Retinoblastoma, deletion 13q14, and esterase D: Application of gene dosage effect to prenatal diagnosis

Retinoblastoma, Deletion 13q14, and Esterase D: Application of Gene Dosage Effect to Prenatal Diagnosis Claudine Junien, Suzy Despoisse, Catherine Turleau, Henriette Nicolas, Franqois Picard, Bernard Le Marec, Jean-Claude Kaplan, Jean de Grouchy

ABSTRACT: Esterase D (ESD) gene dosage studies were performed on amniotic cells from a fetus at risk for del 13q14. The mother was a balanced carrier of an insertion in chromosome #20: 46,XX,ins(20;13)(p12;q1307q14.3). She had already given birth to a monosomic child with retinoblastoma (Rb) and to a phenotypically normal child trisomic for the same 13q14 segment. Both sibs displayed the expected proportionate gene dosage effects for ESD. A 153% value of ESD activity was found in the amniotic cells indicating unambiguously that the fetus was not monosomic for segment 13q14 and therefore not at increased risk for Rb. The mother delivered a phenotypically normal child who was confirmed to be trisamic for segment 13q14 by cytogenetic analysis and by gene dosage studies for ESD in cord blood cells and in lymphoblastoid cells. INTRODUCTION Retinoblastoma is a rare tumor which occurs in early infancy. Its prevalence is roughly 1 in 20,000 births with 95% sporadic and 5% familial cases. Only a few percent are due to a chromosomal aberration [1] (for review see [2]). Thirty-eight cases of retinoblastoma (Rb) with the interstitial deletion of 13q14 have been reported [3-11]. These cases i n c l u d e isolated cases, familial cases due to malsegregation of a parental rearrangement [6, 9-12], and cases of mosaicism [5, 6]. Gene dosage studies have s h o w n that the locus of h u m a n esterase D (ESD EC 3.1.1.1) and that of del 13q-Rb syndrome both lie in the m i d d l e of b a n d 13q14 [13]. The smallest region of overlap [SRO] was further refined to s u b b a n d 13q14.2 and the adjacent part of 13q14.1 [14]. A half-normal ESD value was found in a patient with Rb, but without visible chromosome #13 deletion [8]. This suggests that the loci for Rb and ESD are very closely linked. It also suggests that determination of ESD activity might be a more precise and simpler diagnostic tool than cytogenetic From the Institut de Pathologie Mol6culaire, Inserm U. 119, Paris (C.J., S.D., J.C.K.); Cytog6n~tique Humaine et Compar~e,ER.149 CNRS,U.173 INSERM,Paris (C.T.,J.deG.); Groupe de Recherche de Biologie Pr6natale, Centre Internationalde l'Enfance,U.73 INSERM,Paris (H.N.); Laboratoire de Cytog6n~tique,Facult6 de M6decine,Rennes (F.P.);and Consultationde G~n6tique,H6pital Pontchaillou,Rennes,France (B. LeM.). Address requests for reprints to Claudine Junien, Institut de Pathologie Mol~culaire, U.129 INSERM, 124 rue du Faubourg Saint Jacques F-75014 Paris, France. Received February 25, 1982; accepted April 14, 1982.

281 © Elsevier Science PublishingCo., Inc., 1982 52 VanderbiltAve., New York, NY 1 0 0 1 7

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c. Junien et al. analysis. This assay should, therefore, be applied to all cases of Rb to ascertain those families in whom a prenatal diagnosis would be possible. More or less precise regional chromosomal assignments of at least 24 enzymatic markers have been made by gene dosage studies [15]. The reliability of this method has been demonstrated on cultured cells derived from aborted embryos [15-17] and by the identification of deletion or duplication of a given chromosome segment on cells from patients with unbalanced chromosomal rearrangements [18, 19]. In a previous report we described a case of Rb due to malsegregation of a maternal insertion that was also responsible for a sib born trisomic for the same chromosome segment [10]. When the mother became pregnant for a third time, amniocentesis was performed for cytogenetic investigation. In view of the possible difficulties in chromosome analysis, we also decided to determine ESD activity.

