Molecular cytogenetic study of supernumerary marker chromosomes in an unselected group children

American Journal of Medical Genetics 56:106-111 (1995)

Molecular Cytogenetic Study of Supernumerary Marker Chromosomes in an Unselected Group of Children

Claus H@jbjerg Gravholt and Ursula Friedrich Cytogenetic Laboratory, Institute for Basic Research in Psychiatry, Department of Biological Psychiatry, Psychiatric Hospital in Aarhus, Aarhus University Hospital, Risskov (C.H.G.), and Institute of Human Genetics, The Bartholin Building, Aarhus University, Aarhus (U.F.), Denmark

We report on an unselected group of 24 children with small supernumerary marker chromosomes, found in a large sample of 34,910 consecutive newborns karyotyped at birth. Sixteen of these were available for re- examination. With the use of in situ hybridization with a-satellite centromere probes and satellite 111, ribosomal and p-satellite DNA probes, we have characterized these markers. In 14 of the 16 cases we have been able to determine the chromosomal origin of the marker. 'helve of the markers are derived from the acrocentric chromosomes. Of these 12 markers, 4 are derived from chromosome 14, 4 from chromosome 22, 3 from chromosome 15 and one is from either chromosome 13 or 21. Ten of these markers were initially ascertained with the satellite I11 DNA probe, taking advantage of the fact that satellite I11 DNA is found in the centromeric region of the following chromosomes: 1, 5, 9, 13, 14, 15, 16, 20, 21, 22, andY. Two markers were derived from chromosomes 4 and 8. The origin of the last 2 markers could not be determined with the techniques employed. Only one of these children is psychometrically retarded and has a peculiar appearance. Unfortunately we were not able to determine the origin of the marker in her case. All other children developed normally. o 1995 Wiley-Liss, Inc.

KEY WORDS: small supernumerary marker chromosomes, fluorescence in situ hybridization, Q-, R- and C-banding, a-satellite

Received for publication February 17, 1994; revision received June 29, 1994.

Address reprint requests to Dr. Claus Hojbjerg Gravholt, Cytogenetic Laboratory, Aarhus Psychiatric Hospital, DK-8240 Risskov, Denmark.

0 1995 Wiley-Liss, Inc.

DNA probes, satellite I11 DNA probe, p- satellite DNA probe, ribosomal DNA probe

INTRODUCTION Until recently the identification of marker chromo-

somes has been dependent on morphological and staining characteristics. The introduction of molecular cytogenetic techniques has greatly increased the possibility for full characterization of supernumerary marker chromosomes. This is important for the evaluation of possible phenotypic effect.

In a large multicenter study 1/2,500 de novo small marker chromosomes were detected among 377,353 amniocenteses during a 10-year period. Extra chromosomes of identified origin such as i(12p) and i(18p) were not included in this risk calculation. Abnormal phenotypic outcome was seen in 13% of de novo markers. No significant risk difference between satellited and nonsatellited marker chromosomes was observed [Warburton, 19911. This presents a major diagnostic problem especially when a de novo marker is detected prenatally.

Several researchers have presented data using in situ hybridization with chromosome specific a-repeat centromeric probes to characterize marker and ring chromosomes [McDermid et al., 1986; Crolla et al., 1989, 1992; Callen et al., 1990a,b, 1991,1992; Lin et al., 1990a; Plattner et al., 1991, 1993; Cooper et al., 1992; Rauch et al., 1992; Stetten et al., 1992; Blennow et al., 1992, 1993; Suijkerbuijk et al., 1992; Koch et al., 1993; Michalski et al., 19931. So far, markers have been found originating from all chromosomes, except from chromosomes 5, 7, 10, and 17. Distinct syndromes are associ- ated with the occurrence of markers which are made up by i(12p), $ 1 8 ~ ) and tetrasomy of 22qll.

The present study characterizes marker chromosomes from 16 children found at random in a prospective study [Nielsen and Wohlert, 19911. The chromosomal origin was identified by in situ hybridization with chromosome specific a-repeat centromeric probes. The surrounding heterochromatin was further character-

Marker Chromosomes in Children 107

ized with satellite 111-, p-satellite- and ribosomal-DNA probes.

It is expected that the accumulated knowledge about the exact nature of different marker chromosomes and the possible delineation of new syndromes will become a useful tool in genetic counselling.

