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. Author manuscript; available in PMC: 2006 Jan 25.
Published in final edited form as: Eur J Hum Genet. 2001 May;9(5):364–374. doi: 10.1038/sj.ejhg.5200627

High-resolution physical and transcript map of human chromosome 2p21 containing the sitosterolaemia locus

Kangmo Lu 1, Mi-Hye Lee 1, John D Carpten 2, Mundeep Sekhon 3, Shailendra B Patel 1,*
PMCID: PMC1350990  NIHMSID: NIHMS4368  PMID: 11378825

Abstract

Sitosterolaemia (phytosterolaemia) is an autosomal recessive disorder characterised by the presence of tendon xanthomas in the face of normal or mildly elevated plasma cholesterol levels, premature atherosclerotic disease and has diagnostically elevated plasma and tissue plant sterol concentrations. Affected individuals show an increased absorption of both cholesterol and sitosterol from the diet, decreased bile clearance of these sterols and their metabolites resulting in markedly expanded whole body cholesterol and sitosterol pools. The defective gene is therefore hypothesised to play a crucial role in regulating dietary cholesterol absorption, and its elucidation may shed light on these molecular processes. We have previously localised the defective gene to human chromosome 2p21, between microsatellite markers D2S1788 and D2S1352, a distance of approximately 15 cM. Recently, the disease locus interval has been narrowed to lie between D2S2294 and D2S2291/D2S2174. We have constructed a high-resolution YAC and BAC contigs by using known STSs and generating novel STSs from the minimal interval. Eight previously identified genes and 60 ESTs were mapped to these contigs. The BAC contig contains 60 BAC clones and 108 STSs and encompasses a physical distance of approximately 2.0 cM between microsatellite markers D2S2294 and D2S2291. These results will not only facilitate cloning of the sitosterolaemia gene, but also other disease genes located in this region, and accelerate sequencing of the corresponding genomic clones.

Keywords: BAC contig, mapping, positional cloning, atherosclerosis genes

Introduction

Sitosterolaemia (also known as phytosterolaemia, MIM number 210250) is a rare autosomal recessively inherited metabolic disorder, which was described in 1974 in two affected sisters.1 Sitosterolaemic patients develop tendon and tuberous xanthomas, haemolytic episodes, arthralgias and arthritis, and premature coronary and aortic atherosclerosis leading to cardiac fatalities.1–5 Affected individuals have very high levels of plasma plant sterols (sitosterol, campesterol, stigmasterol, avenosterol) and their 5α-saturated stanols, particularly sitostanol, but their blood cholesterol levels may be normal or only moderately increased.1,4 Increased intestinal absorption and decreased hepatic excretion of sitosterol (the major plant sterol) may be responsible for the accumulation of these non-cholesterol sterols in plasma and tissues of affected patients.4,6–10

In addition to the proposed defects of absorption and excretion of sitosterol, reduced whole body cholesterol synthesis has also been noted.2,11,12

Linkage analyses of 10 well-characterised pedigrees localised the genetic defect to human chromosome 2p21, between microsatellite markers D2S1788 and D2S1352.13 Recently, we have narrowed this interval to lie between microsatellite markers D2S2294 and D2S2291 (Lee et al, manuscript submitted). To refine the minimal critical region and isolate candidate gene(s), we have constructed high-resolution YAC and BAC contigs by using known STSs and by generating novel STSs from this region. We have mapped a number of ESTs to this interval, building a partial transcript map that should aid identification of the defective gene. Additionally, this may facilitate the identification of other disease loci mapped to this region, such as a QTL for serum leptin levels,14 as well as a locus for gingival fibromatosis.15

Materials and methods

Selection and STS contents of YAC clones

YAC clones were identified through the YAC databases developed by CEPH16,17 and the Whitehead Institute18 using all of the known markers and STSs in sitosterolaemia region (D2S2291, D2S2174, D2S1830, D2S1485, D2S2298, D2S119, D2S2294, D2S414). The YAC clones were purchased from Research Genetics, Inc (Birmingham, AL, USA). Single YAC colonies were grown at 30°C for 48 h in 15 ml of selective YPD medium. Total YAC DNA was prepared as described previously.19 The STS contents of the YACs were determined by using PCR amplifications.

Inter-Alu PCR

Inter-Alu PCR was performed using YAC DNA as template and the following primers: CL1, (5′TCCCAAAGTGCTGGGATTACA), CL2 (5′CTGCACTCCAGCCCTGGG) and used as CL2 alone or CL1 and CL2 combined primers.20,21 The PCR products were isolated and cloned into plasmid, pBluescript (Stratagene, La Jolla CA, USA) using TA cloning, as previously described,22 and sequenced using T3 and T7 primers. The sequences were scanned against the databases, using BLAST23 (http://www.ncbi.nlm.nih.gov/BLAST/) and the RepeatMasker program (http://ftp.genome.washington.edu/RM/webRepeatMasker.html). Unique sequences were used to design primers for further mapping (Table 1). Confirmation of chromosome 2 specific sequences was verified by PCR, using chromosome 2 specific humanhamster hybrid somatic cell line DNAs (Corell Cell Repository, Camden, NJ, USA).

