Molecular Cytogenetic Characterization of the Critical Deletion at 17P11.2 in Japanese Patients with Smith-Magenis Syndrome (SMS)

Yamagata Med J 2003；21（2)：111-120

Molecular Cytogenetic Characterization of the Critical Deletion at 17p11.2 in Japanese Patients with Smith-Magenis Syndrome (SMS)

Toshiyuki Kimura, Kiyoshi Hayasaka, Johji Inazawa ＊

Department of Pediatrics, Yamagata University School of Medicine, Yamagata, Japan * Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan (Accepted March 24, 2003 )

ABSTRACT

Smith-Magenis syndrome (SMS) is a contiguous gene syndrome caused by an interstitial deletion of chromosome 17p11.2. The clinical SMS spectrums include short stature, brachydactyly, developmental delay, dysmorphic features, mental retardation, hyperactivity, self-injury, seizures, and sleep abnormalities (especially, reduced REM sleep). Here we attempted to define the minimum common deletion at 17p11.2 in 8 Japanese patients with SMS using molecular cytogenetic approaches, including a prophase fluorescence in situ hybridization (FISH) ordering system and a stretched DNA fiber FISH. Our precise deletion mapping constructed by FISH revealed that one patient with SMS showed a much smaller deletion at 17p11.2 as compared with the other 7 patients with SMS. LLGL1 and FLII, previously mapped within the SMS critical deletion, were mutually nested, and retained on both chromosomes 17 in two patients. ZNF179, a RING finger protein family gene, on the SMS critical region was deleted on one of the two homologues 17p11.2 in 6 out of 8 patients with SMS in the present study. ZNF179 is a "neuronal gene" mapped on the SMS critical deletion and seems to be one of the candidates most likely to be affected with an impairment of the CNS in SMS patients. However, ZNF179 was retained on both 17p11.2 in two SMS patients exhibiting clinical findings characteristic of SMS, suggesting that ZNF179 might not be associated with the neurobehavioral impairments in SMS. Furthermore, DNA fragments from the region at 17p11.2 contained highly repetitive sequences, probably including the low-copy repeats (LCRs) associated with the deletion/duplication mechanism through non-allelic homologous recombination in the patients with SMS.

Key words：Smith-Magenis syndrome, 17p11.2, deletion map, LLGL1, FLII, ZNF179

Address for Correspondence：Toshiyuki Kimura, Department of Pediatrics, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan

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Duchenne muscular dystrophy (DMD ; MIM INTRODUCTION 310200), which was discovered through study- ing a patient with concomitant muscular Smith-Magenis syndrome (SMS) is a con- dystrophy, McLeod syndrome (DMD ; MIM tiguous gene syndrome caused by an intersti- 308350), and chronic granulomatous diseases tial deletion of chromosome 17p11.21),2). There due to a X chromosome deletion that had is no deviation in the parent origin of a deleted removed a set of contiguous genes. This chromosome, and no participation of genomic evidence indicates that the minimal common imprinting. From Caucasian statistics, 1/25,000 deletions in patients with SMS might harbor people are affected with SMS. On the other genes affected with characteristic neurobehav- hand, there seems to be a lesser frequency of ioral abnormalities. this disease in Japan, because only a limited Here we attempted to define the minimum number of Japanese SMS patients have been common deletion at 17p11.2 in 8 Japanese reported so far3). The spectrum of clinical patients with SMS, using molecular cytoge- findings of SMS include short stature, brachy- netic approaches including a prophase fluores- dactyly, developmental delay, dysmorphic fea- cence in situ hybridization (FISH) ordering tures, mental retardation, hyperactivity, self- system10) and the stretched DNA fiber FISH11). injury, seizures, and sleep abnormalities (espe- Our precise deletion mapping constructed by cially reduced REM sleep)4), 5). FISH revealed that one patient with SMS The genomic materials involved in the showed a much smaller deletion at 17p11.2 as chromosomal deletion at 17p11.2 are recog- compared with the other 7 SMS patients. nized to be physically different among SMS LLGL1 and FLII, previously mapped within patients6),7). Recently, Chen et al. identified 3 the SMS critical deletion, were mutually copies of a low-copy repeat (LCR) located nested and retained on both chromosomes 17 within and flanking the SMS common deletion, in this patient. Furthermore, DNA fragments and suggested that homologous recombination from the region at 17p11.2 contained highly of a flanking repeat cluster is a mechanism of repetitive sequences associated with the this microdeletion syndrome8). In particular, deletion/duplication mechanism through non- recombination between repeated sequences at allelic homologous recombination. various regions of the human genome are known to give rise to DNA rearrangements SUBJECTS AND METHODS associated with many genetic disorders including SMS 9). Patients Chromosomal abnormalities have provided All of 8 SMS patients examined here were important clue in the mapping of a number of Japanese, and exhibited clinical findings genes, including some of particular interest to characteristic of SMS. Details of the clinical pediatricians. Positional cloning strategy based manifestations were obtained from only 4 on "deletion mapping" has been applied to the patients (Pt 1, 2, 5 and 6) as listed in the Table task of gene identifications affecting human 1. We could not obtain any information about diseases. An important example is the gene for whether these patients showed an absence of

