Am. J. Hum. Genet. 46:888-895, 1990

Molecular Studies of DiGeorge Syndrome Wendy J. Fibison, * Marcia Budarf, * Heather McDermid, * and Beverly S. Emanuel*

Frank Greenberg,t

*Departments of Pediatrics and Human Genetics, University of Pennsylvania School of Medicine, and Division of Human Genetics and Molecular Biology, Children's Hospital, Philadelphia; and tlnstitute for Molecular Genetics, Baylor College of Medicine, Houston

Summary DiGeorge Syndrome (DGS) is often associated with loss of a portion of the proximal long arm of chromosome 22. Using a probe for the D22S9 locus, we have examined DNA from eight DGS cell lines and from one balanced-translocation carrier parent of a DGS proband. The D22S9 locus is deleted in four DGS patients, with deletion of 22pter-wqll because of unbalanced translocation. The locus is not deleted from three DGS probands with normal chromosomes or from two DGS probands with interstitial deletions of 22q11. The interstitial deletion DGS probands are also heterozygous for D22S43, another proximal 22q11 locus. This suggests that D22S9 and D22S43 are in a flanking but not critical region for DGS. One of the interstitial deletion DGS probands is monosomic for BCRL2 but has two copies of the flanking BCRL4 and BCR loci. Thus, the region critical to DGS (DGCR) may be in proximity to the BCRL2 locus.

Introduction

DiGeorge syndrome (DGS) is a developmental defect of the third and fourth pharyngeal pouches. It is characterized by a variation in the severity of symptoms and by a wide spectrum of involvement. DGS patients manifest several congenital malformations, including absent or hypoplastic thymus, absent or hypoplastic parathyroids, and cardiac malformations of the conotruncal variety, such as truncus arteriosus, interrupted aortic arch, and tetralogy of fallot. In addition, facial dysmorphology and defects in other organ systems have been reported (DiGeorge 1965; Robinson 1975; Conley et al. 1979; Greenberg et al. 1988). The basic etiology of DGS is unknown. The strongest evidence for a genetic etiology is an association with loss of a portion of chromosome 22. Approximately 10%-15% of reported DGS cases are associated with chromosome abnormalities, of which the greatest Received May 23, 1989; revision received December 14, 1989. Address for correspondence and reprints: Beverly S. Emanuel, Ph.D., Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104. 1. Present address: Department of Genetics, University of Alberta, Edmonton, Alberta, Canada. i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4605-0004$02.00

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majority have unbalanced translocations and demonstrate a loss of the short arm and proximal long arm of chromosome 22,22pter-qll, resulting in monosomy for this region. The remaining long-arm material, 22q11-qter, is translocated to a number of different autosomes (Back et al. 1980; de la Chapelle et al. 1981; Kelley et al. 1982; Greenberg et al. 1984, 1988; Augusseau et al. 1986; Bowen et al. 1986; Faed et al. 1987). Most recently, two patients with DGS and interstitial deletion of 22q11.21--q11.23 have been reported (Greenberg et al. 1988; Mascarello et al. 1989). It has been hypothesized that the deletion of contiguous genes located on chromosome 22 results in DGS and that the region critical to DGS (DGCR) lies in 22q11 (de la Chapelle et al. 1981; Kelley et al. 1982; Schmickel 1986). In order to gain more insight into the genetic etiology of DGS and to begin to define a DGCR, molecular studies of 22q11 were undertaken. A number of genes and anonymous DNA probes have been assigned to 22q11. Several proximal loci are D22S9, D22S43, BCR, BCRL2, BCRL3, and BCRL4. Southern blotting and DNA dosage analysis were performed, using probes for these loci, on cell lines from DGS patients in order to determine whether these cell lines were monosomic or disomic for D22S9, D22S43, BCR, and the three BCR-like sequences. The results of these studies begin to delineate a DGCR.

