American Journal of Medical Genetics 41:4!54-459 (3991)

Relative Order and Location of DNA Sequences on Chromosome 21 Linked to Familial Alzheimer Disease Stefan-MatthiasPulst, Teresa Yang-Feng, and Julie R. Korenberg Division of Neurology (S.-MP.), Medical Genetics Birth Defects Center, Ahmanson Departments of Pediatrics (J.R.K.) and Medicine (S.M.P.), Cedars-Sinai Medical Center, University of California, Los Angeles; Department of Human Genetics (T.Y.-F.),Yale University, New Haven, Connecticut

Recently, a gene causing familial Alzheimer disease (FAD) was linked to DNA probes on chromosome 21 by genetic analysis. To investigate the precise physical location of these DNA probes, we have constructed a physical map of this region of chromosome 21 by using quantitative Southern blot analysis of cell lines aneuploid for parts of chromosome 21. The following DNA sequences were investigated: D21S16, D21S13, FB68L (cDNA probe for the amyloid protein precursor [APP] gene),and D21S1. We find that all DNA probes are located in the same region of chromosome 21, in q11.2-q21.05. We further show that D21S16 must be centromeric to D21S13, because D21S16, but not D21S13 is present in one copy in a cell line with deletion of the region 21pter-21q 11.2. High resolution chromosome analysis is presented to define this breakpoint. This new panel of aneuploid cell lines will allow the rapid mapping of new DNA probes in the vicinity of the FAD gene.

KEY WORDS: human chromosome 21, physical mapping, genetic markers INTRODUCTION Intense interest has focused on the genetic and physical map of human chromosome 21. Not only is chromosome 21 the smallest autosome and therefore particularly suited for mapping studies, but defects of it greatly affect human welfare. There are 2 major concerns. Trisomy of this chromosome (Down syndrome) is the most common cause of mental retardation and congenital heart defect [reviewed in Epstein, 19861. In addi~~

Received for publication November 21, 1990; revision received January 21, 1991. Address reprint requests to Julie R. Korenberg, Ph.D., M.D., Medical Genetics, A.S.B. 3 Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, Calif 90048.

0 1991 Wiley-Liss, Inc.

tion, 2 genes involved in the pathogenesis of Alzheimer disease have been mapped to this chromosome. One is the gene coding for the amyloid precursor protein (APP) [Tanzi et al., 1987;Korenberg et al., 19891. The second is a gene coding for familial Alzheimer disease (FAD) [St. George Hyslop et al., 19871. A major unresolved question is whether the genetic causes of FAD are heterogeneous that is, whether FAD may be due to genetic defects at more than one locus in the human genome. A detailed study of single FAD families using genetic linkage analysis with restriction fragment length polymorphisms (RFLPs) has proven to be difficult for 3 reasons [St. George-Hyslopet al., 1987; Goate et al., 1989; Schellenberg et al., 19891. First, in FAD families, only a small number of probands can confidently be diagnosed or excluded as having AD. Second, some of the currently available DNA sequences are not highly polymorphic and their limited informativeness has led to differing estimates of their genetic distances. Third, only one DNA marker has been mapped to the region between the centromere and the loci D21S13 and D21S16 (D.Kurnit, personal communication). Consequently, the true physical location of the FAD gene has yet to be determined. In view of these problems, it is clear that there exists an immediate need for physical mapping studies in the FAD region to align the genetic and physical maps of this region and to provide a means to isolate new DNA probes which map closer t o the FAD locus. This task has been slow in part due to the lack of a fine-structure map of proximal chromosome 21, the lack of efficient mapping techniques, and the lack of probes. We have now overcome some of these difficulties and developed a detailed mapping panel for this region.

MATERIALS AND METHODS Cell Lines We used cell lines which are aneuploid for parts of chromosome 21 (Fig. 1).These include DELBlJC, a lymphoblastoid cell line derived from a patient with an interstitial deletion of chromosome 21 including the region 21q11.2-q21.3 [Korenberg et al., 19911, and a fibroblast cell line from a patient carrying an un-

Order of FAD Linked DNA Probes CHROMOSOME 2 ‘ I CELL LINES TRlSOMlC GM1413

GM1399

1 I I I I I I I I I I I

T I I I I I

1

I

PROBES

MONOSOMIC

1 GM0692

DELLIJC

n

-

12

__

1

APP, D21S46

22.3 3 D21S39,D21S42 1 Fig. 1. Physical map of human chromosome 21: location of chromosome 21-specificprobes and breakpointsof cell lines used in the present study.

