GENOMICS
13, 601-606
(19%)
Isolation and High-Resolution from the Pericentromeric D. L. MILLER,* *Department
F.
Mapping of New DNA Markers Region of Chromosome 10
J. DILL,* J. B. LIcHnR,t K. K. KIDD,t
November
The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been localized to the pericentromeric region of chromosome 10. Several markers that fail to recombine with MEN2A have been identified, including Dl 021, D10S94, 010597, and DlOS102. Meiotic mapping in the MEN2A region is limited by the paucity of critical crossovers identified and by the dramatically reduced rates of recombination in males. Additional approaches to mapping loci in the pericentromeric region of chromosome 10 are required. We have undertaken the generation of a detailed physical map by radiation hybrid mapping. Here we report the development of a radiation hybrid panel and its use in the mapping of new DNA markers in pericentromeric chromosome 10. The radiation-reduced hybrids used for mapping studies all retain small suhchromosomal fragments that include both D10S94 and DlOZl. One hybrid was selected as the source of DNA for cloning. One hundred five human recombinant clones were isolated from a X library made with ppllA DNA. We have completed regional mapping of 22 of these clones using our radiation hybrid mapping panel. Seven markers have been identified and, when taken together with previously meiotically mapped markers, define eight radiation hybrid map intervals between DlOS34 and RBPS. The identical order is found for a number of these using either the radiation hybrid mapping panel or the meiotic mapping panel. We believe that this combination cloning and mapping approach will facilitate the precise positioning of new markers in pericentromeric chromosome 10 and will help in refining further the localization of MEN2A. Academic
Press,
Inc.
INTRODUCTION
There is considerable interest in the generation of a high-resolution map of the pericentromeric region of ’ To dressed lumbia, couver,
P. J. GOODFELLOW**’
of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; and tDepartment Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06570 Received
(cl 1992
AND
whom correspondence and reprint requests should be adat Department of Medical Genetics, University of British Co226 Wesbrook Building, 6174 University Boulevard, VanB.C., Canada V6T 123.
of
1, 1991
chromosome 10. The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been shown to map near the centromere (Wu et al., 1990a). It is flanked by the gene for the p subunit of the fibronectin receptor (FNRB) and D10S34 on the short arm and the gene for the interstitial retinol binding protein (RBP3) and DlOS15 on the long arm (Wu et al., 199Ob; Mathew et al., 1991). Two related syndromes, medullary thyroid carcinoma without pheochromocytomas (MTCWP) and multiple endocrine neoplasia type 2B (MEN 2B), also map to this region of chromosome 10 (Narod et al., 1989; Carson et al., 1990; Norum et al., 1990; Lairmore et al., 1991). Meiotic mapping of new markers in the pericentromerit region of chromosome 10 is limited by a reduction in rates of recombination, most obvious in male meioses (Wu et al., 1990a). Our meiotic mapping panel includes sufficient recombination events for the ordering of markers and generation of a genetic map of chromosome 10 as a whole. The reduced rates of recombination in the pericentromeric region, however, lead to a relative scarcity of critical crossovers (Lichter et al., 1992a). Physical mapping by fluorescence in situ hybridization (FISH) to metaphase chromosomes and comparison of the relative meiotic and physical map positions of a number of markers have confirmed that there is a repression of recombination in the pericentromeric region in male meioses, and to a lesser extent in females (Lichter et al., 1991b). Ordering markers by FISH mapping is difficult, however, as the pericentromeric region is small enough that confidence intervals for map positions often overlap extensively. An independent means of determining the order of markers in this region is therefore essential to the generation of a high-resolution map. Radiation-reduced hybrids have been demonstrated to be extremely useful as cloning resources and in ordering genes and DNA sequences within a given region of interest. Examples include mapping in the WAGR complex on chromosome 11 (Glaser et al., 1990; Rose et al., 1990) and in the Huntington disease region on chromosome 4 (Cox et al., 1989; Pritchard et al., 1989). In this report we demonstrate the use of radiation-reduced hybrids in the generation and mapping of new markers from the pericentromeric region of chromosome 10. The
601 All
Copyright 0 1992 rights of reproduction
0888.7543/92 $5.00 by Academic Press, Inc. in any form reserved.
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AL.
of these studies, more than 50 chromosome 10 markers were examined, and only 3 (DIUZZ, D10S94, and pAlS-6.~65) were found to be present in ppllA DNA (unpublished observations). Somatic cell hybrids retaining translocation and derivative chromosomes used for regional assignment of chromosome 10 sequences have all been described previously (Brooks-Wilson et al., 1990; Brooks-Wilson, personal communication). Characterization of ppllA by in situ hybridization. The human content of ppllA was characterized further by FISH with cloned chromosome 10 alphoid repeats (DlOZl) and total human DNA. Chromosomes were prepared from rapidly growing cultures of ppllA using standard techniques with the addition of colcemid (0.02 or 0.2 pg/ml) for 2-6 h. All reagents for in situ hybridizations were purchased from Oncogene in kit form. Hybridizations, posthybridization washes, and signal detection were all performed as recommended by the manufacturer. Slides were examined using a Carl Zeiss epi-fluorescence microscope with a KP 490 excitation filter, a 510 beam splitter, an LB 530 barrier filter, and a Planachromatic oil immersion objective (100X/1.3). Both metaphase and interphase cells were scored for the presence of fluorescent signal. Photographs were taken using Fujicolour Super HG film for color prints (400 ASA).