Molecular Studies of DGS

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Material and Methods Material

Cell lines. - Seven cell lines were obtained from the Human Genetic Mutant Cell Repository (Coriell Institute for Medical Research, Camden, NJ). Three of the cell lines, established from individuals with DGS, are associated with unbalanced translocations; these cell lines are GM2944A (Kelley et al. 1982), GM3577 (Kelley et al. 1982), and GM5401 (Greenberg et al. 1984). In each of these cell lines, there is only one normal chromosome 22 and a derivative autosome with a portion of the long arm of 22 translocated to it. Each cell line is monosomic for 22pter--q11 (fig. 1). GM2944A is a fibroblast line with karyotype 46,XY,-22,+der(20) t(20;22)(qll;qll). GM5401 is a fibroblast line with karyotype 45 ,XY,-4 -22, +der(4)t(4;22)(q35.2;qll). GM3 577 is a fibroblast line with karyotype 45,XY,-3, -22,+der(3)t(3;22)(q29;qll). GM5878 is a lymphoblastoid line established from the balanced-translocation carrier parent of a DGS child. The karyotype of this cell line is 46,XYt(10;22)(q26.3;qll.21) (Kelley et al. 1982; Cannizzaro and Emanuel 1985). Three DGS cell lines with normal karyotype-i.e., (1) GM5876, a fibroblast line, 46,XY; (2) GM7215, a fibroblast line, 46,XX; and (3) GM7939, a lymphoblastoid line,

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Lost

11

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12 13

11

12

q

q1i -q ter Figure I Idiogram showing portion of the short arm and proximal long arm of chromosome 22 that is lost in the DGS probands with unbalanced translocations of chromosome 22. Remaining longarm material (qll-qter) translocates to an autosome.

46,XY-were also studied. In addition, two DGS cell lines with 22q11 interstitial deletions were studied. Patient 27 (Greenberg et al. 1988) was given the lab number 7248. It is a fibroblast line with the karyotype 46,XY,del(22)(qll.21q11.23). A lymphoblastoid line (Mascarello et al. 1989), lab number KM4987, has the karyotype 46,XX,del(22)(qll.21q11.23). Probes-p22/34 (D22S9) is a 1.8-kb EcoRI-HindIIl, single-copy, anonymous human genomic fragment isolated from a flow-sorted chromosome 22 library and localized to 22q11 by chromosomal in situ hybridization (McDermid et al. 1986), linkage analysis (Rouleau et al. 1989), and somatic cell hybrids (Budarf et al. 1989). It is inherited in a Mendelian fashion, is polymorphic for TaqI in human genomic DNA, and recognizes two alleles-one of 5.8 kb and one of 3.2 kb. p22/34 gives a single 5.2-kb band when hybridized to genomic DNA cut with HindIII. The probe used as an internal control for p22/34 was 13NS2, a unique human genomic 3-globin fragment derived from the second intervening sequence of the P-globin gene. It is approximately 920 bp and gives a 7.8-kb band when used as a probe against human genomic DNA digested with HindIII (gift of S. Surrey). ,BNS2 maps to the short arm of chromosome 11, an autosome not affected in DGS, and gives a band distinct in size from the test probe on Southern blot analysis of human genomic DNA. The probe used for the BCR-related genes is a 160bp, cDNA, HindIll/EcoRI fragment from the 3' end of BCR (Lifshitz et al. 1988). It recognizes four loci: BCR, BCRL2, BCRL3, and BCRL4. HindIII digestion of human genomic DNA produces bands of 9 kb (BCR), 13 kb (BCRL2), 19.5 kb (BCRL3), and 23 kb (BCRL4) with this 3' BCR probe. The 9-kb BCR band was used as an internal control for the 13-kb BCRL2 band. The pH32 probe (D22S43) was isolated from the flow-sorted HindIll chromosome 22 library (LL22NS01) (Budarf et al. 1986). It is polymorphic with TaqI, producing alleles of 5.0, 4.0, and 3.5 kb in human genomic DNA. Methods In situ hybridization was carried out, using methods described elsewhere (Cannizzaro et al. 1985), with the p22/34 (D22S9) probe. Banding was achieved using a modified Wright-Giemsa staining method (Cannizzaro and Emanuel 1984). Fifty metaphase spreads with morphologically identifiable chromosomes were analyzed. Only metaphase spreads with grains situated on nonoverlapping chromosomal regions were included in the present study.

Fibison et al.

