SPECIAL FEATURES CENTROMERE
MEETING REPORT
D22S24
Proceedings of the Second International Chromosome 22 Workshop
D22S9 D22S43 TOP1P2 D22S264"
GuY A. ROULEAU AND BEVERLY S. EMANUEL 1
BCR
The Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, Pennsylvania 19104
CRYB2A* D22S1
This summary outlines the progress made toward constructing a physical map of h u m a n chromosome 22 at a workshop held in Montebello, Quebec, September 10-13, 1991. This meeting was supported by the National Institutes of Health (U.S.A.), the Medical Research Council of Canada, the Fonds de Recherche en Sante du Quebec, and the National Cancer Institute of Canada and was endorsed by HUGO. The highlights of this meeting included: (1) the presentation of the cloning of the Ewing sarcoma t(11;22) breakpoint; (2) the evidence that there is a good start toward YAC and cosmid contig construction; (3) the presentation of extensive longrange maps and the assignment of a set of temporary reference markers, two of which fulfill the criterion of being of index marker quality (Fig. 1). At present there are five cosmid contigs that cover 1.5 Mb, and a 1.8-Mb YAC contig around D22S1. The most extensive pulsed-field gel (PFG) map extends 7.4 Mb. This represents approximately 20% of chromosome 22. The first scientific session addressed the construction of a genetic map of chromosome 22. J. D u m a n s k i (Stockholm) presented a map consisting of 45 polymorphic markers typed in C E P H pedigrees, including 35 markers generated at his institute. The total span of the map is 110 cM with an average distance of 5 cM between markers. Twenty-two loci, including haplotypes of multiple markers at the same locus, were ordered with a probability of over 1000:1. J. Haines (Boston) described a genetic linkage map of h u m a n chromosome 22, including 28 polymorphic markers using the Venezuelan Reference Pedigree. The map extends 128 cM (sex averaged) from D22S24 to D22S45. The sex-specific maps are of similar length, although there is variation in individual regions. J. Haines also reported the existence of eight dinucleotide repeat polymorphisms, six of which are included in his map. These polymorphisms are D22S156, D22S257, D22S258, PDGFB, CRYB2, CYP2D, IL2RB, and TOP1P2. The second scientific session dealt with the cytogenetic analysis of chromosome 22. L. Kearney (London) presented data on a flow-sorted chromosome 22 cosmid library constructed at the ICRF in collaboration with Dr. H. Lehrach. A total of 9216 clones have been gridded a n d are available for probing. Of the seven random clones from this library that were mapped by in situ hybridization, four localized to chromosome 22 (approxi1To whom correspondence should be addressed at Room 5033B, The Children's/Hpspital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104. 0888-7543/92 $5.00 Copyright © 1992by AcademicPress, Inc. All rights of reproductionin any form reserved.
D22S32 D22S29 KI436 CYP2D D22S22 D22S23 D22S21 D22S55 ARSA TELOMERE
FIG. 1. The ordered set of chromosome 22 reference markers determined at the Montreal Chromosome 22 Workshop held in September 1991. The asterisks indicate index markers that have over 70% heterozygosity. These markers are available upon request from Dr. Marcia Budarf, The Human Genome Center, Children's Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104.