MATERIALS AND METHODS The study comprises an unselected group of 24 chil-

dren with small marker chromosomes, found among 34,910 consecutively liveborn children [Nielsen and Wohlert, 19911. The incidence was 1 in 1,500 births. We have been able to reexamine 16 of these children. The rest could not be reached or did not want to participate. All 24 children were seen at least once in connection with genetic counselling and the 16 children parti- cipating in this study have been examined 2-6 times during the follow-up studies of children with supernumerary marker chromosomes. They were physically examined, but no psychological tests were performed. Twenty-two of the 24 children developed normally. Of the nonparticipants, 4 were mosaics, 4 cases had inherited the marker chromosome from one of their parents, 3 were de novo and in one case the mode of inheritance could not be established.

One child (case 6013) was born at 29 weeks of gesta- tion and died 3% hours after birth; the autopsy showed: intracranial hemorrhage, ruptured cerebellar ten- torium, subtotal atelectasis of the lungs, polycystic de- generation of the right kidney and infarcts in the cotyle- dons of the placenta. The weight was 1,060 g and the length was 38 cm. Most of the changes were judged to be caused by the premature and complicated delivery. Her mother and several members of the family have the same marker chromosome and a normal phenotype. In- stead of the deceased child, the mother has been reex- amined in this study and her marker, which is identical to that of the proposita, has been characterized.

Chromosome Preparations The chromosome preparations were from 72-hour

PHA-stimulated lymphocyte cultures of peripheral blood. The slides were air-dried and preserved in 90% ethanol until use. Some of the slides were made from chromosome suspensions stored in fixative (methanol/ acetic acid 3:l) a t -20°C for up to 2 years.

DADAPI-, Q-, R- and C-banding were performed according to standard procedures. Prometaphase analysis on acridine orange-stained preparations was performed on selected patients in an attempt to establish the exact nature of the extra chromosome. In all cases it could be resolved that a marker chromosome and not a ring chromosome was involved.

In Situ Hybridization Labelling and in situ hybridizations were performed

according to a modified protocol [Gravholt et al., 19921. DNA probes were labelled with biotin [Gibco BRL 8247 SA, Gaithersburg, MD). Hybridization mixture was with 50% formamide, 10% dextran-sulfate and 4 X SSC. The slides were denatured at 72°C for 10 minutes, cooled on ice and hybridization was performed for 1-3

days at 37"C, washed at 52°C in 0.4 x SSC/O.l% Tween 20 and then at 37°C in 0.1 x SSC/O.1% Tween 20. When using low-stringency conditions, hybridization and washing were performed at room temperature.

Avidin-FITC (Vector A-2001) was used for probe detection. In cases with faint biotin signal, the signal was amplified by biotinylated anti-Avidin (Vector BA- 0300) diluted 1:200 in 2 x SSC and another layer of Avidin-FITC for up to three rounds. A mixture of Propidiumiodid (Sigma P 4170) and Antifade (10 ml so- lution consisting of 230 mg DABCO (1,4-diazabicyclo- [2.2.2.loctan), 800 pl HzO, 200 pl 1 M Tris-C1 (pH 81, 9 ml glycerin) was placed on the slides as counterstain- ing. In cases where a marker was found to be derived from chromosome 14 or 22, we subsequently hybridised at low stringency with probe aRI(680)-534. Using this probe under these conditions, it hybridizes only to chromosomes 13,14,18, and 21. If there was any signal on the marker it was concluded that its origin was from chromosome 14 and if not, from chromosome 22 [Callen et al., 19923. In one case where the marker was found to be derived from chromosome 4 or 9, it was subsequently concluded to be of chromosome 4 origin since it gave no signal with pMR9A (9 cen). FISH (fluorescence in situ hybridization) with chromosome specific probes was performed according to supplier's instructions (Angewandte Gentechnologie Systeme GmbH, 6900 Heidelberg, Germany) with minor modifications.

The DNA probes employed are listed in Table I.

RESULTS The more detailed characterization of the markers is

described in Tables I1 and 111. Cases 40, 28239, 32830 and 41934 were derived from chromosome 14. Cases 531, 36319, 39906 and 45539 were derived from chromosome 22. Cases 348, 6013 and 45604 were derived from chromosome 15. Case 348 was DADAPI-negative but showed 2 signals with FISH. Cases 6013 and 45604 were DADAPI-positive. Case 6013 showed 2 signals with FISH and one DMDAPI band, while case 45604 showed only a single signal with FISH, but 2 DADAPI bands. The configuration of these markers derived from chromosome 15 excludes the presence of an inverted duplication (inv dup(l5)), which often goes along with phenotypic abnormalities, since a small segment of euchromatin is bordered on either end by 15p material. One (case 42618) was either from chromosome 13 or 21, no more precise determination of origin being possible. Case 38587 was derived from chromosome 8 and case 39997 was from chromosome 4. Two cases (27621 and 27883) were unidentifiable, despite using an entire panel of a-satellite DNA probes from all chromosomes. In C-banding they did not show any signal (Fig. 1).