Table 1.

Primer sequences used in YAC/BAC contigs

aSTS Forward primer (5′–3′) Reverse primer (5′–3′) Size (bp) bAccession no.
C-506D15.F CCAGTGGCATTTAGTACATTA AATGCCACTGAATCACACAC 207 AZ051294
C-506D15.R AAAAAGGCTGCCCACCTTTA GTCAAGAGGTAGATGAAATGC 206 AZ051295
C-498C3.F TCTTGTTTTCCGATTCTGTTC GTCAATTTCTACAGTGTAGCT 203 AZ051296
C-528A6.F CCCAGCACCAAATACAGTGAA CAGTACATCTTCCGGCTCTA 218 AZ051297
C-528A6.R CCCTGATATTTACCCAGCTC CAAGAAGAATGAGATCTGGC 266 AZ051298
C-569J16.F TACCTGAGCTTCCTAGGATG CAGACAGCCTCAGTCGCTA 306 AZ051299
R-990A23.F AACGCATGGCTCTATAGAGG AGGACAGCAGTGTCATTTAC 412 AQ702225
R-990A23.R CTGGATGGAACGCTCACTA CAGAAGCCTCAGGGTAAGA 268 AQ668383
R-1081G2.F GATAAAGTTTAAGGTTATCTC GAAATAAGCTTAGCCTGTAC 275 AQ740126
R-316B10.F CACAACTATGACCACTTTGAC CGTGAGGTTCTCCTTTCCC 299 AQ541175
R-316B10.R ACAGCTAAGGATAATGAGGCA GTGTTTATCCCCCAAGCACT 288 AQ507714
R-32814.R GGTGAGAATTCTAGCAAGCTA CTATGGATAGAACTCTCAGTG 256 AQ539167
R-959M3.R ACAACACTGACAGTCTATCCG CCAGATAGATAGTATGAGTTCA 215 AQ667844
R-646H10.R AGGATAATGAGGCATGTGAAG TCGTGGGTTCACATAGCACA 372 AQ516454
R-72C11.R CAGTGGTGCTTGTAGCAGG CCCATAGTGATCAAGCACTA 260 AQ285023
R-203O10.R CACGTGCATTGAAGGCTAATA TTGATCTAGCTAAGCTAGTCC 214 AQ418643
C-520L5.F AGAGTTTCTGCTCTCTATGG AGTGATTCTTTGATGGGCAG 301 AZ051300
C-520L5.R ATTCTCCTCTAGGCCTCTAG TCAGCCTCTGCCTCTTGGAA 274 AZ051301
R-92L13.F GCCTAACAGCCAATCTGAG TAACCCTACATGTGTTCCCA 285 AQ322533
R-92L13.R TATAGGGATCCAACAGTACC GAGCTACATGATGGCCTTCA 288 AQ322528
R-2415.R TGGTACTCGCATCTCCTTG GTCAAGGAGTCTTCTTGGG 243 AQ013398
Y888G9.L CTACCTAAAAGGCTTGGTTATC CTCTGCAGAGTATTGCCACA 207 AZ051302
R-161J6.R AACTGAAGTGGTACTGACAG AAGACGGCAGATGTATCCTG 185 AQ376733
C-535K2.F AGGAAAGTCAAGCTCCAGAG TTAAAGAGAGCCTTCAGCTTC 233 AZ051303
C-535K2.R CTTGTGCCACTACTGCACTC TTGTGCTGTCCATTCCTAGAA 244 AZ051304
C-325O15.F AGGACATTCTTACAGGCTACA CAGCTAGTTATCTGAAGCTGA 253 AZ051305
C-441A7.F TATTTGCTCATTTAATGAGCCTA CCTTACATAGTTCTCATCCTC 224 AZ051306
C-441A7.R TGATGGGGGAAAGCCACAAA CTGTGGCCTCCCAAATTTC 306 AZ051307
C-2094M11.R GGAAACTGTGCAAGTGAAGA TTAACAACAGGAGTCCCGCT 177 AQ566340
C-2117A16.R TAGCAAATCCTGTGCCATTC TATAAAGGAAGGTCCTGACC 318 AQ753530
R-117H6.F TCTGTGGACTGACCTAGTAG TGGGCGCAAGATTTCTGAG 257 AQ350077
C-285H9.F CCCACTACTGTGCAAACTTC ATAAGAGCCATCCGGATTGC 208 AZ051308
42C1 GAACAAGATCTGTAAGGGGT TTCAGTAACATTGCATATTTTTCT 138 AZ051309
42B2 ATGGAAGAGGGTTGGATGTTG TGAGTGTCTGCCGGTGTA 163 AZ051310
42 E7 TGAGACCTTTCTGCTTCTATCC CACTGGAGAGTTGGCTGTG 314 AZ051314
42B7 GCTGAGAATATCACTTTACTCC GATTTCCAAGGTTACAATGTGTA 151 AZ051315
45A12 ACTTGCTTGGTTTTGGTAAT ACAGTCTCTTTGTGATCTT 217 AZ051316