mately 60%). DNA from a sorted normal chromosome 22, a constitutional translocation t(11;22) chromosome, and a CML t(9;22) chromosome were successfully used for whole chromosome and regional p a i n t i n g by fluorescence in situ hybridization (FISH). A. Aurias (Paris) presented F I S H data with the precise mapping of D22S1 to the 22qll.32-q12.1 interface, VIIIF2 to 22q12.1, a n d LIF to the 22q12.1-q12.2 interface. Testing of DiGeorge patients showed the presence of a single chromosome 22 with cosmid 506 (from Dr. P. Scambler). Cosmid 2271 (P. Scambler) displayed m a n y loci on 22q11.2, suggesting the presence of multiple duplicated sequences in this chromosomal region. M. Budarf (Philadelphia) described the development of new fluorescent nucleotides for use in in situ hybridization experiments. These probes can be introduced into DNA by nick-translation or random priming and yield signal intensities similar to t h a t obtained with biotin-avidin. Use of these nucleotides eliminates the need for most posthybridization steps, a n d different fluorescent labels can be used simultaneously. The third scientific session dealt with the presence of repeat sequences on chromosome 22. G. Rouleau (Montreal) presented data identifying six separate G G T loci. Three loci map
1124
GENOMICS14, 1124-1126 {1992)
SPECIAL FEATURES to chromosome 22: one between the B C R region and the Ewing sarcoma breakpoint, a n d two between the centromere and the B C R region. T h r e e additional G G T loci each map to chromosomes 18, 19, a n d 20. F I S H confirmed the presence of multiple copies of G G T on chromosome 22q11.2. P. Scambler (London) identified small probes t h a t detect chromosome 22-specific repeat sequences p r e s e n t in 2 to 30 copies. Some of these duplications give rise to closely linked loci, while others are more dispersed throughout 22ql 1. H e speculated t h a t the presence of these repeats might make the region meiotically unstable, leading to the high frequency of chromosome aberrations in the region. T h e fourth session dealt with chromosome 22 translocations a n d hybrid m a p p i n g panels. G. T h o m a s (Paris) described 31 well-characterized somatic cell hybrids t h a t identify 29 separate regions on chromosome 22, a level of resolution of 1-2 Mb. T h e hybrids described included 2 chromosome 22-only cell lines, 3 pairs of hybrids t h a t are derived from the same translocation b u t which contain the different derivative chromosomes, 12 hybrids containing a single distal fragment, 3 hybrids with a single p r o x i m a l fragment, 5 hybrids with a single interstitial fragment, and 3 hybrids with multiple fragments. M. Lipinski (Paris) p r e s e n t e d d a t a on irradiation-reduced hybrids constructed using EYEF3A6. These were characterized using F I S H , Southern blotting, a n d PCR. One hybrid, DA5, contains one large a n d two small h u m a n chromosome fragments, one of which includes the interval from L I F to D22S29. Inter-Alu-PCR was used to generate D N A from this region, which was cloned into pUC18. These clones are under study. M. B u d a r f (Philadelphia) presented d a t a on 22 somatic cell hybrids t h a t subdivide chromosome 22 into 20 intervals. T h e highest density of b r e a k p o i n t s is in 22q11, which is separated into 12 regions. A n u m b e r of loci have been m a p p e d using these hybrids, including ADSL, peripheral benzodiazepine receptor (BZRP), GNAZ, p a r v a l b u m i n (PVALB), acrosin (ACR), catechol-O-methyltransferase (COMT), and an expressed L I N E sequence (LRE1). In the fourth session the isolation of clones from chromosome 22 was discussed. J. T r o f a t t e r (Boston) isolated 3800 cosmids from three separate sources: (1) flow-sorted chromosome 22 D N A (Lawrist 5; 2700 cosmids); (2) chromosome 22-specific cell line 10888 (sCos-1; 400 cosmids); a n d (3) a somatic cell hybrid composed of chromosome 22 and some p a r t s of chromosomes 15 a n d 19 (pWE15; 700 cosmids). T h e library was screened for microsatellite repeat sequences with identification of 600 (GT) n positive clones, 140 (AG) n positive clones, a n d 40 (AAC)n positive clones. These are being m a p p e d to specific regions of chromosome 22 by either F I S H or a somatic cell h y b r i d panel. T h e y are further screening the library with the following oligonucleotide probes: (AT)12, (AGC)7, (AAT)7, (AGG)7, (CCG)7 (AAAG)7, (AAAT)7 (AAGG)6, a n d (AGAT) 7. P. de Jong (Livermore) p r e s e n t e d information on two cosmid libraries constructed from sorted chromosome 22 DNA. T h e first was cloned into Lawrist5, arrayed, a n d tested. It was found to be complete but only 30% of the clones were from chromosome 22. T h e second library has only just been completed a n d seems to be of 85% purity, with 10-fold redundancy for chromosome 22. These libraries have been distributed to numerous investigators. N. Blin (Homburg) presented d a t a on several new polymorphisms. J. Zucman (Paris) described a
