Ring chromosome 22 and neurofibromatosis Tommerup N, Warburg M, Gieselmann V, Hansen BR, Koch J, Petersen GB. Ring chromosome 22 and neurofibromatosis. Clin Genet 1992: 42: 171-177.
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Variable constitutional mosaicism, mos45,XY,- 22/46,XY, -22, mar/ 46,XY, -22, +r(22)/47,XY, -22, -tr(22)+mar/47,XY, -22, r(22)*2, was , found in PHA-stimulated peripheral blood, in a lymphoblastoid cell line
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N. Tommeruula, M. Warburg', V. 6isselmann3, B. R. Hansen', J. Koch' and 6. 1. Petersen' 'The Danish Center for Human Genome Re
search,?he John F. Kennedy Institute, Glostrup, Denmark; 'Department of Medical Geneti. UC levill Hospital, Oslo. Noway. 'Department of Ophthalmology, Division of Pediatric Ophthalmology and Handicaps, Gentofte Hospital, Denmark; 5Depamnent of Biochemistry II. GeorgAugust-University, Gijttingen. Germany. 6Department of Radiology. Hdstebro Hospital, Denmark; 'Institute of Human Genetics, University of Aarhus, Denmark; 'Department of Clinical Genetics. Bsrkop. Denmark
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Key words: acoustic newoma mental retardation neurofibromatosis NF2 ring chromosome 22 somatic mutation testicular carcinoma tumor suppressor Dr. Niels Tommerup. The John F. Kennedy Institute, GI. Landevq 7, DK-2600 Glostrup, Denmark
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Received 13 February, revised 11 May, accepted for publition 9 June 1992
The neurofibromatoses consist of at least two distinct forms: von Recklinghausen neurofibromatosis (NFl) where the main signs are caf&au-lait spots, intertriginous freckling, iris hamartomas (Lisch nodules) and multiple skin neurofibromas (schwannomas) - and neurofibromatosis 2 (NF2) characterized by bilateral acoustic neuromas and peripheral schwannomas (see Martua & Eldridge 1988). Apart from NFl and NF2, other types of neurofibromatoses have been proposed (Riccardi 1982, Riccardi & Eichner 1986, McKusick 1990) but it is not known whether some of these less defined types may be variant forms of NFl or NF2. The observation of specific loss of genes on chromosome 22 in acoustic neuromas as well as in other NF2-associated tumors tentatively assigned the locus for NF2 to chromosome 22 (Seizinger et al. 1986, 1987). This assignment was confirmed by family studies demonstrating linkage to DNA markers on chromosome 22 (Rouleau et al. 1987, Wertelecki et al. 1988). The isolation of the NFl gene on chromosome 17 (Fountain et al. 1989, Menon et al. 1989, O'Connell et al. 1989, 1990, Viskochil et al. 1990) was greatly facilitated by the existence of constitutional
translocations which disrupted the NFl locus (Schmidt et al. 1987, Ledbetter et al. 1989). So far, translocations or visible deletions have not been reported in NF2 patients. However, one constitutional chromosome anomaly, a ring chromosome 22 - r(22) - has been reported in association with a type of neurofibromatosis which did not fully meet the diagnostic criteria of either NFl or NF2 (Duncan et al. 1987). We report here another constitutional chromosome rearrangement involving chromosome 22 in a male who developed neurofibromatosis, an intracranial tumor and testicular seminoma.