FAMILY CASE REPORT Retinoblastoma was discovered in a 13-month-old child referred for evaluation of mental retardation. The patient's karyotype was described as 46,XY,del(13)(q1307 q14.3). The proximal break point was estimated at a point seven-tenths of the width of band q13; the distal break point, at the interface between subbands q14.2 and q14.3. The ESD activity was 46% and 61% of normal in red blood cells and fibroblasts, respectively [14]. The mother was found to be a balanced carrier of an insertion in chromosome #20: 46,XX,ins(20;13)(p12;q1307q14.3); her ESD activity was normal. A 4-year-old sister had inherited the abnormal chromosome #20: 46,XX,der(20),ins(20;13)mat, thus being trisomic for segment 13q1307--~13q14.3, with an ESD level 149% of normal (Fig. 1). She was however phenotypically normal with an IQ of 117 [10]. When the third pregnancy occurred, amniocentesis was performed at 16 weeks of gestation for cytogenetic and biochemical investigations.

MATERIAL Amniotic cells and fibroblast cell lines derived from a skin biopsy were cultured and lymphoblastoid cell lines were established as previously described [20, 21]. At birth, cord blood was collected in heparin for ESD determination and chromosome analysis.

METHODS Cytogenetic Investigations Karyotype analyses were performed after RHG and GTG banding. Prometaphase chromosomes were obtained by thymidine synchronization. R-banding was obtained after BrdU incorporation [22] and FPG staining (RTBG) and G-banding after treatment with trypsin (GTTG).

Enzymatic Investigations Fibroblasts, amniotic cells, and red blood cells were extracted in 0.7 mM mercaptoethanol and 0.27 mM EDTA. Glucose-6-phosphate dehydrogenase (G6PD), phosphoglucose isomerase (GPI), 6-phosphogluconate dehydrogenase (6PGD), pyruvate kinase (PK), lactate dehydrogenase (LDH), and enolase (ENO) were then estimated [23]; ESD activity was measured by the fluorescent method [24]. All enzyme activities were tested in triplicate.

283

A

mother -

patient

sister

RTBG

B

13

20

it| m

F i g u r e 1 (a) Chromosome pairs #13 and # 2 0 showing the insertion in the mother who is a balanced carrier 46,XX,ins(20;13)(p12;q1307q14.3); in the patient who is monosomic for 13q1307q14.3; and his sister who is trisomic for the same segment. (bJ Chromosome pairs #13 and # 2 0 of the sib reported in this paper.

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C. ]unien et al.

RESULTS

In order to eliminate variations due to nonspecific effects, we compared the ESD activity with six control enzymes encoded by chromosomes other than chromosome #13. Determinations were made simultaneously on (1) cultured amniotic cells from the fetus at risk, (b) fibroblasts derived from embryonic tissues of a regular trisomy 13 spontaneous abortion, and (c) cultured amniotic cells from a normal control. The normal range of ESD activities was also determined in four other control amniotic cell strains. The value obtained was 0.78 ± 0.20 IU/mg protein. As shown in Table 1, the ESD activity exhibited a sesquialteral gene dosage effect (1.5 x normal) in the fetus at risk as well as in the regular trisomy 13 embryo. Hence the fetus was considered trisomic for the segment that carries the ESD locus. However, this partial trisomy was regarded as harmless and it was decided not to interrupt the pregnancy (see Discussion). Cytogenetic investigations a few weeks later confirmed that the fetus, like the elder sister, had inherited the abnormal maternal chromosome #20 with the interstitial insertion of 13q1307-*q14.3. At birth, cord blood enzymes were assayed with age-matched controls. The ESD activity was increased in the red blood cells and in the lymphoblastoid cell line (Table 2). The level was compatible with the hypothesis of a partial trisomy for the segment carrying the gene for ESD. Chromosome banding analysis on cord blood lymphocytes confirmed the karyotype to be 46,XY,der(20),ins(20;13)mat. DISCUSSION

This is the first report of prenatal diagnosis of chromosome imbalance by gene dosage studies on amniotic cells. Normal values for enzyme activities in fibroblasts and cultured amniotic cells often show wide variation, and it could be supposed that this might hinder the search for gene dosage effects useful for prenatal diagnosis. However, in preliminary studies on amniotic cells from normal controls we found that the ESD activity did not vary more than that of other enzymes and that it was

Table 1 Enzyme activities in cultured amniotic cells from the fetus at risk and from normal control amniotic cells and in fibroblasts from a trisomy 13

Ratio

Enzymatic activities

{IU/mgprotein) Enzyme G6PD 6PGD GPI ENO PK LDH

SRO °

Fetus

T r i s o m y 13

Control

Fetus control

T r i s o m y 13 control

Xq28 lp34-pter 19pter-q13 1p34-p36 15q22-qter 11p123-p128

0.229 0.138 1.38 0,58 2.53 5.97

0.207 0.071 1.52 0.78 1.62 4.80

0.203 0.113 1.37 0.66 2.18 6.24

1,13 1,22 1.01 0,87 1.16 0.096

1.02 0.63 1.11 1.18 0.74 0.77

1.06

0.91

13q14

1.72

1.51

1.06

1.62

1.42

1.53

1.56

Mean ratio ESD ESD ratio m e a n ratio °SRO, smallest region of overlap (chromosomal assignment).