FIG. 1. Characterization of radiation-reduced hybrid ppllA by in situ hybridization. (A) Hybridization with total human genomic DNA. (B) Hybridizat,ion with chromosome 10 alphoid repeats. Note the presence of two hybridizing marker chromosomes in metaphase spreads and zero, one, or two hybridization signals in interphase cells.
panel of hybrids we have developed serves to confirm the proposed map order based on meiotic and FISH mapping for a number of markers. It also allows the determination of map order for markers that could not be precisely positioned using FISH or genetic mapping techniques. One hybrid was used as a source of DNA for the isolation of additional markers from the pericentromeric region of chromosome 10, two of which fall into previously undefined map intervals. MATERIALS
AND
METHODS
Somatic cell hybrids. Seven high-dose radiation-reduced hybrids were selected from a larger series (Goodfellow et al., 1990b) for use in mapping in the proximal region of the long arm of chromosome 10. All seven (pplA, pp5A, pp7A, pplOA, pplOC, ppllA, and ppl6C) retain DIOS94 and DZOZl (Brooks-Wilson, personal communication). The DlOS94/DIOZI-positive hybrids were expanded in culture to high cell numbers, and large quantities of DNA were prepared for mapping studies. ppllA was selected as a cloning source for the generation of new markers from the MEN2A region based on its limited human content and potential to enrich for the region of interest. At the outset
Library construction and generation of new markers from the MENZA region. ppllA DNA was used for the construction of a recombinant library in the LambdaGEMvector (Promega). Briefly, library construction was as follows: ppllA DNA was partially digested with SauSAI (1 h at 0.00375 U/pg DNA) and size-selected on a sucrose gradient. Fragments between 15 and 20 kb were partially filled with dATP and dGTP and ligated into XhoI-digested and partially filled vector arms according to the manufacturer’s specifications. Phage were packaged using the Packagene in uitro packaging system (Promega). Recombinant clones (500,000) were plated on Eseherichia coli NM621 cells, and plaques were lifted onto Hybond-N (Amersham) and screened for the presence of human sequences by hybridization with 32P-labeled total human DNA. DNA from human recombinant clones was prepared from lo-ml liquid lysates (LE392 host strain) by the method of Grossberger et al. (1987) after two to three rounds of plaque purification. Phage DNA was digested with EcoRI, HindIII, or EcoRI and Hind111 in combination, separated on 1% agarose gels, and transferred to Hybond-N (Amersham). Southern blots of phage DNA were hybridized with “P-labeled total human DNA to confirm their human origin and to identify candidate unique sequences for mapping purposes. Radiation hybrid mapping in pericentromeric chromosome 10. EcoRI-digested, somatic cell hybrid, hamster and human control DNAs were size-separated on 1% agarose gels and transferred to GeneScreen Plus (NEN DuPont). The resulting Southern blots were hybridized with a series of markers from the pericentromeric region of chromosome 10 (DlOS34, probe TB14.34; FNRB, probe pGEM-32; DlOZl, probe pnRP8; DIOS94, probe 0.95Eco/Sac; D10S97, probe KW63Sac1, DIOS102 probe MEN203 WITI; RBP3, probe pH.4IRBP) to test for the presence or absence of markers in each of the radiation-reduced hybrids. The presence or absence of RETwas determined by PCR amplilication using gene-specific primers under the following conditions: denature at 94°C 30 s, anneal 60°C 30 s, extend 72°C 30 s for 25 cycles. The resulting synthesis product was run on a 1% agarose, 3% NuSieve (FMC) GTG gel and examined for the presence of a 229.bp humanspecific product. Oligonucleotide primers were developed from sequence from the 3’ untranslated region of RET (GenBank Accession No. M57964). Primers are at base positions 3520-3540 and 37323749. Unique or low-copy inserts from phage derived from the library described above were purified by twice running into a 1% low-meltingtemperature agarose gel and then being excised. The inserts were labeled in the agarose by random priming (Feinberg and Vogelstein, 1984). After labeling, 15 ng of insert was preassociated with 500 pg of sheared total human DNA at 65°C for 1 h in the presence of heterologous DNA in 1 ml of hybridization solution before being added to Southern blots.
MARKERS
IN
PERICENTROMERIC
CHROMOSOME
603
10
HYBRID
Chromosome localization
I
locus/probe
PP~A
PP~A
pp7A
pplOA
pplOC
ppllA
ppl6C
TraxK2
64034 ,cr,.,n
CY5
CY6
CHOKl
-2-28
p3-3
liDM11,35 IDM146,152
hDM145
1DM17,23,215, 216,31,A33,51 IDM21,417, 513
FIG. 2. Presence or absence of chromosome 10 DNA markers in a panel of numbers or gene symbols) have all been previously localized in the pericentromeric content of conventional hybrids: TraxK2, qll.2-qter; 64034p61c10, ten-qter; q11.2:q22.1-qter. Three groups of markers (XDM11,35, XDM146,152, and XDM1451 lies. Two groups of short arm markers could not be ordered with respect to reference present; 0 not present; 0 not tested. CY5, CY6, or CHOKl-Z-28. RESULTS
Cytogenetic Characterization Hybrid ppll A
of the Radiation-Reduced
Analysis of G-banded metaphase chromosomes prepared from ppllA resulted in the identification of a very small marker chromosome present in approximately 50% of the spreads (not shown). In situ hybridization with biotinylated total human DNA and cloned chromosome 10 alphoid repeats revealed that the small marker chromosome identified in G-banded spreads was largely, if not entirely, human-derived (Fig. 1). A second humanderived fragment was identified as positive for hybridization with both total human DNA and chromosome 10 alphoid repeats. Two hundred one metaphase spreads and 276 interphase cells were scored for hybridization signals with the human and alphoid probes. Forty-four percent of all spreads showed one hybridization area with total human DNA and 8% showed two distinct hybridization signals. Using the cloned alphoid repeats as a probe, 42% of the spreads gave a single hybridization
somatic cell hybrids. Markers with locus designations (D region by meiotic mapping, FISH, or both. Chromosome 10 CY5, pter-q26.3; CY6, pterq24.3; CHOKl-Z-28, pterwere derived from 1Oq outside the region in which MEN2A markers. These short arm markers were not tested against
signal and 10% gave two signals. Two percent of the metaphase spreads revealed three sites of hybridization with total human DNA. Generation and Mapping of New Markers Derived From the ppllA Hybrid Library and Radiation Hybrid Mapping of the Pericentromeric Chromosome 10 Region Screening 500,000 recombinant clones (one to two genome equivalents) of the unamplified ppllA library for the presence of human repeat sequences resulted in identification of 105 human clones (0.02%). Twenty-two recombinant clones from the ppllA library were regionally localized on chromosome 10 by Southern blot hybridization using a panel of seven radiation-reduced hybrids and five conventional somatic cell hybrids (Fig. 2). Six markers mapped in proximal 1Oq in the interval between DlOZl and RBP3 (see below). Eleven were assigned to the short arm of the chromosome (XDM12, XDM17, XDM21, XDM23, XDM31, XDMA33, XDM51, XDM215, XDM216, XDM417, and XDM513) and five to
604
MILLER
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AL.