Materials and methods Case report The proband is a severely mentally retarded 25year-old male, born to healthy parents when the mother was 19 years old and the father 24 years old. Body weight was 3000 g, and body length 50 cm.The reports from the neonatal and early childhood period are scanty but include severe mental retardation without development of speech, general hypotonia, unsteady gait, congenital rightsided hydronephrosis, hydroureter and an unspeci171
Tommerup et al. schwannomas, and the diagnosis neurofibromatosis (von Recklinghausen) was tentatively assigned. A progressive bilateral deafness associated with balance problems was gradually noted. At the age of 22 years, he had an ophthalmological examination. Vision was hand movements bilaterally, but he was disinterested in Teller acuity cards. Fixation and following large objects was present. There was no nystagmus and no ptosis. OFC was 58 cm, i.e. he was not microcephalic. The distance between the inner canthi, 35 mm, and between the outer canthi, 95 mm was normal. The ears were large and flat, they were not low-set. The nose was prominent. There was a scar from nephrectomy in the left flank, but there were no mapleleaf-shaped depigmentations of the skin, no cafe-au-lait spots and no intertriginous freckling. He was able to walk without support, but ran into every obstacle. There were no signs of cardiological problems. Slit-lamp examinations disclosed no cataract or smaller lenticular opacities. There were no Lisch nodes in the irides. By direct and indirect ophthalmoscopy the discs appeared atrophic, there were normal macular and peripheral retinal features. Phacomata were not observed. Refraction was close to emmetropia.
fied cardiac defect. At the age of 3 years he underwent unilateral nephrectomy due to repeated infections of the urinary tract. Pictures of his face from that time show a prominent occiput and a facial dysmorphism with ridged metopic sutures, downward slanting palpebral fissures, left esotropia and epicanthus, large low-set ears, tented upper lip (Fig. I), a small umbilical hernia, bilateral calcaneovalgus deformity and puffy feet. A high-vaulted palate, enamel defects of the incisors, and bilaterally undescended testes positioned in the inguinal canals were also reported. At the age of 17 years, he was surgically treated for a unilateral testicular tumor (6 x 5 x 4.5 cm);at that time both testes were descended, but occasionally positioned in the inguinal canals. Histology of the tumor showed grade I seminoma; a separate 5-mm large tumor was also detected and penetration of tunica or infiltration into rete testis was not detected. Subsequently there have been no signs of relapse. Reexamination of the histological sections was not possible; the original histological report did not mention signs of testicular dysgenesis or dysplasia. At the age of 19 years, five cutaneous tumors (0.61.5 cm) were noted. Due to suspicion of metastases from the seminoma, three of the cutaneous tumors were removed, histologically classified as
b
a
Fig.
172
I. The proband at the age of a) 3 years and b) 25 years.
Ring chromosome 22 and neurofibromatosis
Fig. 2. CT-scan of the brain. Arrows on the tumor.
The finding of optic atrophy associated with a chromosome 22 abnormality prompted us to examine whether he had an acoustic: neurinoma. Brain stem-evoked response audiometry suggested bilateral audiological affection. CT scanning of the skull revealed a large (4 cm)unilateral tumor in the left cerebellopontine area a.nd a slight hydrocephalus (Fig. 2). Surgical intervention was decided against; judged by the CT scan it could be a unilateral acoustic neuroma or a cystic process. Both parents were healthy, admittedly without cafi-au-iait spots or any other signs of neurofibromatosis. This included normal slit-lamp examinations.
digested with the appropriate restriction endonucleases (Boehringer Mannheim) under the recommended conditions, separated on 0.7% agarose gels, and transferred to Hybond N + filters. The following 32P-labeIledprobes (Feinberg & Vogelstein 1983) were used: a 1.7 kb arylsulfatase A cDNA probe (HT14ICP8) (Stein et al. 1989), pMS3-18 (D22S1) (Barker et al. 1984) and W23C (D22S28) (Rouleau et al. 1989). X-ray films were exposed for 3-5 days. As controls for dosage analysis, filters were rehybridized either with a 464 bp zeta-globin cDNA probe (probe 4p7-7) from chromosome 16 (Cohen-Solal et al. 1982) or with CRI-P137 (D7S65) (Barker et al. 1987). Autoradiograms were scanned with a Shimadzu CS-9000 dual-wavelength flying spot scanner.