m e t h o d using Alu-PCR-amplified D N A from defined somatic cell hybrids in F I S H for regional chromosomal p a i n t i n g and for hybridization to gridded chromosome 22 cosmids. Cosmids identified by this technique were found to m a p in the expected regions of the chromosome. Using this m e t h o d a n d the 31 somatic cell hybrids of G. Thomas, it is possible to identify cosmids t h a t map to any of 29 subregions of chromosome 22. M. Aubry (Montreal) described the cloning and mapping of six novel zinc-finger motif-containing cosmids to chromosome 22. Two m a p to the short arm a n d four to 22q11.2. M. Sanson (Montreal) described the isolation and mapping of 45 NotI linking clones to the long arm of chromosome 22. He estimates t h a t this represents less t h a n half the sites present on chromosome 22. W. Fiedler (Erlangen) described a microdissection library generated for chromosome 22q12-q13.2. Twenty-seven clones were regionally mapped: 10 proximal and 17 distal to the Ewing sarcoma translocation. T h e most extensive session was devoted to the physical m a p p i n g of chromosome 22. H. M c D e r m i d (Edmonton) presented a p a r t i a l long-range restriction m a p for 22ql 1 containing a p p r o x i m a t e l y 7400 kb. She also presented d a t a on the precise mapping of a congenital deletion of 22q13 with the proximal b r e a k p o i n t near D22S29. A. M e n o n (Boston) presented d a t a on the fragment sizes detected by 13 probes t h a t are within the D22S1-D22S29 interval for NotI, NruI, MluI, a n d other infrequently cutting enzymes. T h e probes D22S1 a n d D22S33 were found to map to the same 200~kb SfiI fragment. N. Blin (Homburg) showed t h a t D22S16 and P D G F B map to the same 900-kb NruI fragment. W. Fiedler presented d a t a on the m a p p i n g of 23 microclones from 22q12-q13.1 using the enzymes NotI, SfiI, MluI, and BssHII. T h e lengths of the BssHII fragments added up to approximately 5000 kb. EAN20 and L I F were located on the same SfiI fragment; EAN09 and EAN78 were located on common NotI, SfiI, a n d BssHII fragments. O. Delattre (Paris) described the use of the Lawrence Liver~ more cosmid library in the construction of cosmid contigs by r a p i d walking. In all, 1500 kb of D N A in five large contigs has been constructed in the D22S1-D22S15 interval. I. D u n h a m (London) and J. Collins (London) described their efforts to isolate chromosome 22 YACs. T h e y have screened the S a i n t Louis (average YAC size 250 kb) and the I C R F (T. Monaco; average YAC size 600 kb) YAC libraries using a pool of chromosome 22 probes obtained from J. Dumanski. A total of 604 YACs have been identified by this method. T h e y have also developed an a u t o m a t e d system for placing 1536 YAC clones on one nylon m e m b r a n e for screening by hybridization. This density will allow the entire array of chromosome 22 YACs to be included on the same filter. T h e y have identified, by hybridization, 105 YACs for specific loci, including 51 for GGT. Twenty-six S T S s have been designed and are being u s e d to identify cosmids. Two YACs for N A G A were found to be positive for CYP2D, suggesting a close physical association. K. P a t e l (London) reported the identification of a L I F YAC (200 kb), a D22S15 YAC (450 kb), a D22S1 YAC (900 kb), and YACs for each end of the D22S1 YAC (500 a n d 550 kb each). This D22S1 YAC contig spans 1800 kb. These YACs were oriented by in situ hybridization using end cosmids. He also described the isolation of 11 zinc-finger motif-containing cosmids t h a t map, by in situ hybridization to 22p (two clones),
1125
SPECIAL FEATURES 2 2 q l l (six clones), 22q12 (one clone), and 22q13 (two clones). NotI linking clones are also being constructed using a PCRbased strategy in which Alu a n d NotI primers are used. Eight unique fragments have been isolated from the interval D 2 2 S 1 D22S15 using irradiation-reduced hybrids. C. Denny (Los Angeles) described his investigations into the causes of cocloning in YACs. He found t h a t the net crossover rate was small (3-4%), suggesting t h a t coligation is the most frequent source of scrambled clones. He discussed various strategies to avoid this problem. H. Shizuya (Pasadena) presented d a t a demonstrating the instability of cosmid clones. He then described a new cloning vector, called bacterial artificial chromosome (BAC), which is derived from the F-factor. T h e average insert size is 100 kb a n d recombinant clones containing h u m a n D N A are very stable. A library using chromosome 22-sorted D N A is being constructed. N. Shepherd (Wilmington) described a new vector system based on P1 phage. The main