Of the 16 markers, 12 were of acrocentric origin. Ten of these had retained satellite I11 DNA and 7 had retained ribosomal DNA and p-satellite DNA. Two of the markers which had retained ribosomal DNA did not show any satellites; the other five did show satellites.

In 7 cases the marker chromosome was inherited from one of the parents. In 9 cases it was due to a de novo mutation (Table 11).

The phenotypic appearance of the children is sum- marized in Table 11. Case 531 was found to have only

108 Gravholt and Friedrich

TABLE I. DNA Probes

Name Type of DNA Chromosomal location

D1Z5 a-repeat 1 pBS4D a -repeat 2 pAE0.68 a-repeat 3 pG-Xba 111340 a-repeat 4 and 9 pG-Al6 a-repeat 5 and 19 p308 a-repeat 6 p7tl a-repeat 7 pJM128 a-repeat 8 pMR9A a-repeat 9 pAlORR8 a-repeat 10 pHS53 a- r e p e a t 11 pBR12 a-repeat 12 aRI(680)-534 a-repeat 13 and 21 aXT (680) a-repeat 14 and 22 pTRA-20 a-repeat 15 pZ16A a-repeat 16 p17H8 a-repeat 17 L1.84 a-repeat 18 pz20 a-repeat 20 p190.22 a-repeat 22 pBamX7 a-repeat X Cy84 a-repeat Y $BE ribosomal 13,14,15,21,22 P2lP p-satellite 9,13,14,15,21,22 pTRS-47 satellite I11 1,5,9,13,14,15,16,20,21,22,Y

Reference

Oncor Rocchi et al. [1990] Baldini et al. 119891 Hulsebos et al. [1988] Hulsebos et al. [1988] Jabs e t al. [19841 Wayne et al. [19871 Donlon e t al. [19861 Rocchi a t al. [19911 Devilee et al. [1988] Yurov et al. [19891 Looijenga et al. [19901 Jorgensen et al. [19871 Jorgensen et al. [1988] Choo et al. [1990bl Rocchi, personal communication Waye and Willard [1986] Devilee et al. [1986] Baldini et al. [1992] Rocchi, personal communication Willard et al. [1983] Wolfe et al. [19851 Sylvester e t al. [1986] Greig and Willard [19921 Choo et al. [1990al

minor anomalies in the form of hypertelorism and a flat nasal bridge, but her intelligence was normal at age 20 years.

One child (case 27621) has a peculiar appearance and psychomotor retardation and shows severe language deficit. She suffers from chronic middle ear infection, is incontinent for urine and stools, understands simple sentences, has a volatile temper and frequently throws herself into aggressive and destructive fits. She has no simian crease, low-set ears or hypertelorism. The size of her marker chromosome is 1/6 G. The patient’s mother, who was 29 years old at the first examination, is slightly mentally retarded. She has the karyotype 46,XX/47,XX,+mar/47,XX,+del(X)(pll) (89/7/5%). Her

karyotype is unstable. On repeated examinations it was as follows: at the age of 30 years: 46,XXl 47,XX,+mar/47,XX,+del(X)(pll) (96/2/2%) and at the age of 38 years: 46,XX/46,X,del(X)(pl1)/47,XX,+mar/ 47,XXX (97/1/1/1%). The maternal grandmother’s karyotype is: 45,X/46,XX/47,XXX (4/91/5%). She is 58 years old and of normal intelligence. The proband’s father and older sister have normal chromosomes. Since we suspected the marker chromosome in the proposita to be of X chromosome origin, we performed whole chromosome painting with an X chromosome probe, but no signal was seen on the marker. Testing with a-satellite DNA probes from all the other chromosomes gave a negative result, as did C-banding.

No.