The patterns of retention of the 22 new markers derived from the ppllA library were similarly examined. Representative hybridizations are shown in Fig. 3, and the results for all hybridizations are summarized in Fig. 2. All seven radiation-reduced hybrids retain sequences corresponding to DlOZl and DlOS94 (Figs. 3A and 3B). One hybrid, pp5A, retains a human chromosomal fragment including DlOZl and D10S94 but lacks RET and the monomorphic sequences recognized by probe KWGASacI (DlOS97-like) (Fig. 2). The probe KWGASacI detects several loci on chromosome 10. The polymorphic EcoRI fragments that define DlOS97 are, however, easily recognized (Lichter et al., 1992b). Another hybrid, pp7A, is DlOZl-, DlOS94-, and RET-positive, but negative for hybridization with the probe pl-3A (Figs. 2 and 3C). Probe pl-3A is retained in pplOC, whereas probe ~3-3 is not (Figs. 2 and 3D). DlOSl02 and DlOS97 were found to map identically to pl-3A. Taken together, the hybridizations define a consistent series of X-ray radiation-induced breaks in the pp hybrids examined. The chromosomal content and proposed breakpoints in the radiation-reduced hybrids are illustrated in Fig. 4. Probe ~3-3 detects two chromosome 10 loci: the smaller band detected in EcoRI-digested DNAs is derived from the proximal lOq11.2 region, whereas the HYBRlD locus Probe
or pplfl
PPSR
pp7tl
pplER
PplBC
ppllfl
ppl6C
ENRBt
D I 0S34* FIG. 3. Representative hybridizations of three meiotically mapped reference markers and two new recombinant clones that define previously unrecognized radiation hybrid map intervals in lOq11.2. (A) p&P8 (DIOZ1). (B) 0.95Eco/Sac (UIOS91). (C) pl-3A. CD) ~3-3. (E) pH.4IRPB (RPBS). Arrows indicate the human-specific bands detected by pH.4IRPB. pH.4IRBP cross-hybridization to hamster sequences serves as an internal control for the amount of DNA loaded in each lane. Lanes are: 1, WT49 (female human); 2, chromosome 10 only hybrid R342-A4; 3, WSGH (hamster); 4, ppl6C; 5, ppllA; 6, pplOC; 7, pplOA; 8, pp7A; 9, pp5A; 10, pplA.
more distal regions of the long arm: three to the distal portion of lOq11.2q22.1 (XDM145, hDM146, and XDM152) and two to lOq24.3-q26.3 o\DMll and XDM35). All seven radiation-reduced hybrids included in the mapping studies retain fragments derived from the proximal region of the long arm of chromosome 10 and, when considered together, allow ordering of probes in proximal lOq11.2. As a means of localizing breakpoints in the radiation hybrids relative to known marker loci, the hybrid DNAs were tested by Southern blot analysis for the presence or absence of chromosome 10 alphoid repeats (corresponding to Dl 021) and an additional five markers from lOq11.2, all of which had previously been demonstrated to be tightly linked to MEN2A and to one another (DlOS94, D10S97, RET, DlOSlO2, and RBP3).
Al-2Al
I
010s94*
BEI,* DlBS97-
like
Pl-3R,
m,* 018s182* p3-3
FIG. 4. Representation of human chromosome 10 content of seven radiation-reduced hybrids. Proposed map order for defined intervals is indicated. *Reference loci for which order has been determined by meiotic mapping. Fragments in pplA, pp5A, and pplOA extend beyond RPB3 on the long arm of chromosome 10. The chromosomal fragment in pplOA also extendsbeyond FNRB on the short arm of chromosome 10.
MARKERS
IN
PERICENTROMERIC
larger fragment detected by ~3-3 and retained in only two of the radiation hybrids (pplA and pplOA) is localized to the distal portion of lOq11.2-q22.1 (not shown). DISCUSSION Fluorescence in situ hybridization using cloned chromosome 10 alphoid repeats and total human DNA probes served to better define the frequency of retention and the organization of the human chromosomal fragments in the ppllA radiation-reduced hybrid. Approximately 50% of hybrid cells retain human-derived material. The in situ hybridization results corroborate our earlier DNA hybridization studies, which suggested that the hybridppllA has a very restricted human DNA content. We interpret the results of our in situ hybridization analyses as indicating that the most frequently retained small marker chromosomes represent chromosomal fragments that overlap substantially. In situ hybridization of a series of recombinant clones derived from the ppllA library to ppllA hybrid chromosomes would allow us to test this hypothesis. For mapping purposes we have considered the two alphoid repeat-containing fragments present in ppllA as a single fragment. We are able to estimate the size of the clonable human material present in the ppllA hybrid by considering the following: (a) the frequency of retention of human fragments detected by in situ hybridization with total human DNA (approximately 50%), (b) the near diploid karyotype of the hybrid cell line, and (c) the 0.02% human content based on hybridization of the recombinant library with total human DNA. Using these figures, we estimate the clonable human content of ppLlA to be less than 3000 kb. We propose a map order for proximal 1Oq as follows: DIOZ1-DIOS94-(RET, DIOS97-like)-(pl-3A,XDM44,XDM55, XDMl21, XDM151 ,DIOS102, DfOS97) -p3 - 3 RBP3. Probes pl-3A and ~3-3 are on the long arm distal to RET, but proximal to RBP3. pl-3A is proximal to ~3-3 based on the observation that pl-3A is retained in pplOC, whereasp3-3 is not. Our map is generated assuming the least number of breaks (Fig. 4) and that markers close together are less likely to be segregated by X-ray irradiation-induced chromosome breaks than those further apart. Of the seven new markers generated in the pericentromerit region from the ppllA library, five map identically to a region distal to RET but proximal to RBP3. The recombinant clone, ~3-3, maps between pl-3A and RBP3. Another clone, XDM12, maps to the proximal short arm, between DlOS34 and DlOZl. We have examined the retention of XDM12 in an additional 20 radiation-reduced hybrids and observe a striking frequency of coretention with chromosome 10 alphoid repeats. Nine of a total of 27 hybrids examined retain XDM12. Eight of these also retain sequences corresponding to DlOZl. The frequency of coretention of XDM12 with alphoid repeats (47%) suggests that XDMl2 originates from very near the centromere, in proximal lop, between DlOS34
CHROMOSOME
605
10
and DlOZl. This map localization has been confirmed by FISH studies (not shown). This is the first marker to be identified in this region. The proposed order for markers from the pericentromerit region of chromosome 10 based on radiation hybrid mapping agrees with that obtained in meiotic mapping experiments (Lichter et al., 1992a). We have identified a total of eight hybrid-defined map intervals in the pericentromeric region. Five of these are in proximal lOq11.2. Two are in proximal 10~11.2. DlOZl falls into the final interval. The radiation hybrid breakpoints described here will be of value in further refining the organization of the pericentromeric region as additional markers become available. The new markers from the chromosome 10 pericentromeric region described in this paper will help to define the localization of MEN2A and other genes in this region. ACKNOWLEDGMENTS This work was primarily supported by Grant MT10563 to P.J.G. from the MRC of Canada and funds from the Canadian Genetic Diseases Network. Additional support came from USPH Grant CA32066 to K.K.K. P.J.G. is an MRC Scholar; D.L.M. is the recipient of an MRC Studentship. J.B.L. is a fellow of the Medical Informatics Training Program, Grant 5-T15-LM07065-05. We thank Angela Brooks-Wilson for D10S94 and for all her technical assistance. We also thank Yusuke Nakamura and Sarah Mole for MEN203 WlTl.