advantage of this system is the large insert size (average 100 kb) and stability of the clones. T h e seventh session was devoted to neoplasia associated with chromosome 22. B. E m a n u e l (Philadelphia) showed t h a t three rhabdoid tumors and a closely related atypical teratoid t u m o r lost chromosome 22 both cytogenetically and molecularly. O. Delattre (Paris) described the cloning of the Ewing sarcoma and neuroepithelioma breakpoint. All the translocations map within a small region on chromosome 22 (a few kilobases) t h a t is flanked by two H T F islands. T h e chromosome 22 gene has not yet been identified. M. Ruttledge (Stockholm) p r e s e n t e d recent work with deletions in meningioma. Their findings are consistent with a meningioma locus distal to D22S28, making this a separate locus from neurofibromatosis type 2 (NF2). E. Zwarthoff (Rotterdam) presented work on a translocation in a meningioma. T h e translocation breakpoint is detected on P F G analysis using the probe NB129 and maps to the interval flanked by D22S1 and LIF. T h e same probe detected a constitutional deletion in a p a t i e n t with multiple meningiomas, cDNAs are being isolated from this candidate region. The possible significance of this finding, in relation to NF2 and the more distal putative meningioma locus, was discussed, although no consensus was reached. G. Rouleau (Montreal) presented genetic d a t a t h a t confirmed the localization of NF2 to the interval flanked by D22S1 and D22S28 and suggested t h a t D22S32 might be a closer telomeric flanking marker. Evidence strongly rejecting the hypothesis t h a t there is nonallelic genetic heterogeneity in NF2 was presented. T h e eighth and final session was devoted to the molecular definition of the DiGeorge a n d Cat Eye syndromes. P. Scambler (London) presented arguments for reclassifying the velocardiofacial syndrome as a subtype of DiGeorge. He found deletions in eight of eight cases with the cosmids 197 and 506, which are frequently deleted in classical DiGeorge. He also described a family in which the same deletion led to DiGeorge in some family members and velocardiofacial syndrome in others. B. E m a n u e l (Philadelphia) presented detailed mapping d a t a in the DiGeorge region. T h e markers N25, 160b, and R32, which are deleted in 14 of 14 patients, map to the interval defined by the somatic cell hybrid breakpoints GL5 a n d G M l 1 2 2 0 t(X;22). A somatic cell hybrid segregating a DiGeorge microdeletion chromosome has been constructed. Four
GENOMICS14, 1126-1132 (1992)
YACs have been isolated for the locus N25 and are being studied. R F L P analysis of p a r e n t s and children with DiGeorge has shown t h a t the deletion occurred in the maternally inherited chromosome in four of five instances. S. Demczuk (Montreal) presented preliminary d a t a on the identification of chromosome 22 homeobox-containing genes as candidates for DiGeorge syndrome. H. M c D e r m i d (Edmonton) m a p p e d the putative r e a r r a n g e m e n t b r e a k p o i n t in a Cat Eye syndrome pat i e n t to within 100 kb of the m a r k e r D22S72. P. P e a r s o n (Baltimore) described the aims and practical uses of the Genome D a t a B a s e (GDB), as well as the practical problems encountered in designing a large database t h a t integrates genetic and physical d a t a generated from different sources. He suggested t h a t a chromosome 22 database should be constructed so t h a t it can interface with GDB. F u t u r e Perspectives It was agreed t h a t there should be another workshop to be held in Philadelphia in 1 year. A new set of reference markers for chromosome 22 was selected. It was felt t h a t these loci should display very informative polymorphisms, preferably of the microsatellite type. Dr. B u d a r f (Philadelphia) volunteered to serve as a distributing lab for these new markers (see Fig. 1). The most effective method for making a complete hybrid cell panel generally available was discussed. Dr. G. T h o m a s (Paris) agreed to make available filters of his detailed mapping panel so t h a t any other investigator can map loci using the same reference breakpoints. G. T h o m a s (Paris) has agreed to serve as a coordinator for d a t a generated using the Lawrence Livermore cosmid library. J. T r o f a t t e r (Boston) agreed to compile all d a t a on chromosome 22 STSs. J. Collins and I. D u n h a m (London) agreed to screen their YAC libraries for anyone interested in identifying a clone. T h e most efficient method for dissemination of information and communication within the group was discussed. T h e r e was general agreement t h a t there should be a newsletter t h a t would be sent to all interested persons by E-mail, preferably using the chromosome 22 bulletin board. Dr. B. E m a n u e l agreed to coordinate this effort.