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Table 1. Clinical Findings in 4 Japanese Patients with SMS Patients Clinical Findings 1 2 5 6 Craniofacial Brachycephaly ＋＋＋＋ Flat midface ＋＋＋＋ upward slant of palpebral fissures ＋＋＋＋ Strabismus ＋－－＋ Unusual mouth shape ＋＋＋＋ Prognathism ＋＋＋＋ Extremities Short hands ＋＋＋＋ Brachydactyly ＋＋＋＋ Scoliosis ＋－－－ Growth, development Short stature －＋－＋ Mental retardation ＋＋＋＋ Hyperactivity ＋＋＋＋ Self-mutilation ＋＋＋＋ Seizure －＋－－ Sleep disturbance －＋－－ Others Cardiac defect －＋－－ Genital anomalies －－－－

REM sleep, although this phenotype had been Mapping of del(17)(p11.2) in 8 patients reported previously12). Chromosome analyses with SMS by standard or high-resolution G-banding EB virus-transformed LCLs of each patient methods showed del(17)(p11.2p11.2) in all were cultured in RPMI1640 supplemented patients. Preliminary cytogenetic and clinical with 10% fetal bovine serum, and metaphase findings in 4 patients (Pt 1, 2, 5 and 6) were cells from each patient were prepared as previously reported by Kondo et al. 3). Epstein- described 14). Normal metaphase chromosomes Barr (EB) virus-transformed lymphoblastoid as the control were prepared from peripheral cell lines (LCLs) were established from the lymphocytes derived from healthy volunteers. peripheral lymphocytes of these patients FISH to metaphase chromosomes was according to the method described by carried out as previously described10) with 24 Fukushima et al13). Before the study, appropri- cosmid markers mapped on 17p11-12 (CI 17- ate informed consents were obtained from the 498, 502, 505, 532, 536, 596, 603, 608, 631, 638, parents of each patient. 681, 688, 693, 695, 705, 712, 724, 729, 802, 814, 825, 827, 828, and 832)15). These cosmids were chosen from a cytogenetic map of chromosome