REFERENCES Brooks-Wilson, A. R., Goodfellow, P. N., Povey, S., Nevanlinna, H. A., De Jong, P. J., and Goodfellow, P. J. (1990). Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element-mediated PCR. Genomics 7: 614-620. Carson, N. L., Wu, J., Jackson, C. E., Kidd, K. K., and Simpson, N. E. (1990). The mutation for medullary thyroidcarcinoma withparathyroid tumors (MTC with PTs) is closely linked to the centromeric region of chromosome 10. Am. J. Hum. Genet. 47: 946-951. Cox, D. R., Pritchard, C. A., Uglum, E., Casher, D., Kobori, J., and Myers, R. M. (1989). Segregation of the Huntington disease region of human chromosome 4 in a somatic cell hybrid. Genomics 4: 397407. Feinberg, A. P., and Vogelstein, B. (1984). A technique for radiolabeling DNA restriction endonuciease fragments to high specific activity: Addendum. Anal. Biochem. 137: 266-267. Glaser, T., Rose, E., Morse, H., Housman, D., and Jones, C. (1990). A panel of irradiation-reduced hybrids selectively retaining human chromosome 11~13: Their structure and use to purify the WAGR gene complex. Genomics 6: 48-64. Goodfellow, P. J., Myers, S., Anderson, L. L., Brooks-Wilson, A. R., and Simpson, N. E. (199Oa). A new DNA marker (DlOS94) very tightly linked to the multiple endocrine neoplasia type 2A (MENSA) locus. Am. J. Hum. Genet. 47: 952-956. Goodfellow, P. J., Povey, S., Nevanlinna, H. A., and Goodfellow, P. N. (1990b). Generation of a panel of somatic cell hybrids containing unselected fragments of human chromosome 10 by X-ray irradiation and cell fusion: Application to isolating the MEN2A region in hybrid cells. Somatic Cell Mol. Genet. 16: 163-171. Grossberger, lambda.
D. Nucleic
(1987). Acids
Minipreps Res. 15:
of
DNA
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bacteriophage
6737.
Lairmore, T. C., Howe, J. R., Korte, d. A., Dilley, W. G., Aine, L., Aine, E., Wells, S. A., Jr., and Donis-Keller, H. (1991). Familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2B map to
606
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the same region of chromosome 10 as multiple type 2A. &nom& 9: 181-192. Lichter, J. B., Wu, J., Brewster, S., Brooks-Wilson, P. J., and Kidd, K. K. (1991a). A new polymorphic tightly linked t,o MEN2A. Am. J. Hum. Genet. Lichter, J. B., Wu, J., Miller, D. L., Goodfellow, (1992a). A high-resolution meiotic mapping tromeric region of chromosome 10. Genomics
endocrine
neoplasia
A. R., Goodfellow, marker (DIOS97) 49: 349.
P. J., and Kidd, K. K. panel for the pericen13: 607-612.
Lichter, J. B., Difilippantonio, M., Wu, J., Ward, D. C., Goodfellow, P. .J., and Kidd, K. K. (1991b). A comparative linkage and physical map of chromosome 10: HGM 11 26971. Cytogenet. Cell Genet., in press. Lichter, J. B., Wu, J., Brooks-Wilson, A. R., Difillipantonio, M., Brewster, S., Ward, D. C., Goodfellow, P. J., and Kidd, K. K. (1992b). A new polymorphic marker (DIOS97) tightly linked to MENBA. Hum. Genet., in press. Mathew, C. G. P., Easton, D. F., Nakamura, Y., Ponder, B. A. J., and the MENZA International Collaborative Group. (1991). Presymptomatic screening for multiple endocrine neoplasia type 2A with linked DNA markers. Lancet 337: 7-11. Narod, S. A., Sobol, H., Schuffenecker, I., Lavoue, M-F., and Lenoir, G. M. (1991). The gene for MENZA is tightly linked to the centromere of chromosome 10. Hum. Genet. 86: 529-530. Narod, S. A., Sobol, H., Nakamura, Y., Calmettes, C., Baulieu, J-L., Bigorgne. J-C., Chabrier, G., Couette, J., De Gennes, J-L., Duprey, .J., Gardet, P., Guillausseau, P-J., Guilloteau, D., Houdent, C., Lefebvre, d., Modigliani, E., Parmentier, C., Pugeat, M., Siame, C.,
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Tourniaire, J., Vandroux, (1989). Linkage analysis without pheochromocytoma. Norum, R. laney, J. Willard, endocrine markers
J-C., Vinot, J-M., and Lenoir, G. M. of hereditary thyroid carcinoma with and Hum. Genet. 83: 353-358.