MEETING REPORT
Third International Workshop on Human Chromosome 21 STYLIANOS E. ANTONARAKIS The Johns Hopkins University School of Medicine, Center for Medical Genetics, Baltimore, Maryland 21287 This report summarizes progress made toward the completion of the mapping of h u m a n chromosome 21 as presented, discussed, and debated at the T h i r d I n t e r n a t i o n a l W o r k s h o p on H u m a n Chromosome 21. This workshop was hosted by The Johns H o p k i n s University School of Medicine at the Maryland Engineering Center in Baltimore, Maryland, on April 2021, 1992, and was supported by the N a t i o n a l Center for Hu-
1126
0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
MEETING REPORT
D22S24
Proceedings of the Second International Chromosome 22 Workshop
D22S9 D22S43 TOP1P2 D22S264"
GuY A. ROULEAU AND BEVERLY S. EMANUEL 1
BCR
The Children's Hospital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, Pennsylvania 19104
CRYB2A* D22S1
This summary outlines the progress made toward constructing a physical map of h u m a n chromosome 22 at a workshop held in Montebello, Quebec, September 10-13, 1991. This meeting was supported by the National Institutes of Health (U.S.A.), the Medical Research Council of Canada, the Fonds de Recherche en Sante du Quebec, and the National Cancer Institute of Canada and was endorsed by HUGO. The highlights of this meeting included: (1) the presentation of the cloning of the Ewing sarcoma t(11;22) breakpoint; (2) the evidence that there is a good start toward YAC and cosmid contig construction; (3) the presentation of extensive longrange maps and the assignment of a set of temporary reference markers, two of which fulfill the criterion of being of index marker quality (Fig. 1). At present there are five cosmid contigs that cover 1.5 Mb, and a 1.8-Mb YAC contig around D22S1. The most extensive pulsed-field gel (PFG) map extends 7.4 Mb. This represents approximately 20% of chromosome 22. The first scientific session addressed the construction of a genetic map of chromosome 22. J. D u m a n s k i (Stockholm) presented a map consisting of 45 polymorphic markers typed in C E P H pedigrees, including 35 markers generated at his institute. The total span of the map is 110 cM with an average distance of 5 cM between markers. Twenty-two loci, including haplotypes of multiple markers at the same locus, were ordered with a probability of over 1000:1. J. Haines (Boston) described a genetic linkage map of h u m a n chromosome 22, including 28 polymorphic markers using the Venezuelan Reference Pedigree. The map extends 128 cM (sex averaged) from D22S24 to D22S45. The sex-specific maps are of similar length, although there is variation in individual regions. J. Haines also reported the existence of eight dinucleotide repeat polymorphisms, six of which are included in his map. These polymorphisms are D22S156, D22S257, D22S258, PDGFB, CRYB2, CYP2D, IL2RB, and TOP1P2. The second scientific session dealt with the cytogenetic analysis of chromosome 22. L. Kearney (London) presented data on a flow-sorted chromosome 22 cosmid library constructed at the ICRF in collaboration with Dr. H. Lehrach. A total of 9216 clones have been gridded a n d are available for probing. Of the seven random clones from this library that were mapped by in situ hybridization, four localized to chromosome 22 (approxi1To whom correspondence should be addressed at Room 5033B, The Children's/Hpspital of Philadelphia, 34th and Civic Center Boulevard, Philadelphia, PA 19104. 0888-7543/92 $5.00 Copyright © 1992by AcademicPress, Inc. All rights of reproductionin any form reserved.
D22S32 D22S29 KI436 CYP2D D22S22 D22S23 D22S21 D22S55 ARSA TELOMERE
FIG. 1. The ordered set of chromosome 22 reference markers determined at the Montreal Chromosome 22 Workshop held in September 1991. The asterisks indicate index markers that have over 70% heterozygosity. These markers are available upon request from Dr. Marcia Budarf, The Human Genome Center, Children's Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104.