40 348 531 6013 27621

27883 28239 32830 36319 38587 39906 39997 41934 42618 45604 45539

~

TABLE 11. Clinical Characterization of Markers

Phenotypic appearance ___ - Inheritance -

Karyot ype ____ 47,XX,+mar Maternal Normal at 21 years (y) 46,XX/47,XX,+mar De novo Normal at 21 y 47,XX, +mar Hypertelorism, flat nasal bridge, normal I& a t 20 47,XX, +mar Died 3 112 hour after birth. No dysmorphic features 46,XX/47,xX, +mar Maternal Peculiar facial features, psychomotorically retarded,

46,xx/47,xx, +mar De novo Normal at 11 v

De nova' Maternal

severe language deficit. 12 y

46;XY/47;XY;+mar 47,XY, + mar 47,XX, +mar 46,XX/47,xX, +mar 47,XX, +mar 46,XYl47,XY, +mar 46,XY/47,XY, +mar 46,XY/47,XY,+mar 46,XY147,XY, +mar 47,XY,+mar

Maternal Paternal De novo De novo Maternal De novo De novo De novo De novo Maternal

Normal at 11 f Normal at 9 y Normal at 8 y Normal at 7 y Normal at 7 y Normal at 7 y Normal at 6 y Normal at 6 y Normal at 5 y Normal a t 5 y

“Mother has the karyotype: 46,XX,inv(9)(pl lq13)/47,XXX,inv(9)(pllq13), (97/3%).

Marker Chromosomes in Children 109

TABLE 111. Cytogenetic Characterization of Markers

Comparable Rib0 Sat. I11 p-sat No. Karyotype size Percentage" Centromere DNA DNA DNA DAPI C band

40 348 531

6013 27621 27883 28239 32830 36319 38587 39906 39997 41934 42618 45604 45539

47,XX,+mar 46,XX/47,XX, +mar 47 ,XX, +mar 47,XX,+mar 46,XX/47,XX, +mar 46 ,XX/47 ,XX, +mar 46,XY/47,XY, +mar 47,XY,+mar 47,XX,+mar 46,XX/47,XX, + mar 47,XX, + mar 46,XY147,XY, +mar 46,XY/47,XY, +mar 46,XYl47,XY, +mar 46,XY147,XY, +mar 47,XY,+mar

112 G, Nb 112 G, N Almost G, s" 112 G, N 116 G, N 116 G, N 115 G, N 112 G, S 115 G, N Almost G, N 112 G, S 116 G, N 113 G, N 116 G, N G, s Almost G, S

100

100 100

22-24

51-40 19-17 61-59

100 100

100 48-67

76-71 47-67 39-45 50-58

100

14 15 15 22 22 15 15

? ? 14 14 22 8 22 4 14

1312 1 15 22

++ + -

-

NA NA + ++ NA ++ NA

-

- -

++ ++

++ ++ ++ -

-

NA -

++ -

NA ++ NA - -

++ +

N A ~ + + NA - -

+ +

NA + + + + +

NA + *Percentage cells with marker chromosomes at birth and at the latest examination. bN, nonsatellited. 'S, satellited. dNA, not available + +: the presence of two separate signals; +: the presence of one signal; -: no signal seen on marker.

DISCUSSION The phenotypic consequences of a de novo marker

chromosome are difficult to foresee. Originally most of the markers were thought to originate from chromosome 15 [Buckton et al., 19851. This observation was based on DADAPI staining which usually gives a clear fluorescent signal in the centromere regions of chromosomes 1, 9, 16, Y, and in the short arm of chromosome 15. However, several researchers have also found DADAPI fluorescence on other acrocentrics and the heterochromatic region of the Y chromosome [Babu et al., 1986; Buhler and Malik, 1988; Lin et al., 1990bI. Also, DADAPI-negative chromosome 15 has been described [Perez-Castillo e t al., 19871, comparable to our case 348.

In situ hybridization techniques now disclose that markers can be derived from a variety of chromosomes. Until now, marker chromosomes have been identified originating from all the acrocentrics, the sex chromosomes and almost all of the other autosomes. The marker chromosomes in this study have been detected in an unselected sample of consecutively newborn children. Of 24 children originally found with marker chro-

Fig. 1. C-banding chromosomes a: Case 27883. b Case 27621.

mosomes, 16 participated in the present study. To the best of our knowledge the 8 nonparticipating children were physically and psychologically normal. In our study, 15 of 16 children with marker chromosomes were normal. Only one child had a peculiar appearance and mental retardation. Unfortunately, this was one of 2 cases where the marker could not be characterized by the in situ hybridization probes employed. The marker was maternally derived in this case. The slightly mentally retarded mother had a mosaic X chromosome ane- uploidy with a deleted X chromosome. Therefore, this marker would be expected to be derived from X chromosome material. However, when painted with a probe covering the entire X chromosome, the marker did not show any signal. Isolation of the marker by flow-cytom- etry and subsequent reversed painting procedures are in progress to solve this problem.