455

iments. Plasmid inserts were isolated by preparative gel electrophoresis and labelled with p32 dCTP by oligonucleotide priming to a specific activity of 2-5 x lo9 cpm/Fg according to the manufacturer’s specification (Amersham).

Southern Blot Hybridization A total of 8 separate agarose gels were run. In each, 510 pg of each DNA was digested with Eco RI according to the manufacturer’s directions (Bethesda Research Laboratories), electrophoresed in duplicate or triplicate lanes in 1.0% agarose gels with 1X Tris-Borate buffer (TBE) [Maniatis et al., 19821, and transferred to nylon membranes (Hybond, Amersham) by standard techniques [Southern, 19751. Membranes were baked at 80°C for 1 hour. After pre-hybridization, membranes were hybridized simultaneously with 2-4 DNA sequences including a reference probe of known location and copy number (see above). Re-hybridization and hybridization were carried out at 55°C in 15%formamide, 4% SDS, 1%bovine serum albumin, 1mM EDTA, 100 pg/ml salmon sperm DNA, and 0.2 M sodium phosphate, pH 7.2. After overnight hybridization, membranes were washed in a solution of sodium sodium citrate (SSC), 1% SDS, 0.1% sodium pyrophosphate, in which the SSC concentration varied from 2 x -0.2 x and the temperature from 22°C to 65°C. The final wash was in 0.2 x SSC at 65°C for 15 minutes. The membranes were exposed to Kodak XAR-5 X-ray film with intensifying screens. Exposures were chosen such that the densities of all bands fell into the most linear part of the X-ray film as judged by comparison to a standard scale. The standard scale was generated by densitometric analysis of XAR-5 film exposed with a National Bureau of Standards penetrometer (data not shown).

balanced translocation involving chromosome 21 (GM0692) and consequently deleted for the region 21pter-q21.05. Also included are GM3268, a fibroblast cell line derived from a patient carrying an unbalanced translocation t(9;21) resulting in deletion of chromosome 21 including 2lpter-qll and duplication of chromosome 9pter-qll (see below for cytogenetic breakpoint determination); and GM1413 [Williams et al., 19751trisomic for 21q21.05-qter and GM1399, from the same family and therefore involving the same chromosome breakpoint, trisomic for 21pter-q21.05. Cell lines GM0692, GM3268, GM1413, and GM1399 were obtained from the NIGMS cell Repository, Camden, New Jersey and the breakpoints in GM1413 and GM1399 were defined both by Williams et al. and NIGMS. Two human diploid placental DNAs were used as controls. Cell lines were grown to confluency in Earle’s minimal Cytogenetics essential medium with 10%fetal calf serum and L-gluChromosomeswere prepared from fibroblast cultures tamine (2 mM) before DNA isolation. High molecular (GM3268) using standard techniques for high resolution weight DNA was prepared from cell lysates treated overnight at 50°C with sodium dodecyl sulfate (SDS) chromosome analysis. (1.0%) and proteinase K (100 pg/ml) followed by 2-3 Data Analysis phenollchloroformextractions and ethanol precipitation Autoradiograms were scanned with a Helena EDC [Maniatis et al., 19821. densitometer and the area under the curve integrated DNA Probes by computerized linear interpolation. The copy number FB68L is a cDNA probe corresponding to the 3’ end of of a given DNA probe was calculated by normalization of the APP gene [Tanzi et al., 19871.SF13A and SF43 are 2 its hybridization signal with that of a reference probe single copy DNA probes defining 2 loci, D21S39 and (SFlSA, SF43, HHH202) as described under Results. D21S42, both mapped to 21q22 [Korenberg et al., 19871, The data were analyzed by one-tailed t-test. and more recently to 21q22.3 [Korenberg and FalikRESULTS Borenstein, unpublished]. SF85, defining the locus Figure 2 shows a typical autoradiogram of DNAs from D21S46 has previously been mapped to 21q11.2-q21.05 [Korenberg et al., 19871. The location of these probes is cell lines GM1399, GM0692, DELBlJC, and diploid plasummarized in Fig. 1. Probes GSE9 and GSM2l defin- centa simultaneously hybridized with 3 DNA probes. A ing the loci D21S16 and D21S13 are random single copy total of 5 additional Southern blots, each with 2 controls sequences from chromosome 21 that have been localized and 2-3 lanes for each of the test DNAs (GM0692, to 21qll-q21 [Stewart et al., 19851. PW228C defining DELBlJC, GM1399, GM1413) were hybridized with a the locus D21S1 has been mapped to 21qll-q22.1 [Van probe mix containing either D21S16 or D21S13, in addiKeuren et al., 19861.The DNA probe HHH202 defining tion to two others D21S1 or FB68L (APP). The actual the locus D17S33 and mapping to 17q11.2-q21[White et number of determinations of copy number for each probe al., 19871was used as the reference probe in some exper- and cell line is shown in Figure 3. All autoradiograms

456

Pulst et al.

Fig. 2. Copy number determination of probe GSM2l (D21S13). EcoRZ digested DNA's of normal placenta and cell lines with partial aneuploidy of chromosome 21 (DELBlJC, GM0692, GM1399) hybridized simultaneously to probe M21 and 2 chromosome 21-specific DNA sequences. Note the apparently decreased density of bands for D21S13 and D21S46 relative to that of D21S39 in DEL2lJC and GM0692 vs. placental DNA and the increased density of these bands in GM1399.

3 GM 1 3 9 9

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Fig. 3. Copy number determinations of chromosome 21-specific DNA sequences in 4 cell lines with partial aneuploidy for chromosome 21. The copy numbers were determined by comparison with a reference probe of known copy number and location in these cell lines.

were scanned by densitometer and the hybridization signal quantified by computerized integration. The gene copy number was determined in the following way: in order to account for the loading of slightly different amounts of DNA and the possibility of differential transfer between lanes, a probe of known copy number and location (DNA probes for the loci D21S39,

D21S42, or D17S33) was chosen as the reference probe for each lane. First, for each Southern blot the ratio of the hybridization signal for each probe to the reference probe was calculated for each lane ofplacental DNA and the results averaged. This averaged ratio represented the control ratio for that experiment. Second, the average ratio of each probe to the reference probe was calcu-

Order of FAD Linked DNA Probes

457

TABLE I. Gene copy number for 6 DNA probes in 5 partially aneuploid cell linest Cell lines Probes S16

S13

s1 S46

X

GM0692 0.98*

.08

.22

.06

X

3.08* .16 3.30* .13 3.01* .10 3.14** .18 1.90 .ll

2.09 .31 2.09 .07 n.d.

1.00* .09 0.81* .05 0.88 .03 n.d.

s.e X

s.e. X X

s.e. s39

GM1413 1.78

s.e.

s.e. APP

GM1399 2.96*

X

s.e.

2.00 .19 2.97* .12

DEL2lJC

2.03 .21

0.88** .11

0.76** .12 0.81*

GM3268 0.95* .15 2.16 .15 n.d.

.08

0.72

n.d.

n.d.

n.d.

n.d.

n.d.

*different from 2.00 by t-test analysis P < 0.01. **different from 2.00 by t-test analysis P < 0.002. t Abbreviations: x, mean; s.e., standard error of the mean.

lated from the lanes of the test cell lines. Each of these was then divided by the control ratio for the respective probe and averaged. This “standardized ratio” was then multiplied by 2 and yielded the DNA copy number for 4 each probe in each experiment. The individual values for each probe in each cell line 24 are shown in Figure 3. For each probe the DNA copy 23 22 numbers were averaged and the mean copy number P 21 tested for differencefrom 2.0 by t-test (TableI).We found 13 13 P 12 the probes for D21S16, D21S13, D21S1, and APP were -11.2 -11 21 present in single copy in cell lines GM0692 and in 11 1 11:2 DEL21JC. This establishes their location in the chromo12 q 21 some region deleted in both of these cell lines, i.e., 13 22 21q21.11.2-q21.05 (Fig. 1).The distal border of this as21 signment was confirmed by analysis of cell lines GM1399 and GM1413. The probes for D21S16, D21S13, q 22 D21S1, and APP are present in 3 copies in GM1413 and in 2 copies in GM1399 and therefore map centromeric to 34 the breakpoint in 21q21.05. The location of D21S16 and D21S13 was further refined by analysis with cell line GM3268. The chromo9 somal rearrangement carried by this cell line was previously characterized by the NIGMS using standard cytogenetic techniques as a fusion joining the short arm of chromosome 9 with the long arm of chromosome 21 at the centromeres of the respective chromosomes. Higher resolution chromosome analysis, as shown in Figure 4, showed the translocation breakpoints to be in 9 q l l and in 21qll. Although the region is very small and consequently difficult to define, most ofband 21q11.2 appears to be present, suggesting the breakpoint in chromosome 21 is close to the border of 21qll.l-q11.2. This results in a deletion that includes 21pter-q11.2. The molecular analysis showed that the probe for D21S16 was present in single copy, whereas the probe for D21S13 was present in the normal 2 copies (Fig. 5A, Table I). Since the probes for D21S16 and D21S13 give bands of similar size, initial hybridizations were either done on different Southern blots or in subsequent hybridizations of the same membrane. To allow compariFig. 4. Ideogram and chromosomes showing 9;21 translocation carson of the D21S16/D21S13 hybridization signal on the ried by cell line GM3268. Chromosomes 9,21, and the t(9;21) are shown same Southern blot, DNAs were electrophoresed €or an for 2 cells.

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Fig. 5. Copy number determinations of probes GSM2l (D21S13) and GSE9 (D21S16) in cell line GM3268. A Copy number is determined by comparison with a reference probe (SF43A) of known location and copy number in GM3268. B: Copy number is determined by direct comparison of the hybridization signal of both probes in GM3268 and diploid placenta.

extended time period in subsequent experiments. The resulting Southern blots were then simultaneously hybridized with both probes. The ratios of the hybridization signal for D21S13 divided by that for D21S16 in diploid DNA and in DNA from GM3268 are shown in Fig. 5B. In diploid DNA the mean ratio is 1.16, but 2.16 in GM3268. The mean ratios are highly significantly different by t-test analysis (P < 0.001) supporting that D21S16 is present in one copy in GM3268. This confirms the physical location of these probes as 21q11.2 and their order as centromere-D21S16-D2lSl3.

DISCUSSION We have constructed a panel of aneuploid cell lines that divides the proximal region of chromosome 21 from the centromere to band q21 into 4 regions. Using this panel, we have physically mapped 4 DNA probes to a small chromosome region. Further, we have confirmed the order of the 2 DNA sequences most tightly linked to FAD as D21S16 centromeric to D21S13. Finally, from these results we have identified cell lines carrying chromosome breakpoints that may bracket the gene for FAD. We have previously mapped the APP gene to 21q11.2q21.05 using quantitative Southern blotting of DNA probe FB68L and cell lines DEL2lJC and GM0692 [Korenberg et al., 1989, 19911. This placed the APP gene clearly centromeric to the minimal region causing the facial and cardiac changes of the Down syndrome phenotype [Korenberg et al., 19901. This map position was in agreement with some previous assignments [Jenkins et al., 1987;Tanzi et al., 19871,but in conflict with others [Blanquet et al., 1987;Zabel et al., 1987;Patterson et al., 19881. Because of the biologic implications of a map position within the Down syndrome region, we reexamined the map position of the APP gene using 2 additional cell lines, GM1399 and GM1413. These cell lines carry reciprocal duplications of chromosome 21 with the breakpoint in midband q21 (Fig. 1) [Williams et al.,

19751.The APP gene, as well as the probe for D21S1 map centromeric to this breakpoint (Fig. 3, Table I). Thus, the APP gene is not likely to be involved in the generation of the facial and cardiac abnormalities of Down’s syndrome. We analyzed the proximal border of map location for the loci D21S1, D21S13, and D21S16 using the breakpoint in cell line DEL2lJC (Fig. 1).DNA probes for both loci were present in only one copy in this cell line suggesting that the most proximal location ofthese loci may be within band 21q11.2 (Fig. 3, Table I). Our results also confirm recent physical maps using a different set of aneuploid cell lines and pulsed-field gel electrophoresis [Gardiner et al., 1990; Owen et al., 19901 in mapping D21S16 centromeric to D21S13. Our studies order the previously undefined breakpoint in cell line GM3268 as lying between the proximal breakpoint in cell line DEL2lJC and the breakpoints in cell lines GM0692/1399/1413.Combining our data with those of Gardiner et al. [19901 and Owen et al. [19901 suggests that the breakpoints in cell lines GM3268,6:21 and 6918 are all clustered in a region encompassing not more than 600 kb. This panel of cell lines now provides a tool to establish additional polymorphic DNA markers needed to study the pericentromeric region of chromosome 21. These are necessary to strengthen the linkage of single FAD pedigrees to this region, to establish the orientation of the FAD gene with respect to the established markers D21S13, D21S16, D21Sl/S11, and to analyze non-allelic heterogeneity in FAD families [Schellenberg et al., 19891. We have recently used this panel of cell lines to map new DNA markers into the FAD region including one DNA sequence centromeric to D21S16 (Korenberg and Pulst, unpublished). This cell line panel should facilitate the ordering of pulsed-field fragments detected by these probes and the direct analysis of DNA from patients with the inherited and non-inherited forms of AD. Because these cell lines have breakpoints between the FAD locus and the APP gene, they may provide useful biological models to examine regulatory effects for the FAD and APP genes and other genes mapped into this region of chromosome 21.

ACKNOWLEDGMENTS This work was supported by grants from the American Health Assistance foundation, the Steven and Lottie Walker foundation, Cormer and Louis Warschaw endowment fund, and the SHARE’SChild Disability Center. J.R.K. is the recipient of a National Down Syndrome Scholar Award and an Alzheimer’s Association Scholar Award; S.M.-P. is the recipient of a Young Investigator Award from the National Neurofibromatosis Foundation. We thank R. West and T. Kojis for technical assistance and Sue Jane Wang for assistance in the statistical analysis. REFERENCES Blanquet V, Goldgaber D, Turleau C, Creau-Goldberg N, Delabar J, Sinet PM, Roudier M, Grouchy J (1987): The beta amyloid protein (AD-AP) cDNA hybridizes in normal and Alzheimer individuals near the interface of 21q21 and q22.1. Ann Genet (Paris) 30:68-69.

Order of FAD Linked DNA Probes Epstein CJ (1986): “The Consequences of Chromosome Imbalance: Principles, Mechanisms and Models.” New York Cambridge University Press. Gardiner K, Horisberger M, Kraus J, Tantravahi U,KorenbergJR, Rao V, Reddy S, Patterson D (1990): Analysis of human chromosome 21: correlation of physical and cytogenetic maps: gene and CpG island distribution. EMBO J 9:25-34. Goate AM, Haynes AR, Owen MJ, Farrall M, James LA, Lai LYC, Mullan MJ, Roques P, Rossor MN, Williamson R, Hardy JA (1989): Predisposing locus for Alzheimer’s disease on chromosome 21. Lancet 1:352-355. Jenkins EC, Devine-Gage EA, Yao XL, Houck GE, Brown WT, Robakis NK, Wisniewski HM (1987): Alzheimer disease-associated gene sublocalized by in situ chromosome hybridization. Am J Hum Genet 41:A125. Korenberg JR, Croyle ML, Cox DR (1987): Isolation and regional mapping of DNA sequences unique to human chromosome 21. Am J Hum Genet 41:963-978. Korenberg JR, Kalousek DK, Anneren GA, Pulst SM, Hall J, Epstein CJ, Cox DR (1991): Deletion of chromosome 21 and normal intelligence: Molecular definition of the lesion. Hum. Genet. in press. Korenberg JR, Kawashima H, Pulst SM, Ikeuchi T, Ogasawara N, Yamamoto K, Schonberg SA, West R, Allen L, Magenis E, Ikawa K, Taniguchi N, Epstein CJ (1990): Molecular definition of a region of chromosome 21 that causes features of the Down syndrome phenotype. Am J Hum Genet 47:236-246. Korenberg JR, Pulst SM, Neve RL, West R (1989): The Alzheimer amyloid precursor protein maps to human chromosome 21 bands q21.105-21.05. Genomics 5124-127. Maniatis T, Fritsch EF, Sambrook J (1982): “Molecular cloning, a Laboratory Manual.” New York: Cold Spring Harbor Laboratory, pp 280-281. Owen MJ, James LA, Hardy JA, Williamson R, Goate AM (1990): Physical mapping around the Alzheimer disease locus on the proximal long arm of chromosome 21. Am J Hum Genet 46:316-322. Patterson D, Gardiner K, Kao F-T, Tanzi R, Watkins P, Gusella JF (1988): The mapping of the gene encoding the beta amyloid precursor protein and its relationship to the Down syndrome region of chromosome 21. Proc Natl Acad Sci USA 85:8266-8270.