mately 60%). DNA from a sorted normal chromosome 22, a constitutional translocation t(11;22) chromosome, and a CML t(9;22) chromosome were successfully used for whole chromosome and regional p a i n t i n g by fluorescence in situ hybridization (FISH). A. Aurias (Paris) presented F I S H data with the precise mapping of D22S1 to the 22qll.32-q12.1 interface, VIIIF2 to 22q12.1, a n d LIF to the 22q12.1-q12.2 interface. Testing of DiGeorge patients showed the presence of a single chromosome 22 with cosmid 506 (from Dr. P. Scambler). Cosmid 2271 (P. Scambler) displayed m a n y loci on 22q11.2, suggesting the presence of multiple duplicated sequences in this chromosomal region. M. Budarf (Philadelphia) described the development of new fluorescent nucleotides for use in in situ hybridization experiments. These probes can be introduced into DNA by nick-translation or random priming and yield signal intensities similar to t h a t obtained with biotin-avidin. Use of these nucleotides eliminates the need for most posthybridization steps, a n d different fluorescent labels can be used simultaneously. The third scientific session dealt with the presence of repeat sequences on chromosome 22. G. Rouleau (Montreal) presented data identifying six separate G G T loci. Three loci map
1124
GENOMICS14, 1124-1126 {1992)
SPECIAL FEATURES to chromosome 22: one between the B C R region and the Ewing sarcoma breakpoint, a n d two between the centromere and the B C R region. T h r e e additional G G T loci each map to chromosomes 18, 19, a n d 20. F I S H confirmed the presence of multiple copies of G G T on chromosome 22q11.2. P. Scambler (London) identified small probes t h a t detect chromosome 22-specific repeat sequences p r e s e n t in 2 to 30 copies. Some of these duplications give rise to closely linked loci, while others are more dispersed throughout 22ql 1. H e speculated t h a t the presence of these repeats might make the region meiotically unstable, leading to the high frequency of chromosome aberrations in the region. T h e fourth session dealt with chromosome 22 translocations a n d hybrid m a p p i n g panels. G. T h o m a s (Paris) described 31 well-characterized somatic cell hybrids t h a t identify 29 separate regions on chromosome 22, a level of resolution of 1-2 Mb. T h e hybrids described included 2 chromosome 22-only cell lines, 3 pairs of hybrids t h a t are derived from the same translocation b u t which contain the different derivative chromosomes, 12 hybrids containing a single distal fragment, 3 hybrids with a single p r o x i m a l fragment, 5 hybrids with a single interstitial fragment, and 3 hybrids with multiple fragments. M. Lipinski (Paris) p r e s e n t e d d a t a on irradiation-reduced hybrids constructed using EYEF3A6. These were characterized using F I S H , Southern blotting, a n d PCR. One hybrid, DA5, contains one large a n d two small h u m a n chromosome fragments, one of which includes the interval from L I F to D22S29. Inter-Alu-PCR was used to generate D N A from this region, which was cloned into pUC18. These clones are under study. M. B u d a r f (Philadelphia) presented d a t a on 22 somatic cell hybrids t h a t subdivide chromosome 22 into 20 intervals. T h e highest density of b r e a k p o i n t s is in 22q11, which is separated into 12 regions. A n u m b e r of loci have been m a p p e d using these hybrids, including ADSL, peripheral benzodiazepine receptor (BZRP), GNAZ, p a r v a l b u m i n (PVALB), acrosin (ACR), catechol-O-methyltransferase (COMT), and an expressed L I N E sequence (LRE1). In the fourth session the isolation of clones from chromosome 22 was discussed. J. T r o f a t t e r (Boston) isolated 3800 cosmids from three separate sources: (1) flow-sorted chromosome 22 D N A (Lawrist 5; 2700 cosmids); (2) chromosome 22-specific cell line 10888 (sCos-1; 400 cosmids); a n d (3) a somatic cell hybrid composed of chromosome 22 and some p a r t s of chromosomes 15 a n d 19 (pWE15; 700 cosmids). T h e library was screened for microsatellite repeat sequences with identification of 600 (GT) n positive clones, 140 (AG) n positive clones, a n d 40 (AAC)n positive clones. These are being m a p p e d to specific regions of chromosome 22 by either F I S H or a somatic cell h y b r i d panel. T h e y are further screening the library with the following oligonucleotide probes: (AT)12, (AGC)7, (AAT)7, (AGG)7, (CCG)7 (AAAG)7, (AAAT)7 (AAGG)6, a n d (AGAT) 7. P. de Jong (Livermore) p r e s e n t e d information on two cosmid libraries constructed from sorted chromosome 22 DNA. T h e first was cloned into Lawrist5, arrayed, a n d tested. It was found to be complete but only 30% of the clones were from chromosome 22. T h e second library has only just been completed a n d seems to be of 85% purity, with 10-fold redundancy for chromosome 22. These libraries have been distributed to numerous investigators. N. Blin (Homburg) presented d a t a on several new polymorphisms. J. Zucman (Paris) described a