A., Lafreniere, R. G., O’Neal, L. W., Nikolai, T. F,, DeP., Sisson, J. C., Sobol, H., Lenoir, G. M., Ponder, B. A. J., H. F., and Jackson, C. E. (1990). Linkage of the multiple neoplasia type 2B gene (MENPB) to chromosome 10 linked to MENBA. Genomics 8: 313-317.
Pritchard, C. A., Casher, D., Uglum, E., Cox, D. R., and Myers, R. M. (1989). Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene. Genomics 4: 408-418. Rose, E. A., Glaser, T., dones, C., Smith, C. L., Lewis, W. H., Call, K. M., Minden, M., Champagne, E., Bonetta, L., Yeger, H.: and Housman, D. E. (1990). Complete physical map of the WAGR region of 11~13 localizes a candidate Wilms’ Tumor gene. Cell 60: 495-508. Wu, J., Carson, N. L., Myers, S., Pakstis, A. J., Kidd, J. R., Castiglione, C. M., Anderson, L., Hoyle, L. S., Genel, M., Verdy, M., Jackson, C. E., Simpson, N. E.. and Kidd, K. K. (1990a). The genetic defect in multiple endocrine neoplasia type 2A maps next to the centromere of chromosome 10. Am. J. Hum. Cenet. 46: 624-630. Wu, J., Myers, S., Carson, N., C. M., Hoyle, L. S., Lichter, and Kidd, K. K. (199Ob). A around the pericentromeric 461-468.
Kidd, d. R., Anderson, L., Castiglione, J. B., Sukhatme, V. P., Simpson, N. E., refined linkage map for DNA markers region of chromosome 10. Genomics 8:
13, 601-606
(19%)
Isolation and High-Resolution from the Pericentromeric D. L. MILLER,* *Department
F.
Mapping of New DNA Markers Region of Chromosome 10
J. DILL,* J. B. LIcHnR,t K. K. KIDD,t
November
The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been localized to the pericentromeric region of chromosome 10. Several markers that fail to recombine with MEN2A have been identified, including Dl 021, D10S94, 010597, and DlOS102. Meiotic mapping in the MEN2A region is limited by the paucity of critical crossovers identified and by the dramatically reduced rates of recombination in males. Additional approaches to mapping loci in the pericentromeric region of chromosome 10 are required. We have undertaken the generation of a detailed physical map by radiation hybrid mapping. Here we report the development of a radiation hybrid panel and its use in the mapping of new DNA markers in pericentromeric chromosome 10. The radiation-reduced hybrids used for mapping studies all retain small suhchromosomal fragments that include both D10S94 and DlOZl. One hybrid was selected as the source of DNA for cloning. One hundred five human recombinant clones were isolated from a X library made with ppllA DNA. We have completed regional mapping of 22 of these clones using our radiation hybrid mapping panel. Seven markers have been identified and, when taken together with previously meiotically mapped markers, define eight radiation hybrid map intervals between DlOS34 and RBPS. The identical order is found for a number of these using either the radiation hybrid mapping panel or the meiotic mapping panel. We believe that this combination cloning and mapping approach will facilitate the precise positioning of new markers in pericentromeric chromosome 10 and will help in refining further the localization of MEN2A. Academic
Press,
Inc.
INTRODUCTION
There is considerable interest in the generation of a high-resolution map of the pericentromeric region of ’ To dressed lumbia, couver,
P. J. GOODFELLOW**’
of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; and tDepartment Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06570 Received
(cl 1992
AND
whom correspondence and reprint requests should be adat Department of Medical Genetics, University of British Co226 Wesbrook Building, 6174 University Boulevard, VanB.C., Canada V6T 123.
of
1, 1991
chromosome 10. The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been shown to map near the centromere (Wu et al., 1990a). It is flanked by the gene for the p subunit of the fibronectin receptor (FNRB) and D10S34 on the short arm and the gene for the interstitial retinol binding protein (RBP3) and DlOS15 on the long arm (Wu et al., 199Ob; Mathew et al., 1991). Two related syndromes, medullary thyroid carcinoma without pheochromocytomas (MTCWP) and multiple endocrine neoplasia type 2B (MEN 2B), also map to this region of chromosome 10 (Narod et al., 1989; Carson et al., 1990; Norum et al., 1990; Lairmore et al., 1991). Meiotic mapping of new markers in the pericentromerit region of chromosome 10 is limited by a reduction in rates of recombination, most obvious in male meioses (Wu et al., 1990a). Our meiotic mapping panel includes sufficient recombination events for the ordering of markers and generation of a genetic map of chromosome 10 as a whole. The reduced rates of recombination in the pericentromeric region, however, lead to a relative scarcity of critical crossovers (Lichter et al., 1992a). Physical mapping by fluorescence in situ hybridization (FISH) to metaphase chromosomes and comparison of the relative meiotic and physical map positions of a number of markers have confirmed that there is a repression of recombination in the pericentromeric region in male meioses, and to a lesser extent in females (Lichter et al., 1991b). Ordering markers by FISH mapping is difficult, however, as the pericentromeric region is small enough that confidence intervals for map positions often overlap extensively. An independent means of determining the order of markers in this region is therefore essential to the generation of a high-resolution map. Radiation-reduced hybrids have been demonstrated to be extremely useful as cloning resources and in ordering genes and DNA sequences within a given region of interest. Examples include mapping in the WAGR complex on chromosome 11 (Glaser et al., 1990; Rose et al., 1990) and in the Huntington disease region on chromosome 4 (Cox et al., 1989; Pritchard et al., 1989). In this report we demonstrate the use of radiation-reduced hybrids in the generation and mapping of new markers from the pericentromeric region of chromosome 10. The
601 All
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of these studies, more than 50 chromosome 10 markers were examined, and only 3 (DIUZZ, D10S94, and pAlS-6.~65) were found to be present in ppllA DNA (unpublished observations). Somatic cell hybrids retaining translocation and derivative chromosomes used for regional assignment of chromosome 10 sequences have all been described previously (Brooks-Wilson et al., 1990; Brooks-Wilson, personal communication). Characterization of ppllA by in situ hybridization. The human content of ppllA was characterized further by FISH with cloned chromosome 10 alphoid repeats (DlOZl) and total human DNA. Chromosomes were prepared from rapidly growing cultures of ppllA using standard techniques with the addition of colcemid (0.02 or 0.2 pg/ml) for 2-6 h. All reagents for in situ hybridizations were purchased from Oncogene in kit form. Hybridizations, posthybridization washes, and signal detection were all performed as recommended by the manufacturer. Slides were examined using a Carl Zeiss epi-fluorescence microscope with a KP 490 excitation filter, a 510 beam splitter, an LB 530 barrier filter, and a Planachromatic oil immersion objective (100X/1.3). Both metaphase and interphase cells were scored for the presence of fluorescent signal. Photographs were taken using Fujicolour Super HG film for color prints (400 ASA).