42F7 GAAAGTAGGCTAAGAGAGTTAAT GTGAGCCACTGCACCCAG 158 AZ051317
87A10 GGTTCTGTTTCATGTGTATGG CAACTAGAATTGGACTAGATACTC 221 AZ051318
45D2 CACTGCTGAATGTGAACTGC CCCATGGTTTGACAAATGATTTC 262 AZ051311
42C5 CACTTCATCATGTAGAACAGG AGGATGATAGAGGGATTGGTTT 269 AZ051313
45B4 ACTGCTGAATGTGAACTGC TGCTACTATTGCAGCCCT 196 AZ051319
42D12 CTTACACATTGTTATGAAGTGCAC GTCTCAGAGAAAGATGTCACA 215 AZ051323
42C9 GTGTAGCCTATTCAGAGAAC AGTCAGTCTTCACGGCCA 181 AZ051326
45D11 GAACGTGGAATAATATAAGACC TATCTCACCACCCACACTG 187 AZ051327
45E 11 GTCAGCTTTATGGATAGGG GAAATACTCAGAATCCAGAAAC 214 AZ051328
45B2 CATTCTGAGGGCCAGATTT AGATGTAATACTTGCAAGCC 219 AZ051329
45B10 ACCAGAAAATGACACCTTC CATAGTATGAGTGCTACTTGACTC 242 AZ051330
42G8 GGCAAACTTTGGCTCATGG GTGCTAGAATCATCAGTTTGTCAT 272 AZ051331
87A7 CAGCCCTCAGAGACAATAGA TGCTGCCAAGCCATCCAA 222 AZ051332
87A5 TGACAGGGTGAGAGTCCATC GCCTTACACTGACTGACAGAT 300 AZ051333
87A3 CCTCAGTGGAGCAGATTGC AAATTTCCTAGGAAAGTTGGG 257 AZ051334
87A2 CACATTATCTCTGAGTAGAG CTATGCTTCTGAATGCCAG 178 AZ051335
41HM9 CCCACCAGCAGTGTATGAG GTTCCACATCACTGGTCATC 153 AZ051344
42C2/T7 CAGACCATAGCATCCTCTTT TCACACTTCACACAAGGTC 234 AZ051337
42F10 CAAGACTGGTTGCCATATGG CATCTCTTTCCTCCCCTC 201 AZ051339
42 E2 CCAGATTTGACCAAAAGCCC AGATGTAATACTTGCAAGCC 209 AZ051340
42D8 CCTACATGTGTTCCCATTGCA TTGCCTTGATGCCTCCCA 175 AZ051341
42D3 AACCACTCTTAACTCCAGGG GCAAGCCTTCTTAAATAGGCATA 237 AZ051342
42B1 AGGTGGATGTCTACAATGGTC GGTTTGCATATAGCCAGTCAC 187 AZ051343
42H11 GCACTCCAGCCTGGGCAA AGAGGTGAAGCTTACTGGAA 183 AZ051338
87A1 GATTACAGGCATCAGCCAC CCAGTCCTCCAAAAATGGTC 175 AZ051336
42A3 AGGCAATCTGGGTTACTAGG CGACTGAACATACAGACACT 210 AZ051312
45F3 CAAGTACTGTTCTAAGGGCT TATGATAGAGGTATGCACTGG 168 AZ051320
45D2 TGGCCACTATCATTATTAGAAA CTCTTCAGAGAGTTTGGACC 255 AZ051321
42B3 AACAGTCAGCTTCTCAAAGG ATGGAGACTTCTTTAGGAGG 217 AZ051325
45A8 CATCTTCATCATCAAGCAGTG AAGTACTGTGCCAAGGCCTG 240 AZ051322
D2S4009 GATCCAGTGTCATTATGCATAC GCCAGTTGTTAATATTTTGCC 219 G64673
D2S4010 CAGCGGTAGTCTCTATGATA TCAGAAGGTTCCTTATACAAGGC 172 G64671
D2S4014 TGCAGACTGTAATTGTGGGCT GACTCCAGATGAGATCTATGACTG 297 G64669
D2S4015 CTCAAATCTCTGACTCCAGATC GGCTATCCACTCAATAATTC 297 G64672
D2S4016 GATAAGCAAGCTGGTCACACTC ATTTGAGCTTCAGAGGTCAA 253 G64670
D2S4019 ATGATCTGCATGAGGGTCAAGG GAGTATATTTAGAAATTTCCATAA 102 G64675
D2S4020 TAGTCTTAATGTTTCCCTTGG GAGACTAGTTTTCTGACTCAAG 189 G64676
D2S4023 GAGATTCTTTTTATTCTGATTTTTTGAG ATGATCTGCATGAGGGTCAA 127 G64677
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Table shows the primer sets for all the unique STS and microsatellite repeats identified in this study and not available in the public databases.