m e t h o d using Alu-PCR-amplified D N A from defined somatic cell hybrids in F I S H for regional chromosomal p a i n t i n g and for hybridization to gridded chromosome 22 cosmids. Cosmids identified by this technique were found to m a p in the expected regions of the chromosome. Using this m e t h o d a n d the 31 somatic cell hybrids of G. Thomas, it is possible to identify cosmids t h a t map to any of 29 subregions of chromosome 22. M. Aubry (Montreal) described the cloning and mapping of six novel zinc-finger motif-containing cosmids to chromosome 22. Two m a p to the short arm a n d four to 22q11.2. M. Sanson (Montreal) described the isolation and mapping of 45 NotI linking clones to the long arm of chromosome 22. He estimates t h a t this represents less t h a n half the sites present on chromosome 22. W. Fiedler (Erlangen) described a microdissection library generated for chromosome 22q12-q13.2. Twenty-seven clones were regionally mapped: 10 proximal and 17 distal to the Ewing sarcoma translocation. T h e most extensive session was devoted to the physical m a p p i n g of chromosome 22. H. M c D e r m i d (Edmonton) presented a p a r t i a l long-range restriction m a p for 22ql 1 containing a p p r o x i m a t e l y 7400 kb. She also presented d a t a on the precise mapping of a congenital deletion of 22q13 with the proximal b r e a k p o i n t near D22S29. A. M e n o n (Boston) presented d a t a on the fragment sizes detected by 13 probes t h a t are within the D22S1-D22S29 interval for NotI, NruI, MluI, a n d other infrequently cutting enzymes. T h e probes D22S1 a n d D22S33 were found to map to the same 200~kb SfiI fragment. N. Blin (Homburg) showed t h a t D22S16 and P D G F B map to the same 900-kb NruI fragment. W. Fiedler presented d a t a on the m a p p i n g of 23 microclones from 22q12-q13.1 using the enzymes NotI, SfiI, MluI, and BssHII. T h e lengths of the BssHII fragments added up to approximately 5000 kb. EAN20 and L I F were located on the same SfiI fragment; EAN09 and EAN78 were located on common NotI, SfiI, a n d BssHII fragments. O. Delattre (Paris) described the use of the Lawrence Liver~ more cosmid library in the construction of cosmid contigs by r a p i d walking. In all, 1500 kb of D N A in five large contigs has been constructed in the D22S1-D22S15 interval. I. D u n h a m (London) and J. Collins (London) described their efforts to isolate chromosome 22 YACs. T h e y have screened the S a i n t Louis (average YAC size 250 kb) and the I C R F (T. Monaco; average YAC size 600 kb) YAC libraries using a pool of chromosome 22 probes obtained from J. Dumanski. A total of 604 YACs have been identified by this method. T h e y have also developed an a u t o m a t e d system for placing 1536 YAC clones on one nylon m e m b r a n e for screening by hybridization. This density will allow the entire array of chromosome 22 YACs to be included on the same filter. T h e y have identified, by hybridization, 105 YACs for specific loci, including 51 for GGT. Twenty-six S T S s have been designed and are being u s e d to identify cosmids. Two YACs for N A G A were found to be positive for CYP2D, suggesting a close physical association. K. P a t e l (London) reported the identification of a L I F YAC (200 kb), a D22S15 YAC (450 kb), a D22S1 YAC (900 kb), and YACs for each end of the D22S1 YAC (500 a n d 550 kb each). This D22S1 YAC contig spans 1800 kb. These YACs were oriented by in situ hybridization using end cosmids. He also described the isolation of 11 zinc-finger motif-containing cosmids t h a t map, by in situ hybridization to 22p (two clones),
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SPECIAL FEATURES 2 2 q l l (six clones), 22q12 (one clone), and 22q13 (two clones). NotI linking clones are also being constructed using a PCRbased strategy in which Alu a n d NotI primers are used. Eight unique fragments have been isolated from the interval D 2 2 S 1 D22S15 using irradiation-reduced hybrids. C. Denny (Los Angeles) described his investigations into the causes of cocloning in YACs. He found t h a t the net crossover rate was small (3-4%), suggesting t h a t coligation is the most frequent source of scrambled clones. He discussed various strategies to avoid this problem. H. Shizuya (Pasadena) presented d a t a demonstrating the instability of cosmid clones. He then described a new cloning vector, called bacterial artificial chromosome (BAC), which is derived from the F-factor. T h e average insert size is 100 kb a n d recombinant clones containing h u m a n D N A are very stable. A library using chromosome 22-sorted D N A is being constructed. N. Shepherd (Wilmington) described a new vector system based on P1 phage. The main advantage of this system is the large insert size (average 100 kb) and stability of the clones. T h e seventh session was devoted to neoplasia associated with chromosome 22. B. E m a n u e l (Philadelphia) showed t h a t three rhabdoid tumors and a closely related atypical teratoid t u m o r lost chromosome 22 both cytogenetically and molecularly. O. Delattre (Paris) described the cloning of the Ewing sarcoma and neuroepithelioma breakpoint. All the translocations map within a small