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Retinoblastoma, Deletion 13q14, and Esterase D Table 2

ESD activities in the cord b l o o d red cells and in the l y m p h o b l a s t o i d cell line from the n e w b o r n patient and from n o r m a l n e w b o r n s or n o r m a l controls Cord blood (IU/g hemoglobin) Newborn patient

ESD Ratio ESD patient control Mean ratio for other enzymes ° Ratio ESD mean ratio for other enzymes

Lymphoblastoid cell line (IU/mg protein)

Normal newborns Newborn patient

94.0

53.3 - 9.3

Normal controls

1.15

0.69

1.77

1.67

1.02

1.25

1.73

1.34

°Other enzymes tested were G6PD, 6PGD, GPI, LDH, ENO, PK.

in the same range as that of skin fibroblasts from normal adults. We also k n e w from previous studies of the Rb patient and his sister [10], and of a case of regular trisomy 13, that the sesquialteral or u n i p l e x gene dosage effect could be unambiguously d e m o n s t r a t e d in fibroblasts as well as in red cells. The level of ESD activity found in the amniotic cells clearly i n d i c a t e d that the fetus was not m o n o s o m i c for 13q14 and therefore not at risk for Rb. Moreover the 153% value of ESD was compatible w i t h a sesquialteral gene dosage effect indicating that the fetus was trisomic for 13q14. This was confirmed by cytogenetic analysis. It s h o u l d be e m p h a s i z e d that prenatal diagnosis of a c h r o m o s o m e i m b a l a n c e by gene dosage effect is feasible only after p r e l i m i n a r y fibroblast investigation in the i n d e x case and, if possible, in other m e m b e r s of the family to d e t e r m i n e the half and the sesquialteral values. Even though the p r o p o r t i o n of familial interstitial deletion or translocation of 13q14 remains low in the etiology of Rb, we believe that qualitative and quantitative assay of ESD should be carried out in patients at risk of d e v e l o p i n g Rb to p e r m i t early treatment. Since Rb can be transmitted b y unaffected carriers in the same family [9, 10, 12], the Rb case reported by M u r p h r e e et al. [8] w i t h half-normal ESD, but w i t h o u t detectable 13q deletion, stresses the necessity of measuring ESD even w h e n c h r o m o s o m a l analysis fails to reveal an abnormal 13q. In two recent surveys of 42 and 46 Rb patients [5, 6] m o s a i c i s m of del(13q14) was found in three cases. Unfortunately, these authors d i d not report ESD levels. The linkage b e t w e e n ESD and Rb is not unique; the vast majority of W i l m s ' tumors (95%), another c h i l d h o o d cancer, a p p e a r to be sporadic cases and o n l y a few percent are due to a c h r o m o s o m a l deletion. Deletion of b a n d 11p13 is k n o w n to be responsible for the WAGR c o m p l e x association: p r e d i s p o s i t i o n to W i l m s ' tumor or g o n a d o b l a s t o m a (W), aniridia (A), genitourinary abnormalities (G), and mental retardation (R) [25]. Gene dosage studies for catalase (CAT EC 1.11.1.6) in patients w i t h partial d u p l i c a t i o n or deletion of 11p s h o w e d that the locus of this enzyme m a p s to band 11p13 close to the locus (or loci) for WAGR [26]. The CAT activity was n o r m a l in 18 patients w i t h W i l m s ' tumor, w h i c h a p p e a r e d to be sporadic cases. It was also n o r m a l in two patients with the autosomal d o m i n a n t form of a n i r i d i a but decreased in three patients w i t h del 11p13 a n d WAGR [27]. F a m i l i a l occurrence of the a n i r i d i a - W i l m s ' t u m o r s y n d r o m e due to malsegregation of an

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C. J u n i e n et al. i n s e r t i o n a l t r a n s l o c a t i o n has b e e n r e p o r t e d in t w o different f a m i l i e s [28, 29]. In these f a m i l i e s catalase m e a s u r e m e n t s h o u l d also p r o v e u s e f u l for p r e n a t a l diagnosis.

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