a

Prefix C is CITB-SHP-C BAC library; R is RPCI-11 BAC library; Y is CEPH YAC library; D2S is a microsatellite marker.

b

Prefixes AZ and G are from this study.

BAC clone screening

PCR-based library screening

The CITB-SHP-C Human BAC library,24 (Research Genetics, Inc., Huntsville, AL, USA) was screened by a PCR-based assay of DNA super-pools and plates according to the vendor recommended procedures. Positive clones were obtained from Research Genetics, Inc., plated on agar plates containing 12.5 μg/ml chloramphenicol and colonies screened by PCR for STS content verification.

Hybridisation-based library screening

High-density gridded filters of BAC libraries (RPCI-11) were obtained from Roswell Park Cancer Institute (Dr. Peter de Jong's laboratory, Buffalo NY, USA), and screened with radioactive probes from the IMAGE cDNA clones of ESTs mapped to the YAC contig. Positive clones were obtained from the Roswell Park Cancer Institute.

Selection from database

All known STS, EST and Alu PCR sequences were checked by a Basic BLAST against the Alu database (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) and masked by RepeatMasker23 and unique sequences thus identified were used as probes. Sequenced BACs in the public databases were identified by a BLAST 2.0 alignment search of the HTGS database25,26 (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) and the complete BAC sequences were obtained from GenBank (http://www.ncbi.nlm.nih.gov/genbank/query_form.html). BACs with known end-sequence information were determined by searching the BAC End Sequence Database at TIGR (http://www.tigr.org/tdb/hum-gen/). The overlapped BACs or BAC contigs were obtained by searching the Washington University Human mapping database (http://genome.wustl.edu/gsc/cgibin/fpchuman.-single.pl) for likely matches to specified clones.

Sequencing of YAC and BAC ends

Isolation of YAC ends was performed using a modified vectorette method, using primers as previously described.27 YAC DNA (0.1 μg) was digested with 10 units of RsaI and AluI in 30 μl reaction buffer. Five microlitres of digested YAC DNA was ligated to vectorette adapters using 10 units of T4 DNA ligase in a total volume of 50 μl by incubation overnight at room temperature. The YAC end fragments were purified by Qiagen PCR column kit and directly sequenced using the left or right internal primers.

To obtain BAC end-sequences, BAC plasmid DNA was prepared using alkaline lysis procedure and tip-500 columns (Qiagen).28 The quality and quantity of DNA samples were tested by HindIII digestion pattern on agarose gels, as well as by the presence of expected STS markers. Direct BAC end sequencing was performed using an automated ABI 373 DNA sequencer. Three micrograms of BAC DNA and 50 pmoles of primer were used in a total volume of 40 μl. The following primers were used: T7 (5′-TAATACGACTCACTATAGGG-3′) and SP6 (5′ATTTAGGTGACACTATAG-3′). PCR reactions were carried out under the following cycle conditions: initial denaturation at 96°C for 4 min; 100 cycles of 96°C for 10 s, 50°C for 10 s, 60°C for 4 min. The end sequences of some BAC clones were obtained by searching BAC End Sequence Database at TIGR.

Transcript map

To identify candidate genes, known genes and ESTs previously mapped to the region between D2S177 and D2S337 were selected from the Human Transcript Map.29 The selected ESTs and genes were tested by PCR amplification against our YAC and BAC contigs and positive clones further characterised, as described above.

Results

Construction of a YAC contig

The initial goal was to construct an extensive YAC contig spanning the sitosterolaemia candidate region on chromosome 2, between markers D2S2174 and D2S2294. Based on the publicly available contig maps (contig WC2.4) from Whitehead Institute/MIT Center for Genome Research (WI/MIT) (http://carbon.wi.mit.edu) and the CEPH-Généthon (CEPH) (http://www.cephb.fr/infoclone.html), 30 YAC clones were identified, using following microsatellite and STSs markers (D2S414, D2S2294, D2S119, D2S2298, D2S1484, D2S1486, D2S1485, D2S1830, D2S2174 and D2S229). Additionally, information from a published partial YAC contig was also available.30 All YAC clones were screened for the markers and confirmed by testing three colonies of each clone for their STS contents (Figure 1). Two gaps were identified at the telomeric ends, and no further YACs were identified, despite additional library screening. However, we subsequently identified a BAC, R-489G24, positive for markers D2S2259 and an EST T71978, that linked YACs 888g9 and 899b1, thus closing one gap (Figure 1). Since the sitosterolaemia locus is located towards the centromeric end, no further attempts were made to close the more distal gap. Using the YAC contig, new markers were generated, employing a combination of YAC end-sequence analyses and inter-Alu PCR. To confirm that the identified STSs were from chromosome 2, all of these markers were screened for their presence in human chromosome 2-specific somatic cell hybrid cell-lines. Sixty ESTs from the databases (Unigene and GeneMap98, see Materials and methods) were screened by PCR against the YAC contig. Six of these were positive for the YAC contig (Figure 1), of which five mapped to within the region of interest. The sixth EST (T71978) was found to be positive on a linking BAC (see above). By performing inter-Alu PCR using YACs 919c10, 930a1 and 761e1 as templates, we generated 35 additional unique sequences from Alu PCR clones for obtaining STS markers and eight microsatellite repeat markers that allowed for further fine-mapping of the sitosterolaemia locus (Lee et al, manuscript submitted). A total of 76 STSs, including 17 new STSs generated from YAC insert-end sequences and inter-Alu PCR products and nine EST markers, were used to order the clones (Table 1 and Figure 1) and span an approximate distance of 5 cM. This physical map provided a resource for the construction of a BAC contig.