Results Cytogenetic studies
A ring G chromosome, with an extra small marker chromosome in most of the cells, was diagnosed in the proband in 1970. Also, in 1970, a mosaic karyotype, mos46,XY /46,XY, -G, + r(G) was reported in the peripheral lymphocytes of the phenotypically normal father, which would indicate a paternal origin of the ring chromosome. However, in 1989, a repeat chromosome analysis of the father and the mother revealed completely normal karyo-
Cytogenetic studies
Due to the severe mental retardation, chromosome analyses had been performed at ages 3 and 5 years. At the age of 25 years, peripheral PHA-stimulated lymphocytes were studied from both the proband and the parents. The following staining methods were applied: plain Giemsa-staining and later on Q-and C-banding as well as AgNOR-staining (see Andersen et al. 1990). An Epstein-Barr-virus-transformed cell line established from peripheral blood lymphocytes and a skin fibroblastic cell line from the proband established from the periphery of an excised cutaneous schwannoma were also karyotyped. h z situ hybridization with a biotin-labelled chromosome 14/22 specific alphoid repeat probe C1 22 (Kslvri et al. 1991) to peripheral blood chromosomes was performed essentially as described in Andersen et al. (1990). DNA studies
DNA was extracted from peripheral blood and from two samples of a cutaneous neurofibroma,
1 3 :
C t
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Fig. 3. Sequential Q- and AgNOR-staining of chromosomes 22 in the family showing the paternal origin of the normal chromosome 22 in the proband (arrow). b) Sequential Q-and C-banding. c) I n siru hybridization with 14/22-alphoid repeat with signals on both the normal chromosome, the ring and the small marker chromosome.
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Tommerup et al. types (100 cells), and sequential Q- and AgNorstudies indicated that the normal chromosome 22 originated from the father (Fig. 3a), suggesting a maternal origin of the aberrant chromosomes. Unfortunately, the early slides from 1970 have been lost. The cytogenetic findings in the proband are summarized in Table 1. The majority ( ~ 7 0 % of ) the metaphases in PHA-stimulated lymphocytes, both at ages 3 and 25 years, and in the lymphoblastoid cell line, contained both the ring chromosome and the small marker chromosome; the large majority of the remaining cells had only lost the small marker. Other types (complete monosomy 23, two rings) were rare. Thus, the mosaic pattern in PHAstimulated peripheral lymphocytes was remarkably stable over time. In contrast, the fibroblastic cell line was more unstable, with 8% of the cells being completely monosomic for all of chromosome 22, and 30%)of the cells being monosomic for that part of chromosome 22 contained in the ring chromosome. Only 14% of the fibroblasts contained both the ring and the marker. Both the ring chromosome and the small marker contained C-band positive material (Fig. 3b), and both were intensely labelled following in situ hybridization with the chromosome 14122 alphoid repeat probe, along with the normal chromosome 22 and both chromosomes 14 (Fig. 3c). DNA studies
The family was not informative for the polymorphisms (TuqI: 5 kb, 3 kb; BamHI: 19 kb, 7 kb) which can be detected with the ARSA cDNA probe. However, the dosage analysis indicated that the proband was hemizygous (Fig. 4). Thus, the mother was heterozygous for both the BumHI (data not shown) and the TuqI polymorphism, whereas the father was double homozygous (BumHI: 19 kb; Tuql: 5 kb) (Fig. 4). Only the BumHI: 19 kb and TuqI: 5 kb alleles were detected in the proband. As exemplified by Fig. 4, the density of
kb
C1 F
Ratio
1
1
M
B T1 T2 C2 C3
1 0.5 0.5 0.5
1
1
Fig. 4. Southern blot o f TaqI digested DNA hybridized with probe (ARSA cDNA) and rehybridized with dosage controf probe (zeta-globin). Ratio: The mean ratio of the densities of ARSA:zeta-globin hybridization signals in the normal controls was set at 1. Lanes: CI-C3, control DNA; F, bther; M, mother; 9, proband, peripheral blood; TI -T2, proband, tumor samples I &2.