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17, which was constructed by high-resolution filter. The order and orientation of the signals FISH with 324 newly isolated cosmids15). In on band 17p11.2 were determined by a pair- addition, cosmids containing each of three wise comparison along the longitudinal axis of genes, FLII, LLGL1, and ZNF179, were used the chromatids of elongated prophase chromo- as probes, since these three genes had been somes. Further, to examine the precise ar- mapped within the SMS critical deletion at rangement of overlapping cosmids, FISH 17p11.216)-18). The 70E2 and 62F2 cosmids, signals on stretched DNA fibers were observed kindly provided by Dr. James R. Lupski, as described11), 20). contained 3' and 5' DNA fragments of the Analysis of the FLII and LLGL1 genes human FLII gene, respectively16). A cosmid Since FLII and LLGL1 were localized in clone, designated as cAR5 for the human close proximity to each other in our prophase LLGL1 gene, was isolated by Koyama et al.17), FISH, we performed stretched DNA fiber and a clone cosBFP containing the human FISH to determine the precise arrangement of ZNF179 gene was isolated by Orimo et al.18). 70E2 and cAR5 on a DNA fiber. Cosmids 70E2 For the precise mapping and ordering of and cAR5 contained 3’genomic fragments of FLII, LLGL1, and ZNF179 by FISH, slides of FLII and LLGL1, respectively. prophase chromosomes and stretched DNA In addition, after 70E2 and cAR5 were fibers were prepared10),11). DNA probes were double-digested with EcoRI and Not I, their labeled with biotin (BIO)-16-dUTP or digoxi- DNA fragments were e lectrophoresed in 0 .8% genin (DIG)-11-dUTP (Roch, Germany) by agarose-gel, and we then constructed EcoRI / nicktranslation. Labeled probes (final concen- Not I restriction maps of 70E2 and cAR5. As tration 25 ng/ml) were added to a hybridization shown in Fig.2, 4 DNA fragments from both solution consisting of 50% formamide, 10% cosmids were completely identical. We sub- dextran sulfate, and 2xSSC. Sonicated salmon cloned these DNA fragmentsinto pBluscript(+) sperm DNA (2μg/ μl), Escherichia coli tRNA and determined the sequences of each clone by (2μg/ μl), and Cot-1 DNA (125 ng/ml), were a Pharmacia ALF express DNA sequencer added to eliminate background-noise due to (Pharmacia Biotech Inc.), according to the repetitive sequences such as Alue and L1. A manufacturer's instruction. hybridization reaction was carried out at 37℃ overnight. After the slides were washed in the RESULTS appropriate condition, a BIO- or DIG-labeled probe was detected with avidin-FITC (Boehrin- Construction of a Deletion Map in Pa- ger - Mannheim) or anti - DIG Rhodamine tients with 17p(11.2-11.2) by FISH (Boehringer - Mannheim), respectively. The A chromosomal deletion at 17p11.2 was chromosome slides were then counterstrained detected in all patients with SMS examined by with DAPI (1μg/ml), and mounted in antifade FISH, although the sizes of the chromosome solution containing 1% DABCO19). Then, materials involved in the deletion were multicolor fluorescent signals were simultane- physically different. Seven patients (Pt 2-7) ously observed using a Nikon FXA fluorescent showed a relatively large deletion involved in microscope equipped with a dual band-pass at least 11 cosmids (cCI17-832, -695, -593/603,

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Fig. 1. Mapping of deletions at 17p11.2 in 8 Japanese patients (P1-8) with SMS by FISH. FISH to metaphase and interphase chromosomes was carried out with cosmids (CI17 series) previously mapped on chromosome bands 17p11-12. The numbers indicate that of each cosmid in cCI- 17 cosmids. CI17-596 and -608 were overlapped, and CI17-681 had a Not1 restriction site. Each opened rectangle indicates a hemizygous deletion of the cosmids. Closed rectangles indicate a retention of the cosmids on both homologous chromosomes 17p. Shaded rectangles indicate a retention of the cosmids containing repetitive sequences. Among the 8 patients, Pt1 showed least deletions, involving only 3 cosmids. FLII, LLGL1, and ZNF179 were previously reported to be localized within the SMS critical deletion, although all of the three were retained in Pt1.

-638, -825, -631, -681, -693, -827, -536, -502, smallest 17p11.2 deletion of overlapping, c70E2, and cAR5). On the other hand, Pt 1 critical to SMS, among our 8 patients was the showed a hemizyogous deletion harboring only deletion involved in Pt 1, as shown in Fig.1. 3 cosmids (-695, -593/603, and -638). In addition, FISH signals generated by cCI17- Physical Ordering of FLII, LLGL1, and 688, -712, -729, and -828 showed a repetitive ZNF179 by FISH pattern at 17p11.2-12 along the longitudinal Our prophase FISH ordering system showed axis of chromosome region 17, strongly the order of three cosmids, FLII, cCI17-502, suggesting that these 4 cosmids contained and ZNF179 from the centromere to the 17p11.2-region-specific repetitive sequences. telomere (Fig.1). However, FISH signals for The ZNF179 gene, previously localized FLII overlapped signals for LLGL1, even on within the critical deletion for SMS at elongated prophase chromosomes, in all the 17p11.221), was retained on both homologous cells examined. Our observation strongly chromosomes 17p in Pt 1 and Pt 8. These suggested that both genes might be localized results taken together showed that the most less than 100kb apart, based on our previous

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Fig. 2. (Top) Stretched DNA fiber FISH to determine the arrangement of the FLII and LLGL1 genes Cosmid clones, 70E2 (☆) and 62F2 (＊), contained 5’- and 3’-FLII genomic fragments, respectively, and were detected as FITC signals. Rhodamine-labeled cosmid cAR5 (◇) contained LLGL1. The yellow signals (arrow-head) were detected as a psudo-color generated by overlapping of the green signals for 70E2, and the red signals for cAR5, unambiguously indicating that FLII and LLGL1were nested. (Bottom) Schematic representation of the same image. Cosmids of 70E2, 62F2 and cAR5 on a DNA fiber.