region on chromosome 22 (a few kilobases) t h a t is flanked by two H T F islands. T h e chromosome 22 gene has not yet been identified. M. Ruttledge (Stockholm) p r e s e n t e d recent work with deletions in meningioma. Their findings are consistent with a meningioma locus distal to D22S28, making this a separate locus from neurofibromatosis type 2 (NF2). E. Zwarthoff (Rotterdam) presented work on a translocation in a meningioma. T h e translocation breakpoint is detected on P F G analysis using the probe NB129 and maps to the interval flanked by D22S1 and LIF. T h e same probe detected a constitutional deletion in a p a t i e n t with multiple meningiomas, cDNAs are being isolated from this candidate region. The possible significance of this finding, in relation to NF2 and the more distal putative meningioma locus, was discussed, although no consensus was reached. G. Rouleau (Montreal) presented genetic d a t a t h a t confirmed the localization of NF2 to the interval flanked by D22S1 and D22S28 and suggested t h a t D22S32 might be a closer telomeric flanking marker. Evidence strongly rejecting the hypothesis t h a t there is nonallelic genetic heterogeneity in NF2 was presented. T h e eighth and final session was devoted to the molecular definition of the DiGeorge a n d Cat Eye syndromes. P. Scambler (London) presented arguments for reclassifying the velocardiofacial syndrome as a subtype of DiGeorge. He found deletions in eight of eight cases with the cosmids 197 and 506, which are frequently deleted in classical DiGeorge. He also described a family in which the same deletion led to DiGeorge in some family members and velocardiofacial syndrome in others. B. E m a n u e l (Philadelphia) presented detailed mapping d a t a in the DiGeorge region. T h e markers N25, 160b, and R32, which are deleted in 14 of 14 patients, map to the interval defined by the somatic cell hybrid breakpoints GL5 a n d G M l 1 2 2 0 t(X;22). A somatic cell hybrid segregating a DiGeorge microdeletion chromosome has been constructed. Four
GENOMICS14, 1126-1132 (1992)
YACs have been isolated for the locus N25 and are being studied. R F L P analysis of p a r e n t s and children with DiGeorge has shown t h a t the deletion occurred in the maternally inherited chromosome in four of five instances. S. Demczuk (Montreal) presented preliminary d a t a on the identification of chromosome 22 homeobox-containing genes as candidates for DiGeorge syndrome. H. M c D e r m i d (Edmonton) m a p p e d the putative r e a r r a n g e m e n t b r e a k p o i n t in a Cat Eye syndrome pat i e n t to within 100 kb of the m a r k e r D22S72. P. P e a r s o n (Baltimore) described the aims and practical uses of the Genome D a t a B a s e (GDB), as well as the practical problems encountered in designing a large database t h a t integrates genetic and physical d a t a generated from different sources. He suggested t h a t a chromosome 22 database should be constructed so t h a t it can interface with GDB. F u t u r e Perspectives It was agreed t h a t there should be another workshop to be held in Philadelphia in 1 year. A new set of reference markers for chromosome 22 was selected. It was felt t h a t these loci should display very informative polymorphisms, preferably of the microsatellite type. Dr. B u d a r f (Philadelphia) volunteered to serve as a distributing lab for these new markers (see Fig. 1). The most effective method for making a complete hybrid cell panel generally available was discussed. Dr. G. T h o m a s (Paris) agreed to make available filters of his detailed mapping panel so t h a t any other investigator can map loci using the same reference breakpoints. G. T h o m a s (Paris) has agreed to serve as a coordinator for d a t a generated using the Lawrence Livermore cosmid library. J. T r o f a t t e r (Boston) agreed to compile all d a t a on chromosome 22 STSs. J. Collins and I. D u n h a m (London) agreed to screen their YAC libraries for anyone interested in identifying a clone. T h e most efficient method for dissemination of information and communication within the group was discussed. T h e r e was general agreement t h a t there should be a newsletter t h a t would be sent to all interested persons by E-mail, preferably using the chromosome 22 bulletin board. Dr. B. E m a n u e l agreed to coordinate this effort.
MEETING REPORT
Third International Workshop on Human Chromosome 21 STYLIANOS E. ANTONARAKIS The Johns Hopkins University School of Medicine, Center for Medical Genetics, Baltimore, Maryland 21287 This report summarizes progress made toward the completion of the mapping of h u m a n chromosome 21 as presented, discussed, and debated at the T h i r d I n t e r n a t i o n a l W o r k s h o p on H u m a n Chromosome 21. This workshop was hosted by The Johns H o p k i n s University School of Medicine at the Maryland Engineering Center in Baltimore, Maryland, on April 2021, 1992, and was supported by the N a t i o n a l Center for Hu-
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0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