FIG. 1. Characterization of radiation-reduced hybrid ppllA by in situ hybridization. (A) Hybridization with total human genomic DNA. (B) Hybridizat,ion with chromosome 10 alphoid repeats. Note the presence of two hybridizing marker chromosomes in metaphase spreads and zero, one, or two hybridization signals in interphase cells.
panel of hybrids we have developed serves to confirm the proposed map order based on meiotic and FISH mapping for a number of markers. It also allows the determination of map order for markers that could not be precisely positioned using FISH or genetic mapping techniques. One hybrid was used as a source of DNA for the isolation of additional markers from the pericentromeric region of chromosome 10, two of which fall into previously undefined map intervals. MATERIALS
AND
METHODS
Somatic cell hybrids. Seven high-dose radiation-reduced hybrids were selected from a larger series (Goodfellow et al., 1990b) for use in mapping in the proximal region of the long arm of chromosome 10. All seven (pplA, pp5A, pp7A, pplOA, pplOC, ppllA, and ppl6C) retain DIOS94 and DZOZl (Brooks-Wilson, personal communication). The DlOS94/DIOZI-positive hybrids were expanded in culture to high cell numbers, and large quantities of DNA were prepared for mapping studies. ppllA was selected as a cloning source for the generation of new markers from the MEN2A region based on its limited human content and potential to enrich for the region of interest. At the outset
Library construction and generation of new markers from the MENZA region. ppllA DNA was used for the construction of a recombinant library in the LambdaGEMvector (Promega). Briefly, library construction was as follows: ppllA DNA was partially digested with SauSAI (1 h at 0.00375 U/pg DNA) and size-selected on a sucrose gradient. Fragments between 15 and 20 kb were partially filled with dATP and dGTP and ligated into XhoI-digested and partially filled vector arms according to the manufacturer’s specifications. Phage were packaged using the Packagene in uitro packaging system (Promega). Recombinant clones (500,000) were plated on Eseherichia coli NM621 cells, and plaques were lifted onto Hybond-N (Amersham) and screened for the presence of human sequences by hybridization with 32P-labeled total human DNA. DNA from human recombinant clones was prepared from lo-ml liquid lysates (LE392 host strain) by the method of Grossberger et al. (1987) after two to three rounds of plaque purification. Phage DNA was digested with EcoRI, HindIII, or EcoRI and Hind111 in combination, separated on 1% agarose gels, and transferred to Hybond-N (Amersham). Southern blots of phage DNA were hybridized with “P-labeled total human DNA to confirm their human origin and to identify candidate unique sequences for mapping purposes. Radiation hybrid mapping in pericentromeric chromosome 10. EcoRI-digested, somatic cell hybrid, hamster and human control DNAs were size-separated on 1% agarose gels and transferred to GeneScreen Plus (NEN DuPont). The resulting Southern blots were hybridized with a series of markers from the pericentromeric region of chromosome 10 (DlOS34, probe TB14.34; FNRB, probe pGEM-32; DlOZl, probe pnRP8; DIOS94, probe 0.95Eco/Sac; D10S97, probe KW63Sac1, DIOS102 probe MEN203 WITI; RBP3, probe pH.4IRBP) to test for the presence or absence of markers in each of the radiation-reduced hybrids. The presence or absence of RETwas determined by PCR amplilication using gene-specific primers under the following conditions: denature at 94°C 30 s, anneal 60°C 30 s, extend 72°C 30 s for 25 cycles. The resulting synthesis product was run on a 1% agarose, 3% NuSieve (FMC) GTG gel and examined for the presence of a 229.bp humanspecific product. Oligonucleotide primers were developed from sequence from the 3’ untranslated region of RET (GenBank Accession No. M57964). Primers are at base positions 3520-3540 and 37323749. Unique or low-copy inserts from phage derived from the library described above were purified by twice running into a 1% low-meltingtemperature agarose gel and then being excised. The inserts were labeled in the agarose by random priming (Feinberg and Vogelstein, 1984). After labeling, 15 ng of insert was preassociated with 500 pg of sheared total human DNA at 65°C for 1 h in the presence of heterologous DNA in 1 ml of hybridization solution before being added to Southern blots.