Figure 1.

Figure 1

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YAC clone contig encompassing the sitosterolaemia locus. The markers are oriented along the X-axis from telomeric end at the left to the centromeric end at the right, and the YAC clones are indicated along the Y-axis. There are two gaps in the contig (boxed areas, see Text). The distal centromeric gap is closed by a linking BAC, R-489G24, giving a contiguous contig from D2S1754 to D2S2227. ESTs mapped to the YAC contig are underlined, microsatellite markers are italicised and STSs are in normal font.

Note also that our YAC contig is a telomeric extension of a published adjoining YAC contig, thus providing a continuous map that spans chromosome 2p15–2p21 (D2S1364–D2S1754, ~14 Mb) and contains several human disease loci.31

Construction of a BAC contig

To construct a BAC map, we used the following strategy; (1) identify BAC clones using both a PCR-based and filter hybridisation-based BAC library screenings, (2) screen all positive BACs for STS content by PCR, (3) search BAC-related databases for updated information, and (4) perform chromosome-walks using selected STSs generated from BAC ends. Initially, PCR was used to screen the CITB-978SK-B human BAC library using six repeat polymorphic markers (D2S2294, D2S119, D2S2298, D2S1830, D2S2174, and D2S2291), a YAC end-sequence (from 888g9L) and an EST marker (T71978). Twelve BAC clones, positive for D2S2294, D2S119, D2S2291, 888g9L and T71978, were identified. For hybridisation-based BAC library screening, high-density filters were hybridised with a mixture of five probes consisting of ESTs T99836, T71978, A007E35, stSG63433, A010A13, previously mapped to the YAC contig. Eight more positive BAC clones were obtained from RPCI-11 BAC library. The BAC end sequences of identified clones were determined by direct automated sequencing or by searching the BAC end sequence database at TIGR. BAC end sequences of the inserts of BAC clones were used to develop further STS markers. All STS markers were tested by PCR amplification against all identified BAC clones, to verify true positives. By searching the databases in an iterative manner, we identified 18 sequenced BACs. In total, we used 118 markers, composed of 29 microsatellite markers, 53 new STSs from BAC/YAC end sequences and inter-Alu PCR sequences, and 36 EST markers. The constructed BAC contig contains 60 BAC clones, which contains a high density of STS markers, at an average of about 20 kb for each marker, and covers a physical distance of about 2.0 Mb (Figure 2). A significant number of these BACs have been sequenced (boxed, Figure 2), but about 500 kb sequence is not publicly available.

Figure 2.

Figure 2

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BAC clone contig encompassing the sitosterolaemia locus. The markers are oriented along the X-axis from telomeric end at the left to the centromeric end at the right, and the BAC clones are indicated along the Y-axis. Prefixes of the BAC clones are as follows, R; from RPCI-11 BAC library; and C; from CITB-SHP-C BAC library. ESTs are underlined, microsatellite markers are shown in italics and STSs are in normal font. A box indicates BACs, for which almost complete sequence information is available in the genome databases. Four gaps (indicates by vertical lines) were identified, but these BACs are indicated, as they contain markers placed on the YAC contig framework (Figure 1). Of these the more centromeric is spanned by YAC 888g9. BAC R-436K12 (not shown) is linked to the contig published by Kirchener et al,31 and links our YAC contig at the centromeric end.

Mapping of known genes and ESTs to the YAC/BAC contigs

We have constructed a transcript map (Table 2) of the BAC contig using two methods. From GeneMap'99, based upon two radiation hybrid panels,32,33 we selected 80 genes and ESTs between anchor markers D2S177 and D2S2291. All ESTs were verified by PCR against the BAC contig. Of the 80 markers, only eight known genes and 30 ESTs mapped unambiguously to our BAC contig. The eight known genes are KIAA0544 protein,34 ERF2 protein,35 3-hydroxyanthranilic acid dioxygenase,36 CGI 60 protein,37 leucinerich protein,38 protein phosphatase 1B (formerly PP2C),39 Na+-independent neutral and basic amino acid transporter (solute carrier family 3, SLC3A1),40 and KIAA0436.41 In the second approach, using the known human genomic sequences from STSs and sequenced BACs between D2S2294 and D2S2291, we identified a further 30 ESTs by a BLASTN search of the EST databases. All of these 30 identified ESTs contain unique sequences, >95% matched to genomic sequences, and have not been previously mapped to a chromosome. A summary of the mapped ESTs to our BAC contig is shown in Table 2. We computed the expression patterns for many of these ESTs (Table 3). Additionally, we screened each of the mapped ESTs against the databases, looking for homologous ESTs/genes identified in other species, on the assumption that highly conserved expressed sequences may reflect proteins that have highly conserved and critical functions, such as selective sterol absorption. Only sequences that had >100 bp of sequence identity and >70% homology are reported (Table 3). Although such analysis is limited by the lack of depth of the EST databases for the other species, we identified 11 ESTs that appear to have homologues in non-human sequence databases (Table 3), although none from the Drosophila database were identified.

Table 2.

ESTs and genes mapped to the YAC/BAC contigs in this study

NCBI No (GeneMap '99) Aliases or synonyms UniGene No. Genbank Accession No. Image Clone ID Known genes Mapping data
WI-20996 stSG41980, T48876 Hs.19280 R26389 132199 KIAA0544 protein R-501O7
SGC33875 T98917 122669 R-78I14
stSG48396 Hs.98023 AA854974 1394041 R-459K11
SGC34340 WI-6575, SGC34340 Hs.78909 X78992 ERF-2 protein R-339H12
STSG16054 A009V10 Hs.17711 R98822 207006 R-339H12
WI-14187 G21943 Hs.16063 AA515534 925219 R-339H12
stSG52431 Hs.165571 AI566776 2168475 R-489G24
T89476 sts-T89476 Hs.16587 AA934036 1551421 R-489G24
stSG15818 R83265 194194 R-489G24
WI-8407 Hs.108441 Z29481 3-Hydroxyanthranilic acid dioxygenase R-489G24
BCD1971 M79071 no image clone R-489G24
T71978 sts-T71978 Hs.168439 AA534545 925906 R-489G24
stSG58568 Hs.58598 AI359618 2013757 R-489G24
stSG30561 Hs.58598 AA169121 594556 506D15
H96893 stSG21270 Hs.32241 AI274775 1986682 R-489K21
stSG21136 H58682 205857 R-489K21
AF151818 CGI 60 protein R-489K21
T99836 Hs.18176 T99836 123200 R-489K21
WI16988 A007E35 Hs.142718 AA034046 429916 R-1081G2
stSG63433 Hs.190354 AA700586 433330 R-1081G2
stSG32054 stSG1757, SHGC-8019 T17102 Hs.182490 M92439 Leucine-rich protein mRNA R-1081G2
Hs.128293 AI223013 1838809 R-559M23
Hs.225721 AI873444 2362159 Trans-prenyltransferase (TPT) R-559M23
Hs.225721 AA889371 1471263 R-559M23
AA457390 838194 R-559M23
AA828868 1374287 R-559M23
Hs.187945 AA937699 1491139 R-559M23
A004I37/H99661 stSG51096 Hs.169652/Hs.5687 AA164383/AA565932 PP2C Protein phosphatase 2C R-24I5
stSG3387 A003R48 R11895 25315 R-24I5
stSG52154 Hs.112916 AA620873 1049335 R-24I5
M95548* SHGC-9884, stSG4626 Hs.198294/Hs.154834 D82326/M95548 Amino acid transporter, SLC3A1 R-24I5
M95548* SHGC-9884, stSG4626 Hs.110 AB007896 KIAA0436 mRNA R-24I5
A009V46 Hs.174862, Hs.220859 H95593 242930 R-194L1
A010A13 WI-18144 Hs.124990 H58934 207758 R-194L1
D29089 D29089 no image clone R-194L1
stSG8383 H60063 205767 R-194L1
Hs.132799 AA922097 1543611 R-194L1
Hs.129473 AA994134 1628550 R-194L1
Hs.213492 AI928677 2466254 R-194L1
Hs.124990 H60592 207898 R-194L1
W80452 415494 R-194L1
stSG26329 H57813 205424 R289E6
Hs.136519 AA601487 1100969 R-442O5
T87425 115418 R-442O5
WI-3495 G02557 Hs.188588 AA583683 1088083 R-442O5
AA835723 1372934 R-442O5
H64341 210718 R-442O5
AA838139 1385549 R-442O5
Hs.170428 AI459058 2149952 R-442O5
Hs.170428 AW206717 2722480 R-442O5
Hs.233172 AW022706 2486137 R-442O5
stSG46410 Hs.97696 AA399659 729207 R-89K21
N24094 N24095 266792 R-576F1
WI-3976 SHGC-17237 Hs.246042 N75945 295200 R-576F1
stSG49702 Hs.167640 H87795 220658 R-436K12
WI-18791 U03911, SHGC-2762, SHGC-10660 Hs.78934 HSU03911 (hMSH2) Mismatch repair protein (MSH2) mRNA R-436K12
stSG60189 Hs.122384 AI015254 1641212 R-436K12
embl-AA007353 sts-AA007353 Hs.256042 AA007353 429281 R-436K12
SGC34683 SHGC-34683, stSG28638, stSG9035 Hs.117085 AA677756 430606 R-436K12
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All ESTs and genes that were mapped to the YAC and BAC contigs (Figures 1 and 2) are shown. For clarity, only the BAC ID is shown in the far right column. BAC R-436K12 is not indicated on the BAC contig (Figure 2), but is contiguous with the centromeric end. Only a representative EST or Image clone is indicated, where multiple clones were identified. The asterisk indicates a GeneMap ID, M95548, which identifies two separate genes that share the 3′ UTR (see text). Additionally, there are two GeneMap98 IDs for the same gene (PP2C) that have been consolidated.

Table 3.

Expression pattern of ESTs and genes

NCBI No (GeneMap'99) GenBank Accession No. Expression pattern Known gene Human Mouse Rat Bovine Porcine Zebrafish Chicken
WI-20996/KIAA0544 R26389 Multiple tissues KIAA0544 protein 57 4 0 0 1 0 0
SGC33875 T98917 Fetal liver, spleen 2 0 0 0 0 0 0
stSG48396 AA854974 Testis 12 0 0 0 0 0 0
SGC34340 X78992 Multiple tissues ERF-2 protein >75 16 15 6 0 1 0
STSG16054 R98822 Fetal liver, spleen 5 0 0 0 0 0 0
WI-14187 AA515534 Multiple tissues 30 0 1 0 0 0 0
stSG52431 AI566776 Brain, eye, heart, pancreas, uterus, thymus 18 0 0 0 0 0 0
embl-T89476 AA934036 Bone, germ cell, prostate 3 0 0 0 0 0 0
stSG15818 R83265 Fetal liver, spleen 40 40 7 1 0 0 0
WI-8407 Z29481 Colon, kidney, lung, placenta, spleen, uterus 3-hydroxyanthranilic acid dioxygenase 6 0 0 0 0 0 0
BCD1971 M79071 Brain 2 0 0 0 0 0 0
embl-T71978 AA534545 Colon, kidney, liver, lung 22 23 1 0 1 0 0
stSG58568 AI359618 Multiple tissues 5 0 0 0 0 0 0
stSG30561 AA169121 Multiple tissues 6 0 0 0 0 0 0
H96893 AI274775 Multiple tissues 32 0 1 0 0 0 0
stSG21136 H58682 Fetal liver, spleen 1 0 0 0 0 0 0
AF151818 Multiple tissues CGI 60 protein 72 8 6 0 0 0 0
T99836 T99836 Fetal liver, spleen 2 0 0 0 0 0 0
WI16988 AA034046 Fetal liver, spleen 6 0 0 0 0 0 0
stSG63433 AA700586 Fetal liver, spleen 2 0 0 0 0 0 0
stSG32054 M92439 Multiple tissues Leucine-rich protein mRNA >100 5 0 0 0 0 0
AI223013 Testis 4 1 0 0 0 0 0
AI873444 Ovary Trans-prenyltransferase (TPT) 3 2 0 0 0 1 0
AA889371 Ovary 3 2 0 0 0 1 0
AA457390 Retina 1 0 0 0 0 0 0
AA828868 Ovary 1 0 0 0 0 0 0
AA937699 Skin 2 0 0 0 0 0 0
A004I37/H99661 AA164383/AA565932 Multiple tissues Protein phosphatase 2C 48 26 8 0 0 0 0
stSG3387 R11895 Brain 4 0 0 0 0 0 0
stSG52154 AA620873 Testis 2 0 0 0 0 0 0
M95548* D82326/M95548 Brain, kidney, pancreas, uterus, colon Amino acid transporter, SLC3A1 36 38 2 1 1 0 0
M95548* AB007896 Multiple tissues KIAA0436 mRNA 100 16 3 0 0 0 0
A009V46 H95593 Fetal liver, spleen 2 0 0 0 0 0 0
A010A13 H58934 Fetal liver, spleen 3 0 0 0 0 0 0
D29089 D29089 Epidermis, keratinocyte 1 0 0 0 0 0 0
stSG8383 H60063 Fetal liver, spleen 1 0 0 0 0 0 0
AA922097 Testis 1 0 0 0 0 0 0
AA922097 Testis 3 5 0 1 0 0 0
AA994134 Tonsil 5 0 0 0 0 0 0
AI928677 Brain 1 0 0 0 0 0 0
H60592 Fetal liver, spleen 3 0 0 0 0 0 0
W80452 Fetal liver, spleen 1 0 0 0 0 0 0
stSG26329 H57813 Fetal liver, spleen 1 0 0 0 0 0 0
AA601487 Adrenal gland 1 2 0 0 0 0 0
T87425 Fetal liver, spleen 2 0 0 0 0 0 0
WI-3495 AA583683 Kidney, nose 5 0 0 0 0 0 0
AA835723 Germinal center B cell 2 0 0 0 0 0 0
H64341 Fetal liver, spleen 1 0 0 0 0 0 0
AA838139 Ovary 1 0 0 0 0 0 0
AI459058 Lung 2 0 0 0 0 0 0
AW206717 Lung 2 0 0 0 0 0 0
AW022706 Ear 1 3 0 0 0 0 0
stSG46410 AA399659 Testis 2 0 0 0 0 0 0
N24095 N24095 Melanocyte 1 0 0 0 0 0 0
WI-3976 N75945 Whole blood 3 0 0 0 0 0 0
stSG49702 H87795 Retina, colon 3 0 0 0 0 0 0
WI-18791 HSU03911 Multiple tissues Mismatch repair protein (MSH2) 63 13 1 1 0 0 3
stSG60189 AI015254 Testis 3 0 0 0 0 0 0
AA007353 AA007353 Lung 3 0 0 0 0 0 0
SGC34683 AA677756 Fetal liver, spleen, neuroepithelium 5 0 0 0 0 0 0
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Expression profiles were determined for the ESTs and genes, based upon the identification of the EST or gene transcript in various cDNA libraries. Thus this profile is a minimal expression pattern. Additionally, homologues for the ESTs and genes were searched for (see Materials and methods) and the number of ESTs thus identified are indicated in the columns on the right. No homologues (based upon parameters specified in the text) were found in the C. elegans or D. melanogaster databases.

Discussion

Positional cloning techniques, combined with computer-assisted data analyses of the sequence rich databases generated by human genome projects,42,43 has considerably facilitated the identification of disease genes. The availability of complete and detailed clone contigs of candidate regions make for efficient positional cloning projects. We first constructed a YAC contig of this region and used it as a resource for the construction of a deep BAC contig. At the centromeric end of our YAC contig, there is a YAC, 972c5, which contains markers D2S2182 and D2S2227, which are also located in a published adjoining YAC contig.31 Thus combined with this published YAC contig, this provides a continuous map that spans chromosome 2p15-2p21 (D2S1364–D2S1754, ~14 Mb) and contains several human disease loci.31

Sixty-seven new STSs were identified by inter-Alu PCR and YAC/BAC end sequencing. The high-resolution physical map generated in this study spans ~2 Mb with complete coverage of the minimal region of sitosterolemia. The data presented here have been parsed for multiple ESTs for single genes represented in the databases and we have attempted to summarise data that are found scattered in a number of different databases, increasing the utility of this information. A summary of the results is provided in Figure 3. Based upon the radiation hybrid mapping databases, our initial YAC contig spans approximately 10 cM. However, this area appears to span only 5 Mb in physical length, suggesting a lower than expected recombination frequency (Figure 3). Assuming that all the non-redundant ESTs mapped to the BAC contig are unique transcripts and taking into account the small number of genes known to map into the BAC contig, we estimate that the gene density is approximately 1 gene per 50 kb of genomic DNA (Figure 3, 40 ESTs and genes mapped with the 2 Mb area).

Figure 3.

Figure 3

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Summary of the YAC, BAC and EST mapping data. The figure shows a summary of the data presented in this study, indicating the genetic distance, physical distance and Genbank Accession numbers for mapped ESTs and genes located in the region of interest. Note that the genetic distance, based upon publicly available databases, spans ~10 cM, but spans ~5 Mb. Although only the Genbank IDs are shown, all identified EST can be obtained by utilising the Unigene or the GeneMap'99 identifiers shown in Table 2. Accession numbers in bold represent known genes, the remaining represent putative ESTs. The exact order of the ESTs at any given map location can not be determined at present and are thus grouped, indicated by the vertical lines.

One of our findings is the mis-assignment of BAC R-35M22. This BAC was previously assigned to chromosome 4 (Genbank accession number AC016338, Birren et al, direct submission), but is positive to DNA sequences from BACs R-24I5 and R-194L1. Additionally, it also contains ESTs A004I37, H99661, stSG3387, stSG52154, M95548, and M95548, 9 of 14 exons of KIAA0436 protein and exon 2 of Na+-independent neutral and basic amino acid transporter, thus placing it firmly on chromosome 2, in the interval D2S119-D2S2291.

Our integrated BAC contig allows for more accurate placement of genes and ESTs than the corresponding region in Genemap'99. In the D2S119-D2S2291 interval from GeneMap'99, 43 ESTs listed, 39 of which are unique. However, only nine of these 39 ESTs actually map to the D2S119-D2S2291 interval into our BAC contig, 30 of 39 map outside of this region. Of the 40 ESTs we have physically mapped to the D2S119-D2S2291 interval of our BAC contig, 31 of these were previously assigned to lie outside of this region. Therefore, the accuracy of GeneMap'99 for the D2S119-D2S2291 interval is only 25%, which is similar to the 30% reported by Kirschener et al for the D2S123-D2S2251 interval, but much lower than 75% in the D2S2291-D2S123 interval reported by the same authors.31

In summary, we have developed 67 new STSs, constructed an integrated YAC and BAC contigs for sitosterolaemia region and mapped eight known genes and 48 ESTs to the contig. These results will facilitate the identification of the sitosterolaemia gene and other disease genes located in this region. Additionally, this information may be useful in ordering some of the sequenced BAC contigs and accelerate sequencing of the corresponding genomic clones.

Acknowledgments

We are grateful to Dr Anand Srivastava for expert advice and critical review of our work, to Starr Hazard and the BioMolecular Computing Resource for assistance with the software. This work was funded by a Scientist Development Award from the American Heart Association grant 9730087N (SB Patel) and by the National Institutes of Health, NHLBI Grant HL60616 (SB Patel) and MO1 RR01070-25 (MUSC GCRC), and by an intramural award from the University Research Committee, Medical University of South Carolina (SB Patel).

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