the TbqI 5 kb allele in the proband was very similar to the density of the 5kb allele in the heterozygotic mother, but of considerably less density than in the homozygotic father, supporting that the proband is actually hemizygotic at the ARSA locus. Furthermore, there was no difference in density between the constitutional tissue (peripheral blood) and the two parts of the schwannoma. The proband was heterozygous at the D22SI locus (BgcII) (Fig. 5a) and had retained this heterozygosity in both schwannoma samples, indicating that this locus had neither been lost during the constitutional chromosome rearrangement nor during tumor development. All three individuals were homozygous at the D22S28 locus (with TuqI, BgnI, S a d ) (Fig. 5b). In the proband, dosage analysis of DNA from peripheral blood did not suggest loss at this locus, either. A reduced intensity of the hybridization signal in one of the tumor samples was not observed in the other sample (Fig. 5b). Discussion
Table 1. Cytogenetic findings in the proband
No. of cells with kafyotype Age (y)
45,xl,-22 +r(22) +r(22)'2 +r(22)+mar
+mar
Total
0
18
50 50
Peripheral blood
3 5 25
5 15
0 0
13 35
14
2
33
0 0
0
12
2
36
0
50
4
24
0
7
15
50
0 0 1
Lymphoblastoid cell line
25 Skin Fibroblasts
25
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Based on the cytogenetid in situ hybridization data, the most likely assumption is that not only the recognizable ring chromosome, but also the small supernumerary chromosome (which may also be a ring) were derived from the missing chromosome 22, either from an original larger ring chromosome which had undergone secondary rearrangements, or by a primary rearrangement with breakpoint within the centromere itself. The demonstration that the short arm Q-band heteromorphism on the normal chromosome 22 originated from the father suggests that the aberrant chromo-
Ring chromosome 22 and neurofibromatosis
a kb
C1
F
M
BTl'l2C2
D22S1
b C1
F
M
B T1 'x2 C2 D22S28
5.4-
Ratio
1
1
1
I
1.1 0.6
1
Fig. 5. Southern blot of u ) g h l digested DNA hybridized with D22SI-probe. b) TuyI digested DNA hybridized with D22S28-
probe and rehybridized with dosage control D7S65-probe (only constilnt band is shown). Ratio: The mean ratio of the densities of D22S28:D7S65 (constant band) hybridization signals in the normal controls was set at 1. Lanes as in Fig. 4.
somes originated from the mother, especially since uniparental paternal disomy can be ruled out by the finding of an allele originating from the mother at the D22S1 locus (Fig. 5a). The only consistent feature of patients with r(22) chromosomes is mental retardation, most often of a severe or profound type (see Schinzel 1984). There is no consistent dysmorphism pattern. Both cardiac and renal malformations have been described in other cases. Several adult patients have been described. Thus, the clinical findings in the present case are not inconsistent with the r(22) diagnosis. The clinical features of the present case do not completely meet the diagnostic criteria of either NF1 or NF2 (see Martuza & Eldridge 1988), but the lack of a family history favors an association between the de nova rearrangement of chromosome 22 and the development of neurofibromatosis. The clinical symptoms of neurofibromatosis in the other described case with r(22) (Duncan et al. 1987) did not meet these criteria either, Since the gene for NF2 has been mapped to chromosome 22, this locus is a likely candidate locus, and the lack of cafe-au-lait spots, intertriginous freckling and iris hamartomas in both cases with r(22), together with the presence of relatively few peripheral
neurofibromas and a tumor mass in the cerebellopontine area in the present case is more in line with NF2 than with NFI. The demonstration of a maternal origin of the constitutional rearrangement in the present case may also be of relevance with respect to the development of N F since the NF2 locus is suspected to be imprinted (see Hall 1990). The function and nature of the NF2 gene are not known, but the loss of genes on chromosome 22 in acoustic neuroma and other tumors associated with NF2 suggests that it is a tumor suppressor gene (Seizinger et al. 1986, 1987). The loci D22S1 and D22S28 have been shown to flank the NF2 locus (Rouleau et a]. 1990). In the present study, DNA analysis of peripheral blood indicated that no loss of heterozygosity had occurred at D22S1 during ring formation, and dosage analysis of D22S28 did not support constitutional loss at this locus, either. Since dosage analysis of D22S28 in tumor DNA gave ambiguous results, we cannot safely draw any conclusions with respect to whether this locus has been subject to allele loss during tumor formation or not. The present study does not exclude the involvement of another N F locus situated somewhere else on chromosome 22. However, apart from the other case with r(22) (Duncan et al. 1987), there is no other evidence of a sepirate N F locus on chromosome 22. if the NF2 locus is associated with the development of N F in the two r(22) cases, the data support either 1) the presence of a tiny constitutional deletion or truncation of the NF2 locus without involvement of the two flanking loci D22S1 and D22S28 in the present case, or 2) that the NF2 locus is present in intact form, but may be subject to loss in association with somatic instability. With respect to the latter possibility, one common factor in the case described by Duncan et al. (1987) and the present case is that they both involve ring chromosomes. Mitotic instability due to breakage/reunion cycles is a well-known feature of ring-formed chromosomes, which may lead to both partial deletions and duplications, as well as complete monosomy (McClintock 1938, Kistenmacher & Punnett 1970). In the present case this is amply illustrated by the different kind of mosaicism found in lymphocytes and fibroblasts, and by the loss, in the majority of fibroblasts, of either one or the other of the aberrant chromosomes. In a recent review of ring chromosome carriers with reported tumors, we have found evidence suggesting that somatic instability may predispose ring carriers to development of tumors associated with those tumor suppressor loci located on the specific chromosome (Tommerup & Lothe, in press). If so, 175
Tommerup et al. the development of a testicular seminoma in the present case may not be fortuitous or merely associated with undescended testes, but may suggest that a gene on chromosome 22 could be involved in testicular tumorigenesis. Since mutations associated with this type of somatic instability would be expected to occur at later stages of life than inherited germline mutations, in a much more erratic fashion and involve only single cells and their progenitors, this hypothesis would predict that the associated clinical symptoms would be less clear than in cases with inherited germline mutations. In this context it is possible that some of the heterogeneity of neurofibromatoses may be caused by somatic mosaicism of mutations involving either the NF1 or NF2 locus. Unlike segmental N F which is a likely candidate for somatic mutation of the N F l gene (Nicolls 1969, Miller & Sparkes 1977), the type of somatic mutations associated with ring chromosomes would not be confined to specific body segments. The potential for clinical variation could be even further augmented by the parental origin of the mutation, by the involvement of different cell types and by the age when the somatic mutation may occur. Acknowledgements Biotin-l I-dUTP was kindly supplied by Dr. N. Gregersen. The zeta-globin cDNA was kindly donated by Dr. Corinne Boehm, Mrs. A. Sand, B. Jespersen and Mr. P. Holst provided expert technical help. This study was supported by The Danish Cancer Society, The Norwegian Cancer Society. The Carlsberg Foundation and The Danish Center for Human Genome Research.