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High-molecular-weight genomic DNA was prepared from cultured cells by using standard techniques. Approximately 2 gg DNA were digested to completion with the restriction enzyme TaqI or HindIII (New England Biolabs) at 651C or 370C, respectively, for 5 h. DNA was electrophoresed through a 0.8% agarose gel and transferred to Gene Screen Plus (New England Nuclear) by Southern blotting (Southern 1975). Filters were hybridized overnight at 420C by using 50% formamide, 3 x SSC, 5 x Denhardt's, 1% SDS, 100 gg herringsperm DNA/ml. Probe was labeled with [a-32P]-dCTP (Amersham) by using a random oligonucleotide priming method (Feinberg and Vogelstein 1984). Filters were washed at 650C with 0.2 x SSC and 0.1% SDS. Filters were exposed to Kodak XAR-5 film with intensifying screens at -70'C for 1-5 d. Data sets for the densitometric analysis consisted of multiple lanes on a gel, one lane containing DNA from a DGS test cell line alternating with one or two lanes containing genomic DNA from normal controls expected to contain two copies of the test probe. The data sets were repeated across the blot to control for variation due to position on the gel or blot and were analyzed from autoradiograms by using a Zeineh Model SL 504 XL densitometer (Biomed Instruments, Fullerton, CA). Results In Situ Hybridization In situ hybridization studies were performed on GM5878, the balanced-translocation carrier parent of a DGS child, by using p22/34 (D22S9) as probe (fig. 2), because cells on the deceased proband were unavailable. The der(22),(22pter--'qll.21::10q26.3--'qter) is retained in every metaphase, as well as the der(10) translocation chromosome. Of 129 grains counted, 15 (12%) localized to the normal 22, and 12 (9%) localized to the der(22). Only one grain localized to the der(10), and this was on the short arm, which is uninvolved in the translocation. This one grain is interpreted as being background hybridization and, therefore, is not significant. These results suggest that D22S9 is proximal to the 22q11 breakpoint in this (10;22) translocation carrier. Since the DGS offspring-45,XX,-10, -22,+der(10)t(10;22)(q26.3;qll.21)pat (Kelley et al. 1982)-of this individual did not receive the der(22), she would have lost one copy of this locus. Presence or absence of D22S9 alleles could be directly determined by Southern blot analysis of the other DGS probands.

46, XY, t(10;22)(q26;qll)

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normal 22

00200

a 0 110,00090

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derivative 22

derivative 10 Figure 2 Distribution of silver grains, after in situ hybridization, over both the normal chromosome 22 and the derivative chromosomes 22 and 10 in the balanced-translocation carrier parent of a proband with DGS (GM5878). p22/34 (D22S9) is the probe.

DNA Analysis-RFLP

Cell lines from DGS probands were analyzed for presence of two alleles by RFLP analysis, and those which demonstrated a single allele were further studied by DNA dosage. The p22/34 probe for the D22S9 locus displays a TaqI polymorphism in human genomic DNA, giving two alleles-one 5.8 kb and one of 3.2 kb. Figure 3 shows the autoradiographic results of Southern blot analysis of genomic DNA digested with TaqI. In the three DGS cell lines with unbalanced translocations-i.e., cell lines GM2944A, der(20), (lane 2); GM3577, der(3), (lane 4); and GM5401, der(4), (lane 5)-only the 5.8-kb allele is present. These are cell lines that are monosomic for the short arm and proximal long arm of 22: pter--'qll. Southern blot data are consistent with either hemizygosity or homozygosity for the 5.8-kb allele of D22S9 in these cell lines. One of the interstitial deletion cell lines, 7248, demonstrates only the 3.2-kb allele for D22S9 (data not shown). This finding is also consistent with either hemizygosity or homozygosity for the 3.2-kb allele of D22S9 in 7248. Two of the DGS cell lines with normal karyotypes (lanes 6 and 7) have both alleles, whereas the third DGS cell line with normal chromosomes-i.e., cell line GM5876 (lane 5)-shows only the 5.8-kb band. GM5878, the balanced carrier of a DGS-related t(10;22), shows both

Molecular Studies of DGS 0 0

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Figure 3 Southern blot analysis of genomic DNA from DGS cell lines and a normal control (lane 1), digested with TaqI and probed with p22/34 (D22S9). Der(20), GM2944A (lane 2); der(3), GM3577 (lane 3); and der(4), GM5401 (lane 4) are DGS cell lines with unbalanced translocations. The remaining three DGS cell lines (lanes 5-7) have normal karyotypes: GM5876 (lane 5), GM7215 (lane 6); and GM7939 (lane 7). Phage X digested with HindIII were used as molecular-weight standards. The upper band is 5.8 kb, and the lower band 3.2 kb.