MARKERS
IN
PERICENTROMERIC
CHROMOSOME
603
10
HYBRID
Chromosome localization
I
locus/probe
PP~A
PP~A
pp7A
pplOA
pplOC
ppllA
ppl6C
TraxK2
64034 ,cr,.,n
CY5
CY6
CHOKl
-2-28
p3-3
liDM11,35 IDM146,152
hDM145
1DM17,23,215, 216,31,A33,51 IDM21,417, 513
FIG. 2. Presence or absence of chromosome 10 DNA markers in a panel of numbers or gene symbols) have all been previously localized in the pericentromeric content of conventional hybrids: TraxK2, qll.2-qter; 64034p61c10, ten-qter; q11.2:q22.1-qter. Three groups of markers (XDM11,35, XDM146,152, and XDM1451 lies. Two groups of short arm markers could not be ordered with respect to reference present; 0 not present; 0 not tested. CY5, CY6, or CHOKl-Z-28. RESULTS
Cytogenetic Characterization Hybrid ppll A
of the Radiation-Reduced
Analysis of G-banded metaphase chromosomes prepared from ppllA resulted in the identification of a very small marker chromosome present in approximately 50% of the spreads (not shown). In situ hybridization with biotinylated total human DNA and cloned chromosome 10 alphoid repeats revealed that the small marker chromosome identified in G-banded spreads was largely, if not entirely, human-derived (Fig. 1). A second humanderived fragment was identified as positive for hybridization with both total human DNA and chromosome 10 alphoid repeats. Two hundred one metaphase spreads and 276 interphase cells were scored for hybridization signals with the human and alphoid probes. Forty-four percent of all spreads showed one hybridization area with total human DNA and 8% showed two distinct hybridization signals. Using the cloned alphoid repeats as a probe, 42% of the spreads gave a single hybridization
somatic cell hybrids. Markers with locus designations (D region by meiotic mapping, FISH, or both. Chromosome 10 CY5, pter-q26.3; CY6, pterq24.3; CHOKl-Z-28, pterwere derived from 1Oq outside the region in which MEN2A markers. These short arm markers were not tested against
signal and 10% gave two signals. Two percent of the metaphase spreads revealed three sites of hybridization with total human DNA. Generation and Mapping of New Markers Derived From the ppllA Hybrid Library and Radiation Hybrid Mapping of the Pericentromeric Chromosome 10 Region Screening 500,000 recombinant clones (one to two genome equivalents) of the unamplified ppllA library for the presence of human repeat sequences resulted in identification of 105 human clones (0.02%). Twenty-two recombinant clones from the ppllA library were regionally localized on chromosome 10 by Southern blot hybridization using a panel of seven radiation-reduced hybrids and five conventional somatic cell hybrids (Fig. 2). Six markers mapped in proximal 1Oq in the interval between DlOZl and RBP3 (see below). Eleven were assigned to the short arm of the chromosome (XDM12, XDM17, XDM21, XDM23, XDM31, XDMA33, XDM51, XDM215, XDM216, XDM417, and XDM513) and five to
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The patterns of retention of the 22 new markers derived from the ppllA library were similarly examined. Representative hybridizations are shown in Fig. 3, and the results for all hybridizations are summarized in Fig. 2. All seven radiation-reduced hybrids retain sequences corresponding to DlOZl and DlOS94 (Figs. 3A and 3B). One hybrid, pp5A, retains a human chromosomal fragment including DlOZl and D10S94 but lacks RET and the monomorphic sequences recognized by probe KWGASacI (DlOS97-like) (Fig. 2). The probe KWGASacI detects several loci on chromosome 10. The polymorphic EcoRI fragments that define DlOS97 are, however, easily recognized (Lichter et al., 1992b). Another hybrid, pp7A, is DlOZl-, DlOS94-, and RET-positive, but negative for hybridization with the probe pl-3A (Figs. 2 and 3C). Probe pl-3A is retained in pplOC, whereas probe ~3-3 is not (Figs. 2 and 3D). DlOSl02 and DlOS97 were found to map identically to pl-3A. Taken together, the hybridizations define a consistent series of X-ray radiation-induced breaks in the pp hybrids examined. The chromosomal content and proposed breakpoints in the radiation-reduced hybrids are illustrated in Fig. 4. Probe ~3-3 detects two chromosome 10 loci: the smaller band detected in EcoRI-digested DNAs is derived from the proximal lOq11.2 region, whereas the HYBRlD locus Probe
or pplfl
PPSR
pp7tl
pplER
PplBC
ppllfl
ppl6C
ENRBt
D I 0S34* FIG. 3. Representative hybridizations of three meiotically mapped reference markers and two new recombinant clones that define previously unrecognized radiation hybrid map intervals in lOq11.2. (A) p&P8 (DIOZ1). (B) 0.95Eco/Sac (UIOS91). (C) pl-3A. CD) ~3-3. (E) pH.4IRPB (RPBS). Arrows indicate the human-specific bands detected by pH.4IRPB. pH.4IRBP cross-hybridization to hamster sequences serves as an internal control for the amount of DNA loaded in each lane. Lanes are: 1, WT49 (female human); 2, chromosome 10 only hybrid R342-A4; 3, WSGH (hamster); 4, ppl6C; 5, ppllA; 6, pplOC; 7, pplOA; 8, pp7A; 9, pp5A; 10, pplA.
more distal regions of the long arm: three to the distal portion of lOq11.2q22.1 (XDM145, hDM146, and XDM152) and two to lOq24.3-q26.3 o\DMll and XDM35). All seven radiation-reduced hybrids included in the mapping studies retain fragments derived from the proximal region of the long arm of chromosome 10 and, when considered together, allow ordering of probes in proximal lOq11.2. As a means of localizing breakpoints in the radiation hybrids relative to known marker loci, the hybrid DNAs were tested by Southern blot analysis for the presence or absence of chromosome 10 alphoid repeats (corresponding to Dl 021) and an additional five markers from lOq11.2, all of which had previously been demonstrated to be tightly linked to MEN2A and to one another (DlOS94, D10S97, RET, DlOSlO2, and RBP3).
Al-2Al
I
010s94*
BEI,* DlBS97-
like
Pl-3R,
m,* 018s182* p3-3
FIG. 4. Representation of human chromosome 10 content of seven radiation-reduced hybrids. Proposed map order for defined intervals is indicated. *Reference loci for which order has been determined by meiotic mapping. Fragments in pplA, pp5A, and pplOA extend beyond RPB3 on the long arm of chromosome 10. The chromosomal fragment in pplOA also extendsbeyond FNRB on the short arm of chromosome 10.