Fig. 3. Restriction map of genomic DNA fragments digested with EcoRI and Not I The bar indicates 4kb. Four DNA fragments each from the c70E2 and cAR5 cosmids were completely identical. These DNA fragments were subcloned into pBluscript(+) and their sequences were analyzed by a DNA autosequencer. The sequences of each fragment were completely identical (data not shown). This data, together with the stretched DNA fiber FISH, clearly indicated that FLII and LLGL1are mutually "nested genes". study21). Indeed, as shown in Fig.2, more from c70E2 and cAR5 were completely precise mapping of these two genes by identical (data not shown). stretched DNA fiber FISH clearly showed that Chromosomal Deletions and Clinical FISH signals of c70E2, a cosmid containing the Findings in Japanese Patients with SMS 3' DNA fragment of FLII, overlapped signal In order to define the possible relationship of generated by cAR5 containing the 3' DNA the difference between the clinical findings and fragment of LLGL1. These results, together the physical size of the17p11.2 deletion in with the results from the restriction maps patients with SMS, we examined the clinical (Fig.3), showed that these two genes were findings in 4 patients (Pt 1, 2, 5 and 6) with nested. Indeed, sequences of DNA fragments respect to their deletions. As listed in the Table

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1, these 4 patients showed a similar clinical murine Llgl, which was originally isolated as a spectrum for SMS, except for "seizures" and homologue of a Drosophila tumor suppressor "sleep disturbance". These two phenotypes, gene l(2)gl (lethal(2) giant larvae). Koyama et associated with an impairment of the central al. isolated LLGL1, and mapped it on 17p11.2, nerve system (CNS), were observed only in Pt which is a critical region of SMS. In the 2, who exhibited a hemizygous deletion of present study, we clearly delineated two cCI17-832 localized on the most distal part of genomic DNA fragments containing parts of the SMS critical deletion, as shown in Fig.1 LLGL1 and FLII at the same position along a DNA fiber, unambiguously indicating that the DISCUSSION 3' end of FLII physically overlapped the 3'end of the LLGL1 gene (Fig.2). It is noteworthy SMS is a multiple congenital anoma- that LLGL1 and FLII were mutually "nested lies/mental retardation syndrome associated genes", as previously reported in human red with an interstitial deletion of chromosome 17 and green color pigment genes on chromosome involving band 17p11.2. It is hypothesized to Xq2822). Furthermore, LLGL1 and FLII were be a contiguous gene syndrome in which the detected on both homologous chromosomes phenotype arises from the haplo-insufficiency 17p11.2 in one SMS patient (Pt 1), who showed of multiple and functionally unrelated genes in representative clinical phenotypes for SMS. close physical proximity, although the true Taken together, haplo-insufficiency at LLGL1 molecular basis of this syndrome has not yet and FLII might not be critical in the spectrum been clarified. Therefore, in the present study, of clinical findings such as short stature, we carried out molecular cytogenetic charac- brachydactyly, developmental delay, dysmorphic terization of chromosomal deletions at 17p11.2 features, sleep disturbances, and behavior in 8 Japanese patients with SMS. problems, all of which were possessed by Pt 1. Previously, two different genes, FLII and We previously mapped the human brain LLGL1, were mapped within the SMS critical finger protein gene, ZNF179, of the RING deletion, and the haploinsufficiency of these finger protein family, on the critical region of genes might be affected with SMS16),17). FLII is SMS at 17p11.2. Indeed, as shown in Fig.1, the human orthologue of Drosophila mela- this gene was deleted on one of the two nogaster flightless-1 gene, fliI, in which homologues 17p11.2 in 6 (Pt 2-7) out of 8 mutations result in the loss of flight ability, and patients with SMS in the present study. when severe, is lethal due to its incomplete ZNF179 is a "neuronal gene"18) and seems to be cellularization with subsequent abnormal the candidate gene most likely to be affected gastrulation. Chen et al. reported that FLII is with an impairment of the CNS in SMS mapped within the SMS critical region, and patients. However, two patients (P t1, 8) har- that this gene was hemizygously deleted in 12 boring ZNF179 on both 17p also showed SMS patients. They also suggested that the clinical symptoms characteristic of SMS haploinsufficiency of this gene might be (Fig.1). These findings strongly suggest that associated with the pathogenesis of SMS. On ZNF179 might not be associated with the the other hand, LLGL1 is the orthologue of the manifestations of the SMS complex phenotypes

－ 117 － Kimura, Hayasaka, Inazawa associated with an impairment of the CNS. with SMS, to understand the pathogenesis of Furthermore, in order to define the possible SMS. Almost all of the genes with known or relationship between the difference in clinical unknown functions have been identified and findings and the region involved in the 17p11.2 localized on the human genome in the post- deletion in patients with SMS, we examined sequence era. Now that information concern- the clinical findings in 4 patients (Pt 1, 2, 5 and ing the human genome is more advanced, 6) with respect to a deletion at 17p11.2. As nonrandom and recurrent genomic alterations, listed in Table 1, these 4 patients showed specific to certain diseases, can provide the key almost the same spectrum of clinical manifesta- exploring novel therapeutic approaches for tions characteristic of SMS, except for incurable diseases and understanding the most "seizures" and "sleep disturbance", which were appropriate management for "personalized observed only in Pt 2. Particularly, Pt 2 medicine". showed a hemizygous deletion of cCI17-832, The stability of the human genome is whereas this cosmid was retained on both influenced to a greater extent by low-copy homologous chromosomes 17p11.2 in the other repeats (LCRs). LCRs are region-specific DNA 3 patients without both manifestations. These blocks usually 10-400kb in size with a >95- results strongly suggest that the genes 97% similarity. Through the process of non- relevant to "seizures" and "sleep disturbance" in allelic homologous recombination, LCRs may SMS are likely to lie on the distal region within lead to DNA rearrangement: deletions, the critical deletion for SMS, surrounding duplications, inversions and translocations. cCI17-832. Recently, Lupski et al. detected LCRs within or In order to identify the critical deletion flanking the SMS critical region and suggested involved in SMS, we tried to minimize the that the SMS deletion was caused by the common deletion among our 8 patients. We mechanism of NAHR 8). Indeed, our FISH first performed high-resolution FISH with a showed that 4 cosmids contained repetitive number of cosmid markers mapped on 17p11.2- sequences, probably including LCRs, within or 12. As shown in Fig.1, 7 out of the 8 patients flanking the region of the SMS critical examined showed relatively large deletions deletion, supporting the idea that dele- involving at least 11 cosmids, whereas one tion/duplication at 17p11.2 is the result of the patient (Pt1) showed a hemizygous deletion reciprocal procedure of an unequal crossing- involving only 3 cosmids. These results over via LCRs, that was recently named SMS- strongly suggest that the most minimal REP.8), 9). deletion responsible for SMS lies within the deletion detected in Pt1, because the clinical We thank the SMS patients and their manifestations of this patient showed almost families for the participation in our study, and all the clinical findings characteristic for SMS. Drs. Yoshimitsu Fukushima and Ikuko Kondo Further studies are necessary to evaluate the for the generous gift of the EBV-transformed genes associated with neurobehavioral abnor- LCLs. We also thank Honorary Professor malities characteristic for SMS, within the Tatsuo Abe and his colleagues for their common and minimum deletions of patients encouragement.

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Hum Genet 1991; 49: 1207-1218 REFERENCES 8. Chen KS, Manian P, Koeuth T, Potocki L, Zhao Q, Chinault AC, et al.: Homologous recombina- 1. Smith ACM, McGavran L, Robinson J, tion of a flanking repeat gene cluster is a Waldstein G, Macfarlane J, Zonona J, et al.: mechanism for a common contiguous gene Interstitial deletion of (17) (p11.2p11.2): report deletion syndrome. Nature Genet 1997; 17: 154- of six additional patients with a new chromo- 163 some deletion syndrome. Am J Med Genet 9. Potocki L, Chen KS, Park SS, Osterholm DE, 1986; 24: 421-432 Withers MA, Kimonis V, et al.: Molecular 2. Fischer H, Oswald HP, Duba HC, Doczy L, mechanism for duplication 17p11.2-the homolo- Simma B, Utermann G, et al.: Constitutional gous recombination reciprocal of the Smith- Magenis microdeletion. Nature Genet 2000; 24: interstitial deletion of 17(p11.2)(Smith-Mage- 84-87 nis syndrome): a clinically recognizable micro- 10. Inazawa J, Ariyama T, Tokino T, Tanigami A, deletion syndrome: report of two cases and Nakamura Y, Abe T: High resolution ordering review of the literature. Clin Pediatr 1993; 205: of DNA markers by multi-color fluorescent in 162-166 situ hybridization of prophase chromosomes. 3. Kondo I, Matsuura S, Kuwajima K, Tokashiki Cytogenet Cell Genet 1994; 65: 130-135 M, Izumikawa Y, Naritomi K, et al.: Diagnotic 11. Parra I, Windle B: High resolution visual hand anomalies in Smith-Magenis syndome: mapping of stretched DNA by fluorescent four new patients with del(17)(p11.2p11.2). Am hybridization. Nature Genet 1993; 5: 17-21 J Med Genet 1991; 41: 225-229 12. Tandan R, Taylor R, Adesina A, Sharma K, 4. Lockwood D, Hecht F, Dowman C, Hecht BK, Fries T, Pendlebury W: Benign autosomal Rizkallah TH, Goodwin TM, et al.: Chromo- dominant syndrome of neuronal Charcot-Marie- some subband 17p11.2 deletion: a minute Tooth disease, ptosis, parkinsonism, and de- deletion syndrome. J Med Genet 1988; 25: 732- mentia. Neurology 1990; 40: 773-779 737 13. Fukushima Y, Ohashi H, Wakui K, Nishida T, 5. Colley AF, Leversha MA, Voullaire LE, Rogers Oh-ishi T: A rapid method for starting a JG: Five cases demonstrating the distinctive culture for the establishment of Epstein-Barr behavioural features of chromosome deletion virus-transformed human lymphoblastoid cell 17 (p11.2p11.2) (Smith-Magenis syndrome). J lines. Jpn J Hum Genet 1992; 37: 149-150 Pediatr Child Health 1990; 26:17-21 14. Kimura T, Arakawa Y, Inoue S, Fukushima Y, 6. Zori RT, Lupski JR, Heju Z, Greenberg F, Kondo I, Koyama K, et al.: The brain finger Killian JM, Gray BA, et al.: Clinical, cytogene- protein gene (ZNF179), a member of the RING tic and molecular evidence for an infant with finger family, maps within the Smith-Magenis Smith-Magenis syndrome born from a mother syndrome region at 17p11.2. Am J Med Genet having a mosaic 17p11.2p12 deletion. Am J 1997 ; 69 : 320-324 Med Genet 1993; 47: 504-511 15. Inazawa J, Saito H, Ariyama T, Abe T, 7. Greenberg F, Guzzetta V, Montes de Oca-Luna Nakamura Y: High-resolution cytogenetic R, Magenis RE, Smith ACM, Richter SF, et al.: mapping of 342 new cosmid markers including Molecular analysis of the Smith-Magenis 43 RFLP markers on human chromosome 17 syndrome: a possible contiguous gene syn- by fluorescence in situ hybridization. Genomics drome associated with del(17)(p11.2). Am J 1993; 17: 153-162

－ 119 － Kimura, Hayasaka, Inazawa

16. Chen KS, Gunaratne PH, Hoheisel JD, Young Inazawa J eds. Rinsyo FISH protocol. Tokyo; IG, Miklos GLG, Greenberg F, et al.: The Syujunsya, 1997: 72-74 human homologue of the Drosophila mela- 20. Ariyama T, Kimura T, Yamakawa K, nogaster flightless-I gene (fliI) maps within the Nakamura Y, Abe T, Inazawa J: Precise Smith-Magenis microdeletion critical region in ordering of 26 cosmid markers on chromosome 17p11.2. Am J Hum Genet 1995; 56: 175-182 region 3p-23->p21.3 by two-color FISH on 17. Koyama K, Fukushima Y, Inazawa J, human prophase chromosomes and stretched Takahashi N, Nakamura Y: The human DNAs. Cytogenet Cell Genet 1995; 70: 129-133 homologue of the murine mgl-1 gene maps 21. Kimura T, Arakawa Y, Inoue S, Fukushima Y, within the Smith-Magenis region in 17p11.2. Kondo I, Koyama K, et al.: The brain finger Cytogenet Cell Genet 1996; 72: 78-82 protein gene (ZNF179), a member of the RING 18. Matsuda Y, Inoue S, Seki N, Hosoi T, Orimo A, finger family, maps within the Smith-Magenis Muramatsu M, et al.: Chromosome mapping of syndrome region at 17p11.2. Am J Med Genet human (ZNF179), mouse and rat genes for 1997; 69: 320-324 brain finger protein (bfp), a member of the 22. Hanna MC, Platts JT, Kirkness EF: RING finger family. Genomics 1996; 33: 325- Identification of a gene within the tandem 327 array of red and green color pigment genes. 19. Kimura T: The counterstrain and fluores- Genomics 1997; 43: 384-386 cence prevention of FISH method. In: Abe T,

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