4;14); B 14 and t(14;4); C 9's

have been determined prior to the event taking place. If the translocation originated in the father, this would have re- quired that his parents be available for study. Unfortunately, samples could not be obtained from the paternal grandparents, and we therefore used the mean relative fluorescence values for chromosomes 4 and 14 found in the series of normal indi- viduals reported by Harris et al. (1986), i.e. 788.8 and 429.3, respectively.

When combined with the observed values for the translo- cation products in the father, a deficiency of 48.9 is recorded. If a similar procedure is followed for the child, the difference in fluorescence shift is 45.9. The average of these two values (47.4) corresponds to 6.0% of chromosome 4, 11.0% of chro- mosome 14, or 11 million base pairs of DNA; this is perhaps the best approximation of the amount of DNA lost as a result of the translocation in these patients.

Discussion

In this report, we have demonstrated that flow cytometry is of value to the clinician in that it provides an alternative method of chromosome analysis. Although balanced reciprocal trans- locations have been associated with an increased risk of men- tal handicap (Funderburk et al. 1977; Fryns et al. 1986), the initial discovery of an apparently balanced 4-14 translocation in the affected child was considered less significant when it

was also identified in the patient's father. However, as sub- sequent assessments indicated that the proband's father also had minor clinical abnormalities, this prompted us to investi- gate the family further by flow cytometry. The initial results from flow analysis of chromosomes from lymphocyte cultures were suggestive of some degree of imbalance in the transloca- tion; confirmation of this was obtained following analysis of samples from lymphoblastoid cell lines.

As the paternal grandparents of the proband were not available for study, there was no possibility of determining the relative fluorescence values of the 4 and 14 homologues prior to their involvement in the translocation (assuming the trans- location had arisen de novo in the proband's father). An esti- mate of the amount of DNA lost in the translocation was made by employing the control mean values for 4 and 14 re- ported by Harris et al. (1986), thus giving a size for the dele- tion of approximately 6% of chromosome 4. If the limits of the normal ranges for the sizes of chromosomes 4 and 14 ob- served by Harris et al. (1986) are used in the calculation rather than their means, the deletion size would range from 2.6% to 13.0% of chromosome 4. The upper limit of this range would have obviously been resolved by microscopy and it seems reasonable to conclude that the value obtained using the con- trol means represents the maximum possible amount of DNA lost. The phenotypic abnormalities present in the proband and his father are not similar to those described in individuals with proximal interstitial deletions of 4q (Beall et al. 1988). How- ever, very few patients with deletions in these regions of either chromosome 4 or 14 have been reported.

The application of this technique is likely to have particu- lar value in families with a dysmorphic and/or mentally hand- icapped child in which an apparently balanced translocation has arisen de novo, thus eliminating the involvement of esti- mated control values in the calculation of results. Whereas flow cytometry is clearly an order of magnitude less sensitive than molecular genetic analysis in detecting quantitative ab- normalities of human chromosomes (lower limit approximate- ly 1 million base pairs), it does provide an intermediate mode of analysis between microscopy and molecular techniques (Emanuel 1988).

Acknowledgements. We thank Dr. John Yates for the initial assess- ment of the proband and instigating flow analysis, and Mairi Clarke for lymphoblastoid cell culture.

References

Beall MH, Falk RE, Ying K-L (1988) A patient with an interstitial de- letion of the proximal portion of the long arm of chromosome 4. Am J Med Genet 31 : 553-557

Cooke A, Tolmie J, Darlington W, Boyd E, Thomson R, Ferguson- Smith MA (1987) Confirmation of a suspected 16q deletion in a dysmorphic child by flow karyotype analysis. J Med Genet 24: 88- 92

Cooke A, Gillard EF, Yates JRW, Mitchell MJ, Aitken DA, Weir DM, Affara NA, Ferguson-Smith MA (1988) X Chromosome de- letions detectable by flow cytometry in some patients with steroid sulphatase deficiency (X-finked ichthyosis). Hum Genet 79 : 49-52

Emanuel BS (1988) Molecular cytogenetics: toward dissection of the contiguous gene syndromes. Am J Hum Genet 43 : 575-578

Fryns JP, Kleezkowska A, Kubien E, Van den Berghe H (1986) Ex- cess of mental retardation and/or congenital malformation in re- ciprocal translocations in man. Hum Genet 72 : 1-8

87

Funderburk S J, Spence MA, Sparkes RS (1977) Mental retardation associated with "balanced" chromosome rearrangements. Am J Hum Genet 29 : 136-141

Harris P, Boyd E, Young BD, Ferguson-Smith MA (1986) Determi- nation of the DNA content of human chromosomes by flow cyto- metry. Cytogenet Cell Genet 41 : 14-21

Martin AO, Northrup H, Ledbetter DH, Trask B, Engh G van den, Le Beau MM, Beaudet AL, Gray JW, Sekhon G, Krassikoff N, Booth C (1988) Prenatal detection of 46,XY,rec(5), dup q, inv(5)(p13q33) using DNA analysis, flow cytometry, and in situ hybridization to supplement classical cytogenetic analysis. Am J Med Genet 31 : 643-654

Raimondi SC, Luthardt FW, Summitt RL, Martens PR (1983) High- resolution chromosome analysis of phenotypically abnormal pa- tients with apparently balanced structural rearrangements. Hum Genet 63 : 310-314

Young BD, Ferguson-Smith MA, Sillar R, Boyd E (1981) High reso- lution analysis of human peripheral lymphocyte chromosomes by flow cytometry. Proc Natl Acad Sci USA 78:7727-7731

Received February 21, 1989