MARKERS
IN
PERICENTROMERIC
larger fragment detected by ~3-3 and retained in only two of the radiation hybrids (pplA and pplOA) is localized to the distal portion of lOq11.2-q22.1 (not shown). DISCUSSION Fluorescence in situ hybridization using cloned chromosome 10 alphoid repeats and total human DNA probes served to better define the frequency of retention and the organization of the human chromosomal fragments in the ppllA radiation-reduced hybrid. Approximately 50% of hybrid cells retain human-derived material. The in situ hybridization results corroborate our earlier DNA hybridization studies, which suggested that the hybridppllA has a very restricted human DNA content. We interpret the results of our in situ hybridization analyses as indicating that the most frequently retained small marker chromosomes represent chromosomal fragments that overlap substantially. In situ hybridization of a series of recombinant clones derived from the ppllA library to ppllA hybrid chromosomes would allow us to test this hypothesis. For mapping purposes we have considered the two alphoid repeat-containing fragments present in ppllA as a single fragment. We are able to estimate the size of the clonable human material present in the ppllA hybrid by considering the following: (a) the frequency of retention of human fragments detected by in situ hybridization with total human DNA (approximately 50%), (b) the near diploid karyotype of the hybrid cell line, and (c) the 0.02% human content based on hybridization of the recombinant library with total human DNA. Using these figures, we estimate the clonable human content of ppLlA to be less than 3000 kb. We propose a map order for proximal 1Oq as follows: DIOZ1-DIOS94-(RET, DIOS97-like)-(pl-3A,XDM44,XDM55, XDMl21, XDM151 ,DIOS102, DfOS97) -p3 - 3 RBP3. Probes pl-3A and ~3-3 are on the long arm distal to RET, but proximal to RBP3. pl-3A is proximal to ~3-3 based on the observation that pl-3A is retained in pplOC, whereasp3-3 is not. Our map is generated assuming the least number of breaks (Fig. 4) and that markers close together are less likely to be segregated by X-ray irradiation-induced chromosome breaks than those further apart. Of the seven new markers generated in the pericentromerit region from the ppllA library, five map identically to a region distal to RET but proximal to RBP3. The recombinant clone, ~3-3, maps between pl-3A and RBP3. Another clone, XDM12, maps to the proximal short arm, between DlOS34 and DlOZl. We have examined the retention of XDM12 in an additional 20 radiation-reduced hybrids and observe a striking frequency of coretention with chromosome 10 alphoid repeats. Nine of a total of 27 hybrids examined retain XDM12. Eight of these also retain sequences corresponding to DlOZl. The frequency of coretention of XDM12 with alphoid repeats (47%) suggests that XDMl2 originates from very near the centromere, in proximal lop, between DlOS34
CHROMOSOME
605
10
and DlOZl. This map localization has been confirmed by FISH studies (not shown). This is the first marker to be identified in this region. The proposed order for markers from the pericentromerit region of chromosome 10 based on radiation hybrid mapping agrees with that obtained in meiotic mapping experiments (Lichter et al., 1992a). We have identified a total of eight hybrid-defined map intervals in the pericentromeric region. Five of these are in proximal lOq11.2. Two are in proximal 10~11.2. DlOZl falls into the final interval. The radiation hybrid breakpoints described here will be of value in further refining the organization of the pericentromeric region as additional markers become available. The new markers from the chromosome 10 pericentromeric region described in this paper will help to define the localization of MEN2A and other genes in this region. ACKNOWLEDGMENTS This work was primarily supported by Grant MT10563 to P.J.G. from the MRC of Canada and funds from the Canadian Genetic Diseases Network. Additional support came from USPH Grant CA32066 to K.K.K. P.J.G. is an MRC Scholar; D.L.M. is the recipient of an MRC Studentship. J.B.L. is a fellow of the Medical Informatics Training Program, Grant 5-T15-LM07065-05. We thank Angela Brooks-Wilson for D10S94 and for all her technical assistance. We also thank Yusuke Nakamura and Sarah Mole for MEN203 WlTl.
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Lairmore, T. C., Howe, J. R., Korte, d. A., Dilley, W. G., Aine, L., Aine, E., Wells, S. A., Jr., and Donis-Keller, H. (1991). Familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2B map to
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the same region of chromosome 10 as multiple type 2A. &nom& 9: 181-192. Lichter, J. B., Wu, J., Brewster, S., Brooks-Wilson, P. J., and Kidd, K. K. (1991a). A new polymorphic tightly linked t,o MEN2A. Am. J. Hum. Genet. Lichter, J. B., Wu, J., Miller, D. L., Goodfellow, (1992a). A high-resolution meiotic mapping tromeric region of chromosome 10. Genomics
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Lichter, J. B., Difilippantonio, M., Wu, J., Ward, D. C., Goodfellow, P. .J., and Kidd, K. K. (1991b). A comparative linkage and physical map of chromosome 10: HGM 11 26971. Cytogenet. Cell Genet., in press. Lichter, J. B., Wu, J., Brooks-Wilson, A. R., Difillipantonio, M., Brewster, S., Ward, D. C., Goodfellow, P. J., and Kidd, K. K. (1992b). A new polymorphic marker (DIOS97) tightly linked to MENBA. Hum. Genet., in press. Mathew, C. G. P., Easton, D. F., Nakamura, Y., Ponder, B. A. J., and the MENZA International Collaborative Group. (1991). Presymptomatic screening for multiple endocrine neoplasia type 2A with linked DNA markers. Lancet 337: 7-11. Narod, S. A., Sobol, H., Schuffenecker, I., Lavoue, M-F., and Lenoir, G. M. (1991). The gene for MENZA is tightly linked to the centromere of chromosome 10. Hum. Genet. 86: 529-530. Narod, S. A., Sobol, H., Nakamura, Y., Calmettes, C., Baulieu, J-L., Bigorgne. J-C., Chabrier, G., Couette, J., De Gennes, J-L., Duprey, .J., Gardet, P., Guillausseau, P-J., Guilloteau, D., Houdent, C., Lefebvre, d., Modigliani, E., Parmentier, C., Pugeat, M., Siame, C.,
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Pritchard, C. A., Casher, D., Uglum, E., Cox, D. R., and Myers, R. M. (1989). Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene. Genomics 4: 408-418. Rose, E. A., Glaser, T., dones, C., Smith, C. L., Lewis, W. H., Call, K. M., Minden, M., Champagne, E., Bonetta, L., Yeger, H.: and Housman, D. E. (1990). Complete physical map of the WAGR region of 11~13 localizes a candidate Wilms’ Tumor gene. Cell 60: 495-508. Wu, J., Carson, N. L., Myers, S., Pakstis, A. J., Kidd, J. R., Castiglione, C. M., Anderson, L., Hoyle, L. S., Genel, M., Verdy, M., Jackson, C. E., Simpson, N. E.. and Kidd, K. K. (1990a). The genetic defect in multiple endocrine neoplasia type 2A maps next to the centromere of chromosome 10. Am. J. Hum. Cenet. 46: 624-630. Wu, J., Myers, S., Carson, N., C. M., Hoyle, L. S., Lichter, and Kidd, K. K. (199Ob). A around the pericentromeric 461-468.
Kidd, d. R., Anderson, L., Castiglione, J. B., Sukhatme, V. P., Simpson, N. E., refined linkage map for DNA markers region of chromosome 10. Genomics 8:
