Original Paper

Eur J Hum Genet 1995;3:78-86

1608211 Marjon van Slegtenhorsta Cosmid Contigs from the Bart Janssen* Mark Nellist* Tuberous Sclerosis Candidate Sarvan Ramlakhana Caroline Hermansa Region on 9q34 Arjenne IIesse ling* Ans van den Ouwelanda David Kwiatkowskib Bert Bussen* Julian Sampsonc Key Words Pieter de Jongd Cosmid contig ■ Chromosome walking • ABO • Dicky Halley* Tuberous sclerosis ■ Surfeit • DBH • VAV2 a MGC Department of Clinical Genetics, Erasmus University Rotterdam and Academic Hospital Rotterdam, Abstract The Netherlands; b Experimental Medicine Division, Tuberous sclerosis (TSC) is a heterogeneous multisystem dis­ Brigham and Women's Hospital, order with loci on 9q34 (TSC1) and 16pl3.3 (TSC2). The Boston, Mass., USA; TSC2 has recently been isolated, while the TSC1 gene has c The Institute of Medical Genetics, University Hospital of Wales, been mapped to a 5-cM region between the markers D9S149 Cardiff, UK; and D9S114. In our effort to localise and clone TSC 1, we have d Roswell Park Cancer Institute, Human obtained three adjacent cosmid contigs that cover the core of Genetics Department, Buffalo, N.Y., USA the candidate region. The three contigs comprise approxi­ mately 600 kb and include 80 cosmids, 2 PI clones, 1 YAC, 5 anonymous markers and 4 sequence-tagged sites. The ABO blood group , the Surfeit gene cluster, the dopamine ß- hydroxylase gene (DBH) and YAV2, a homologue of the vav oncogene, have all been mapped within the contigs. Exon trap­ ping and mutation screening experiments, aimed at identi­ fying the TSC 1 gene, are currently in progress.

Introduction ease shows a high penetrance with variable expression and is known for its locus hetero­ Tuberous sclerosis (TSC) is an autosomal geneity, with one locus mapping to chromo­ dominant multisystem disorder. The brain, some 9q34 (TSC1) and another to chromo­ skin, heart and kidneys are often affected and some 16p 13.3 (TSC2) [2], The number of almost all other tissues and organs may be families linked to each locus is approximately involved, except muscle syncytia [1], The dis- equal and there is no significant evidence for a

Received: November 25, 1994 M.A van Slegtenhorst ö 1995 Revision received- February 9, 1995 Department of Clinical Genetics S. Karger AG, Basel Accepted-February 20, 1995 Dr Molewaterplein 50 1018-4813/95/ NL-3015 GE Rotterdam (The Netherlands) 0032-0078Î8.00/0 third locus [3], The TSC2 gene has been iso­ Materials and Methods lated [4] and both may act as growth or Libraries tumour suppressors, since loss of heterozygos­ The ICI YAC library [22] was accessed through the ity (LOH) has been demonstrated on 9q34 [5- UK Mapping Resource Centre and 7] or 16p 13 [7] in various hamartomatous tis­ sets of primary, secondary and tertiary pools for PCR sues from patients with TSC. screening were provided by R. Elaswarapu. Primary The locus for tuberous scle­ pools from the St. Louis YAC library [23] were sup­ plied by J. den Dünnen from the Department of rosis, TSC 1, is tightly linked to the ABO blood Human Genetics in Leiden. The PI library was made group locus [8] and maps in a gene-rich region from human foreskin fibroblast DNA [24], The library on chromosome 9q34. Since the initial link­ was gridded into 125 96-well plates with approximate­ age report by Fryer et al. [8], the TSC1 region ly 12 different PI clones per well and pools were made has been refined to a region of 5 cM between for PCR screening. The chromosome-9-specific cos- mid library LL09NC0LP' was constructed at the Bio­ D9S149 and D9S114 [3, 9-14], However, medical Sciences Division, LLNL, Livermore, Calif., there is no consensus on the exact position of USA under the auspices of the National Laboratory TSC1 within this interval, since some groups Gene Library Projects sponsored by the US Depart­ have found recombinants in favour of a posi­ ment of Energy. The library was replicated on gridded tion proximal to ABO and the dopamine ß- filters as described [25] at the YAC screening core of the Department of Human Genetics in Leiden. Two hydroxylase gene (DBH), while other groups sets of membranes were used to make pools for PCR have presented data supporting a location dis­ screening [26]. The nomenclature of the cosmids in the tal to these markers [15]. The conflicting ob­ contigs is the same as the nomenclature of the library servations have several possible causes, in­ source from which they were obtained. Cosmid cluding misclassification of individuals with ABO 17 was provided by J. Wolfe. only minor clinical findings or non-linkage of Cosmid Library Screening one or more families. Hybridisation probes were generated by inter-Alu Several genes have been mapped within PCR [27] using primers CL1 and CL2 [28] or by isolat­ the TSC1 candidate region, including ABO, ing end fragments from cosmids in low-melting aga­ DBH, the Surfeit gene cluster and VAV2 [ 16- rose. Probes were randomly labelled, competed with 18], while other disorders genetically linked to total human DNA, hybridised to nylon filters and washed using standard procedures [29]. Cosmid libra­ ABO include torsion dystonia [19] and nail ry screening by PCR was performed by screening two- patella syndrome [20, 21]. dimensional pools of clones as described by Green and In this paper we present the results of a Olson [30]. contig assembly and gene mapping effort, fo­ cused on part of the TSC1 candidate region YACs, PI and Cosmid Clones around ABO and DBH. Our detailed map Cosmid and PI DNA was prepared, isolated and fingerprinted using standard techniques [29]. YACs, spans 600 kb, corresponding to more than 2 PI and cosmid clones were mapped back to 9q34 by cM of the TSC1 critical region. Eight genes fluorescence in situ hybridisation (FISH) [31], and several known and novel genetic markers have been precisely positioned on a genomic Sequence-Tagged Sites (STSs) £coRI map between D9S149 and D9S114. STSs were developed by YAC end rescue inverse PCR or direct sequencing of cosmids. YAC end rescue was performed as described by Silverman et al. [32] and the products were sequenced directly. The Se­ quence was derived from the cosmid clones by cycle sequencing (Pharmacia) with the appropriate vector primers.

79 Results and Discussion and it was not investigated further. It is inter­ esting to note that the TSC2 locus on chromo­ Strategy some 16 was also found to be underrepre­ We aimed to isolate a significant part of sented in YAC libraries [unpubl. results]. the TSC1 critical region between the markers D9S149 and D9S114 on 9q34. Several addi­ Contig Assembly tional markers are known to map between Starting points for cosmid contig assembly these two, but have not been convincingly were ABO, DBH and D9S10 (fig. 1). Cosmids associated with genuine recombination were identified with both the left and right events. The initial strategy was to isolate the end clones of YAC 4DD1 and two contigs region in YACs, PI and cosmid clones. How­ were constructed of 110 and 130 kb, respec­ ever, attempts to obtain YACs were ham­ tively (fig. 2, contigs A and B). The orienta­ pered by underrepresentation of the region in tion of the cosmid contigs was consistent with the available libraries. This prompted us to results from YAC inter-Alu PCR screening of follow an alternative strategy which consisted to cosmid library and with the YAC STS map­ of cosmid walking complemented by screen­ ping experiments. No cosmids could be iden­ ing PI libraries. tified distal to cosmid 255A6 (contig B). Only a single non-rearranged cosmid and a single YAC Library Screening P1 clone were detected at the ABO locus, and Two YACs from the ICI library, 4DD1 no clone could be detected linking the two (120 kb) and 25DG9 (320 kb), were identified contigs. However, from the size of the 4DD1 with primers specific for the ABO locus. STS YAC and direct visual hybridisation (DIR- mapping using primer pairs from both ends of VISH) experiments of streched DNA [33] the YACs indicated that the left ends of both [unpubl. results], we estimated that the gap is inserts overlapped; however, inter-Alu PCR approximately 30 kb. in combination with hybridisation experi­ Cosmids were identified with the DBH ments suggested that the region of overlap was cDNA and probe pMCT136 from the D9S10 small. FISH analysis confirmed the localisa­ locus. DBH and D9S10 map 1 and 2 cM distal tion of both YACs to chromosome 9q34; how­ to ABO, respectively, and were linked by ever, 25DG9 gave an additional signal on chromosome walking, covering a physical dis­ chromosome 18 indicating chimerism. This tance of 150 kb (contig C). This indicates that clone was not investigated further. No addi­ the genetic versus physical distance ratio in tional YACs were identified in the ICI library this region is large. The contig was extended using the end clone STSs from 4DD1 or proximal of DBH by 125 kb, but could not be 25DG9, or with additional markers from the extended further towards ABO. We did iso­ TSC1 candidate region (D9S10, D9S66, late a P1 clone with an STS from the proximal DBH). An STS derived from the left arm of end of 251C9, but could not bridge the gap. YAC 4DD1 was used to screen the St. Louis The distance between clone 251C9 (contig B) YAC library and identified two duplicate and 255A6 (contig C) could not be resolved by clones, 51A7 and 61A10 (200 kb). FISH anal­ interphase FISH, indicating that the gap be­ ysis mapped 51A7 to 9qter; however, STS tween contigs B and C is small. DIRVISH mapping experiments using primers derived DNA mapping experiments are in progress to from the right arm of this clone suggested that estimate the size of the gap. it contained a large deletion (data not shown)

80 van Slegtenhorst et al. Cosmid Contigs in the TSC1 Region D9S149 ABO DBH D9S10 D9S114

V ▼ T A B I C I

[ [ ] //■ // cen tel YAC The TSC1 region K\\\\\\\S cosmid contig on 9q34 ] P1 approximately 50 kb.

Fig. 1. Schematic representation of the TSCl region. The starting points for YAC and cosmid walking are indicated, together with the YAC 4DD1, the cosmid contigs and PI clones.

In regions of overlap, the contigs presented and a Hindlll polymorphism (D9S968) was here were consistent with the cosmid contigs detected immediately proximal of DBH. constructed by Hinfl fingerprinting as de­ The position and orientation, where scribed by Nahmias et al. [34], They need at known, of genes identified within the contigs least 50% overlap between cosmids before the are indicated in figure 2. The role and expres­ clones are joined in a contig. Our data are sion pattern of the ABO blood group transfer­ more detailed and detect smaller overlaps. ase indicate that it is not a good candidate for Additional cosmids have been isolated from TSC 1. The Surfeit gene cluster had been pre­ the flanking locus D9S149. Chromosome­ viously mapped by in situ hybridisation tel- walking experiments are currently focused on omeric to the c-abl and can genes on 9q34 closing the gap between D9S149 and the most [35], A oligonucleotide derived from the Surf- proximal ABO contig (contig A). 3 cDNA sequence detected a 1.2-kb EcoRl fragment in several cosmids, slightly distal to Mapping of Markers and Genes in the ABO in contig B. Cosmid 255A6 was digested Contigs with Xbal to orientate the cluster in the map. RFLPs and unique STSs are listed in ta­ In the mouse this cluster consists of 6 house­ bles 1 and 2. The STSs 180G3-T3 and keeping genes, which are unrelated by se­ 4DD1L map to adjacent EcoRl fragments in quence homology [35]. To date, the Surfeit contig A. Two additional STSs, 4DD1R and genes form the tightest gene cluster known in 251C9-T3 were mapped to contigs B and C, mammals. Since these genes are in the critical respectively. Existing minisatellite repeats region of TSCl and not much is known about (D9S122 and D9S150) [13] were precisely po­ their function, mutation analysis in TSC pa­ sitioned within this contig (fig. 2, contig C) tients must be considered.

81 Contig A 180G3-T3 4DD1L

1.4 1 7 6 15 8 11 14 ■ I 7 161 8 I 5m 14 l-=-f + + + H 278E1 — 178A7 147D1- 62G9 80E6 • 180G3 49F6 — 92F3 70C11 142H8 217F1 148F6 40F1 — — 161A1 220G2 256C3

Contig B ABO surf-5 surf-1 surf-4

surf-3 surf-2 4DD1R

05 3.5 * 1.2 0.9, 8; 8 5 10.5 105 13 8 20 + + + -hH- . 6 I4'2!45! 1°-5 4+ + + AB017 3H6 177E4 — 272H1 60F9 39C8 161G2 - 118B5 174E4- 226D9 219C9 218H3 212E10 255A6 211A7 291E1

Contig C DBH 51C9-T3 D9S968 D9S150 D9S122 0.7 —^—I * ± 0.9 2 2 5.5,7 22 I5-5. 8-i*V. 18 9 20 13 1.8 I 17 1.1 + f ■HH- + + ++ + continued below 251C9 254D11 83C7 47F1 201D9 211H3 14F4 100D2 212B11 245G2 124H8 182E3 278C11 -260D5 102C3 -283C7 - 263A9 275C9 5G7 152F5

VAV2

D9S10 D9S66

* Ì 1.5 35 1.1 15 11 . 7 . 11 .4 5. 11 .6.5. 10 h 21 105 .38 +44_jL H—I- » f 152F5 •7A10 —137B8 104C4 270A5 124E8 84 F7 108A10 272C1 -100F10 267E11 ------245G1 37C7 ------255H4 65G5 — 242B6 79B4 184G11

2

82 van Slegtenhorst et al. Cosmid Contigs in the TSC1 Region Table 1. List of RFLPs in the region

Locus Enzyme Probe Fragment sizes, kb Fleterozygosity, %

D9S10 Pstl* MCT136 2.5 and 2.3 50 Hindlll MCT136 2.2 and 2.0 50 (200 ) (RFLPs show linkage disequilibrium) D9S968 Hindlll RD560 4.5 and 2.6 14(115 chromosomes) DBH (several RFLPs* + (CA)n, all listed in GDB) VAV2 Pstl 5' VAV2 5, 4.2 and 2.2 48 (> 100 chromosomes) (bases 1-865)

All RFLPs marked with an asterisk are already listed in GDB. The heterozygosity percent­ ages of the new RFLPs (without asterisk) have been determined in at least 100 chromosomes from Causasians. The map position of each locus is indicated in figures 1 and 2. The VAV2 RFLP maps within the VAV2 gene, distal to the end of the cosmid contig.