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Schellenberg GD, Bird TD, Wijsman EM, Moore DK, Boehnke M, Bryant EM, Lampe TH, Nochlin D, Sumi SM, Deeb SS, Beyreuther K, Martin GM (1988): Absence of linkage of chromosome 21q21 markers in familial Alzheimer’s disease. Science 241:1507-1510. St. George-Hyslop PH, Tanzi RE, Polinsky RJ, Haines JL, Nee L, Watkins PC, Myers RH, Feldman RG,Pollen D, Drachman D, Growdon J , Bruni A, Foncin J F , Salmon D, Frommelt P, Amaducci L, Sorbi S, Piacentini S, Stewart GD, Hobbs WJ, Conneally PM, Gusella JF (1987): The genetic defect causing familial Alzheimer’s disease maps on chromosome 21. Science 235:885-890. Stewart G, Harris P, Galt J, Ferguson-Smith M (1985): Cloned DNA probes regionally mapped to chromosome 21 and their use in determining the origin of non-disjunction. Nucleic Acids Res 13:41254132. Tanzi RE, Gusella JF, Watkins PC, Bruns GAP, St George-Hyslop P, Van Keuren ML, Patterson D, Pagan A, Kurnit DM, Neve RL (1987): Amyloid beta-protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science 235:880-884. Tanzi RE, Haines JC, Watkins PC, Stewart GD, Wallace MR, Hallewell R, Wong C, Wexler NS, Conneally PM, Gusella JF (1988): Genetic linkage map of human chromosome 21. Genomics 3:129-136. Van Keuren ML, Watkins PC, Drabkin HA, Jabs EW, Gusella J F , Patterson D (1986): Regional localization of DNA sequences on chromosome 2 1 using somatic cell hybrids. Am J Hum Genet 38:793-804. Warren AC, Slaugenhaupt SA, Lewis JG, Chakravarti A, Antonarakis SE (1989): A genetic linkage map of 17 markers on human chromosome 21. Genomics 4:579-591. White R, Nakamura Y, O’Connell P, Leppert M, Lalouel JM, Barker D, Goldgar D, Skolnick M, Carey J , Wallis CE, Slater CP, Mathew C, Ponder B (1987): Tightly linked markers for the neurofibromatosis type 1 gene. Genomics 1:346-367. Williams JD, Summitt RL, Martens PR, Kimbrell RA (1975): Familial Down syndrome due to t(10;21) translocation: evidence that the Down phenotype is related to trisomy of a specific segment of chromosome 21. Am J Hum Genet 27:478-485. Zabel BU, Salbaum JM, Multhaup G, Master CL, Bohl J , Beyreuther K (1987): Sublocalization of the gene for the precursor of Alzheimer’s disease amyloid A4 protein on achromosome 21. Ninth International Workshop on Human Gene Mapping (Abstract) No. 603.