None of the marker chromosomes could have been identified by cytogenetic examination employing Q- or R- banding. Only 2 DADAPI-positive cases later turned out to be of chromosomes 15 origin. The presence of a centromere could not be shown in all cases by C-banding (Table 111). As there is growing evidence that the majority of the marker chromosomes are derived from the acrocentrics, we applied FISH with a satellite I11 DNA probe first, followed by a-satellite probes from the acrocentric chromosomes. We found that altogether 12 of 16 markers were derived from the acrocentric chromosomes and furthermore that 10 of these had retained satellite I11 DNA, as shown with the pTRS-47 probe, making this a potentially valuable probe for ini- tial screening in case of marker chromosomes. As examination protocol, we propose starting with tra- ditional cytogenetics, including DADAPI and C-banding, followed by FISH with satellite I11 DNA and, if positive, continuing with the a-satellite probes from the acrocentric chromosomes under low stringency conditions, thereby subdividing the marker into different

110 Gravholt and Friedrich

a-satellite superfamilies, modifying the approach by Plattner at al. [1993]. Only 2 markers were derived from other autosomes (chromosomes 4 and 8). Unfortu- nately 2 markers could not be identified. One could imagine that these 2 markers had truncated a-satellite regions which may be impossible to visualize with FISH. A functional marker chromosome with no detectable a-satellite DNA, satellite I11 DNA or CENP-B protein has been described [Voullaire e t al., 19931.

As shown previously, the marker mosaics are not sta- ble, but tend to change constantly, at least when white blood cells are examined [Gravholt at al., 19911. Even inherited markers may appear as mosaics. The expla- nation could be that those containing ribosomal DNA proliferate, and those which do not are selected against and disappear. As seen in Table 111, large amounts of ribosomal DNA were seen in 4 of the 7 nonmosaic cases, whereas only one of the 5 mosaic cases examined had comparable signal with the ribosomal DNA probe. Fur- thermore, not all the marker chromosomes which gave a signal with the ribosomal DNA probe showed any satellites as would have been expected.

Prospective studies on unselected material most probably reflect the “true” incidence of markers. This study shows that the major proportion of markers do not have any phenotypical impact on the carrier. Mark- ers which are derived from the acrocentrics are most common and have most often no phenotypic effect. But even markers from the other autosomes can occur with- out being phenotypically deleterious, as seen in our 2 cases where a de novo marker from chromosomes 4 and 8 was diagnosed.

“The risk of physical abnormality or mental retardation to a foetus carrying a marker, is minimal if one of the phenotypically normal parents has that marker in a nonmosaic form. Conversely, the risk is very high if the marker chromosome is a de novo mutation andlor approximates in size to a G group chromosome and con- tains euchromatic material” [Buckton et al., 19851. This still holds true in a general way and most probably the risk for a den novo marker to produce and abnormal phenotype is about 13% [Warburton, 19911. It seems that what makes a marker chromosome deleterious to the phenotype is the presence of euchromatin [Stein- bach et al., 1983; Kaffe and Hsu, 1988; Djalali, 1990; Carrasco Juan et al., 19901, but other factors may play a role. Phenomenae such as isodisomy and imprinting should also be taken into consideration [Engel and DeLozier-Blanchet, 199 11.

ACKNOWLEDGMENTS We are grateful for the generous gift of several probes

from different researchers: pBS4D, pAE0.68, pMRSA, pZ16A, p190.22 and pZ20 from Mariano Rocchi, pG-Xba 111340 and pG-A16 from Dr. T. Hulsebos, p308 from Dr. E.W. Jabs, pTRA-20 and pTRS-47 from Dr. K.A. Choo, ~2113 from Drs. H. Willard and G.M. Greig, aRI(680)-534 and aXT (680) from Dr. A.L. Jor- gensen and pCBE from Dr. A.L. Bak. We also thank Dr. Klaus Weber Mathiessen who has been very helpful with advice and material from time to time. Monna

Caprabi is appreciated for her exquisite technical as- sistance. The involved families are thanked for their kind cooperation.

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155-164.

1244-362.

73~193-198.

7~349-356.

Documents

Molecular cytogenetic study of supernumerary marker chromosomes in an unselected group children