Table 2. List of STSs in the region

STS Primer sequences Product Map position length

180G3-T3 5' GGTGT GGTTC TCCCA AGGG 3' 128 bp distal part of GAGAG AGGCT TCCTG CTTGC contig A 4DD1L 5' CCAAG GGAAG CTGGA GAAGT 3' 97 bp left arm of CCAGA CCCAG CCTAC ATTTC YAC4DD1 4DD1R 5' CATGC TGTTG GCACT GTTGTA 3' 135 bp right arm of TTTCT CTTTG GCTTC CCTCTT YAC4DD1 251C9-T3 5' GGAAA GAGGA GCGAG GAAG 3' 152 bp approx, end of CACAA TCTCA CAGTG AATGCC contig C

A number of polymorphic STSs at ABO, DBH, VAV2, D9S149, D9S150, D9S122, D9S66 and D9S114 have been described previously and are therefore not included in the list.

Fig. 2. Detailed fscoRI restriction map of the three contigs described in this paper. Cos- mids are shown below the the £coRI map. Thin bars represent RFLP markers and vertical arrows indicate STSs and microsatellites. Genes are shown above the restriction map as thick bars. The size of the bars indicates the maximal genomic extent. The direction of transcription is indicated by arrowheads. For DBH, surf-1, surf-2, surf-3 and VAV2, the gene structure was studied by Nahmias et al. [34], Yon et al. [35] and Kwiatkowski et al. [16]. The position and orientation of the genes in the cosmid contigs were deduced from our experimients and pre­ viously published restriction maps [34, 35].

83 Our EcoKl mapping data from the DBH D9S10, previously estimated to be 1 cM locus is consistent with that of Kobayashi et apart, are separated by less than 300 kb, and al. [36]. The direction of transcription is to­ estimate that the physical distance between wards the telomere. The role of DBH in the ABO and DBH is less than 300 kb. conversion of dopamine to noradrenaline and In conjunction with the accompanying ar­ the neurological manifestations of TSC led to ticle [34], we have shown that cosmid walk­ the proposal that DBH could be a candidate ing, using a large chromosome-specific cos- for the TSC 1 gene [37], However, more recent mid library can provide almost complete results suggest that TSC 1 maps either distal or coverage of a large genomic region. This min­ proximal to DBH and consequently DBH is imises the need to search for non-chimeric not such an attractive candidate. non-rearranged YAC clones, which have been Exon trapping [38] efforts using our cos- difficult to obtain from the TSC1 region. mids from the D9S10 locus identified a gene Moreover, our contigs and the associated homologous to the vav oncogene [16]. This maps provide a good tool for generating novel gene, designated VAV2, was considered a markers and cloning additional genes from good candidate for the TSC1 gene. However, this region. It would be of great help to get intensive screening failed to identify any mu­ more excluding data on the recombinants tations, and VAV2 was consequently ex­ within the region, so that the search for TSC1 cluded as a candidate gene for TSC1 [16, 17]. can be restricted to a smaller area. LOH stud­ Eight different genes could be placed on ies in tumours of patients and the develop­ the map. The region is gene dense and al­ ment of new polymorphic CA repeats in the though some genes map extremely close to area, especially between ABO and D9S149, each other, we cannot exclude the presence of could help reduce the critical region. Ulti­ other, as yet unidentified, expressed se­ mately, it is hoped that this work will lead to quences in the same region. Experiments to the identification of the TSC1 gene. identify and characterise additional genes from the TSC1 candidate region are in pro­ gress. Acknowledgements Further efforts are being directed towards extending the contigs and screening TSC pa­ We are grateful to Professors H. Galjaard and D. Lindhout for their continuous support. We would like tients for mutations by pulsed-field gel elec­ to thank Dr. S. Povey, Dr. J. Wolfe, Joseph Nahmias trophoresis using novel probes derived from and co-workers for sharing unpublished data and for our cloned material. The identification of their cooperation. This work was funded by the Dutch large deletions at the TSC2 locus made a sig­ Organisation for Scientific Research (NWO). nificant contribution to the rapid isolation of the TSC2 gene [4], In conclusion, we have identified 80 cos- mids, 2 P1 clones and a single non-rearranged YAC from the TSC1 candidate region on 9q34. We have constructed a detailed restric­ tion map of three adjacent cosmid contigs and oriented the maps with respect to known and previously unidentified genes and DNA markers. We have shown that DBH and

84 van Slegtenhorst et al. Cosmid Contigs in the TSC1 Region References

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86 van Slegtenhorst et al. Cosmid Contigs in the TSC1 Region