the 5.8- and 3.2-kb alleles, as does KM4987, an interstitial 22q11 deletion line (data not shown). The pH32 probe to the D22S43 locus displays a TaqI polymorphism (Budarf et al. 1988; M. Budarf, et al. unpublished data), producing alleles in human genomic DNA of 5.0, 4.0, and 3.5 kb. It maps proximal to the constitutional t(11;22) breakpoint and is not amplified in K562. K562 is a cell line from a blast-crisis CML patient with an amplification of 22q11 which extends proximally from BCR, ending in the region proximal to BCRL2. Southern blot analysis of the interstitial deletion cell lines, KM4987 and 7248, reveals that both are heterozygotes for the D22S43 locus. DNA Analysis-Dosage

For those cell lines showing only a single TaqI allele of D22S9, Southern blotting of HindIll-digested DNA followed by densitometric analysis was performed to determine the copy number. Densitometric analysis of HindIll-digested DNA from the balanced carrier, GM5878, showed two copies of D22S9. This is in agreement both with the Southern blot TaqI polymorphism data, which showed two D22S9 alleles, and with the

in situ hybridization data, which showed hybridization of D22S9 to the normal 22 and der(22) chromosomes. Figure 4 shows a representative autoradiograph used for densitometric analysis. Lane A shows DNA from the unbalanced-translocation cell line, GM2944Ai.e., (46,XY,- 22,+ der(20)t(20;22)(qll;qll) - and lanes B and C show DNA from normal controls. The internal control is ,BNS2. Visual observation shows that the signal for D22S9 is less intense in the A lanes as compared with INS2 in the B or C lanes. With the densitometer, the ratio between the signal from D22S9 and that from 13NS2 was determined (table 1). The ratios of the test probe to the internal control for the unbalanced DGS line are quite different from the ratios for the normal controls. The Mann-Whitney U-test showed these results to be statistically significant. The results are consistent with loss of one D22S9 allele in the chromosomally abnormal DGS cell line GM2944A (table 1). Similar autoradiographs and ratios were obtained for the other two unbalanced translocation lines, GM3577 and GM5401 (table 1). Thus, one copy of the D22S9 locus is deleted from each of the DGS cell lines with unbalanced translocations. D22S9 is present in two copies in the chromosomally normal DGS cell line, GM5876 (data not shown). By densitometric analysis, we have also demonstrated that D22S9 is present in two copies in 7248, one of the interstitial deletion DGS cell lines which was uninformative by TaqI polymorphism analysis. A probe for the 3' end of the BCR gene recognizes three BCR-like sequences in human genomic DNA: BCRL2, BCRL3, and BCRL4 (formerly called BCR2, BCR3, and BCR4, respectively). These have been mapped to 22q11 (Croce et al. 1987), and their order

A

B C

A

B C

A B

C

-_3NS2 - D22S9

Figure 4 Southern blot analysis of genomic DNA digested with HindIII and probed with p22/34 (D22S9) and OiNS2, the internal control. Lanes A, DGS cell line GM2944A, which has an unbalanced translocation with loss of 22pter-qll. Lanes B and C, Normal individuals with normal karyotypes. The J3NS2 band has a molecular weight of 7.8 kb, and the D22S9 band is 5.2 kb. Phage X digested with HindIII were used as molecular-weight standards.

Fibison et al.

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Table I p22/34:NS2 Ratios

Cell Line, Karyotype

Test

GM2944A, 46,XY, - 22, + der(20)t(20;22)(ql 1;ql 1) .....

.6.2.8 .1 .2

.5 .9

.4 1.0

.6 .7 .7

1.1 1.3 1.3 2.0

1.2 2.2

.......

.9

1.5 1.6 1.5

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

.7 1.1 1.9

GM3577, 45,XY, - 3, - 22, + der(3)t(3;22)(q29;q I1) .....

GM5401, 45,XY,-4,-22,+der(4)t(4;22)q(35.2;qll) .....

7248, 46,XY,del(22)(qll.21q1I.23)

.........

Control Control

.........

1.8 1.6

GM5878, 46,XY,t(10;22)(q26.2;q1I.2) ......

has been determined (Budarf et al. 1988a). BCRL2 and BCRL4 are amplified in K562 and flank the constitutional t(11;22) breakpoint, with BCRL2 proximal and BCRL4 distal. These loci are distal to D22S9 (Budarf

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Molecular studies of DiGeorge syndrome.

DiGeorge Syndrome (DGS) is often associated with loss of a portion of the proximal long arm of chromosome 22. Using a probe for the D22S9 locus, we ha...
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