Rsfsrences Andersen LB, Tommerup N, Koch J. Formation of a minichromosome by excision of the proximal region of 17q in a patient with von Recklinghausen neurofibromatosis. Cytogenet Cell Genet 1990: 53: 206-210. Barker D, Schafer M, White R. Restriction sites containing CpG show a higher frequency of polymorphism in human DNA. Cell 1984: 36: 131-138. Barker DF, Green P, Knowlton R, Schumm J, Lander E, Oliphant A, Willard H, Akots G . Brown V, Gravius T, Helms C. Nelson C, Parker C. Rediker K, Rising M. Watt D, Weiffenbach B, Donis-KeHer H. A genetic linkage map of chromosome 7 with 63 RFLP markers. Proc Natl Acad Sci USA 1987: 84: 8006-8010. Cohen-Solal MM, Authier B, deRiel JK, Murnane MJ, Forget BG. Cloning and nucleotide sequence analysis of human embryonic zeta-globin cDNA. DNA 1982: 1: 355-363. Duncan AMV, Partington MV, Soudek D. Neurofibromatosis in a man with ring 22: in situ hybridisation studies. Cancer Genet Cytogenet 1987: 25: 169-174. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high activity. Anal Biochem 1983: 132: 6-13. Fountain JW, Wallace MR, Bruce MA, Seizinger BR, Menon AG, Gusella JF, Michels W, Schmidt MA,. Dewald DW, Collins FS. Physical mapping of a translocation breakpoint in neurofibromatosis. Science 1989: 244: 1085-1087.
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Hall J. Genomic imprinting: review and relevance to human diseases. Am J Hum Genet 1990: 46: 857-873. Kistenmacher ML, Punnett HH. Comparative behavior of ring chromosomes. Am J Hum Genet 1970: 22: 304-318. Kslvri R. Koch J, Gregersen N, Jensen PKA, Jsrgensen AL. Petersen KB, Rasmussen K, Bolund L. Application of fluorescent in siru hybridization in clinical genetics: use of two alphoid repeat probes detecting the centromeres of chromosomes 13 and 21 or chromosomes 14 and 22, respectively. Clin Genet 1991: 39: 278-286. Ledbetter DH, Rich DC, OConnell P, Leppert M, Carey JC. Precise localization of NFI to 17q11.2 by balanced translocation. Am J Hum Genet 1989: 44.20-24. Martuza RL, Eldridge R. Neurofibromatosis 2 (bilateral acoustic neurofibromatosis). N Engl J Med 1988: 318: 684-688. McClintock B. The production of homozygous deficient tissues with mutant characteristics by means of the aberrant mitotic behavior of ring shaped chromosomes. Genetics 1938: 23: 3 15-376. McKusick VA. Mendelian inheritance in man, 9th ed. Baltimore and London: Johns Hopkins University Press, 1990 655-656. Menon AG, Ledbetter DH, Rich DC, Seizinger BR, Rouleau GA, Michels VF, Schmidt MA, Dewald G, DallaTorre CM, Haines JL. Gusella JF. Characterization of a translocation within the von Recklinghausen neurofibromatosis region of chromosome 17. Genomics 1989: 5: 245-249. Miller RM, Sparkes RS. Segmental neurofibromatosis. Arch Derm 1977: 113: 837-838. Nicolls EM. Somatic variation and multiple neurofibrornatosis. Hum Hered 1969: 19: 473-479. O'Connell P, Leach P, Cawthon RM. Culver M. Stevens J, Viskochil D, Fournier REK, Rich DC, Ledbetter DJH, White R. Two NFI translocations map within a 600-kilobase segment of 17ql1.2. Science 1989: 244: 1087-1088. O'Connell P, Viskochil D, Buchberg AM, Fountain J, Cawthon RM, Culver M, Stevens J, Rich DC, Ledbetter DH, Wallace M. Carey JC, Jenkins NA, Copeland NG, Collins FS, White R. The human homolog of murine Evi-2 lies between two von Recklinghausen neurofibromatosis translocations. Genomics 1990 7: 547-554. Riccardi VM. Neurofibromatosis: clinical heterogeneity. Curr Probl Cancer 1982: 7: 1-34. Riccardi VM, Eichner JE, eds. Neurofibromatosis. Baltimore: Johns Hopkins University Press, 1986: 169-183. Rouleau GA, Wertelecki W. Haines JL. Hobbs WJ, Trofatter JA, Seizinger BR, Martuza RL, Superneau DW, Conneally PM, Gusella JF. Genetic linkage of bilateral acoustic neurofibromatosis to a DNA marker on chromosome 22. Nature 1987: 329: 246-248. Rouleau GA, Haines JL, Bazanowski A. Colella-Crowley A, Trofatter JA, Wexler NS, Conneally PM. Gusella JF. A genetic linkage map of the long arm of human chromosome 22. Genomics 1989: 4: 1-6. Rouleau GA, Seizinger BR, Wertelecki W, Haines JL, Superneau DW, Martuza RL, Gusella JF. Flanking markers bracket the neurofibromatosis type 2 (NF2) gene on chromosome 22. Am J Hum Genet 1990: 46:323-328. Schinzel A. Catalogue of unbalanced chromosomal aberrations in man. Berlin, New York: Walter deGruyter & Co., 1984. Schmidt MA, Michels W, Deward W. Cases of neurofibromatosis with rearrangements of chromosome 17 involving band 17ql1.2. Am J Med Genet 1987: 28: 771-777. Seizinger BR, Martuza RL, Gusella JF. Loss of genes on chromosome 22 in tumongenesis of human acoustic neurorna. Nature 1986 322: 644-647. Seizinger BR, Rouleau G, Ozelius LJ, Lane AH, St GeorgeHyslop P, Huson S, Gusella J, Martuza RL. Common pathogenetic mechanism for three tumor types in bilateral acoustic neurofibromatosis. Science 1987: 236: 317-319.
Ring chromosome 22 and neurofibromatosis Stein C, Gieselmann V, Kreysing J, Schmidt B. Pohlmann R, Waheed A, Meyer HE, O’Brien JS. von Figura K. Cloning and expression of the human arylsulfatase A. J Biol Chem 1989: 264: 1252-1259. Tommerup N, Lothe RA. Constitutional ring chromosomes and tumour suppressor genes. J Med Genet, in press. Viskochil D. Buchberg AM, Xu G, Cawthon RM, Stevens J, Wolff RK, Culver M, Carey JC, Copeland NG. Jenkins
NA, White R, O’Connell P. Deletions and a translocation interrupt a cloned gene at the neurotibromatosis type 1 locus. Cell 1990 62: 187-192. Wertelecki W, Rouleau GA, Superneau DW, Forehabd LW, Williams JP, Haines JL, Gusella JF. Neurofibromatosis 2: clinical and DNA linkage studies of a large kindred. N Engl J Med 1988: 319 278-283.
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Variable constitutional mosaicism, mos45,XY,- 22/46,XY, -22, mar/ 46,XY, -22, +r(22)/47,XY, -22, -tr(22)+mar/47,XY, -22, r(22)*2, was , found in PHA-stimulated peripheral blood, in a lymphoblastoid cell line
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N. Tommeruula, M. Warburg', V. 6isselmann3, B. R. Hansen', J. Koch' and 6. 1. Petersen' 'The Danish Center for Human Genome Re
search,?he John F. Kennedy Institute, Glostrup, Denmark; 'Department of Medical Geneti. UC levill Hospital, Oslo. Noway. 'Department of Ophthalmology, Division of Pediatric Ophthalmology and Handicaps, Gentofte Hospital, Denmark; 5Depamnent of Biochemistry II. GeorgAugust-University, Gijttingen. Germany. 6Department of Radiology. Hdstebro Hospital, Denmark; 'Institute of Human Genetics, University of Aarhus, Denmark; 'Department of Clinical Genetics. Bsrkop. Denmark
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Key words: acoustic newoma mental retardation neurofibromatosis NF2 ring chromosome 22 somatic mutation testicular carcinoma tumor suppressor Dr. Niels Tommerup. The John F. Kennedy Institute, GI. Landevq 7, DK-2600 Glostrup, Denmark
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Received 13 February, revised 11 May, accepted for publition 9 June 1992
The neurofibromatoses consist of at least two distinct forms: von Recklinghausen neurofibromatosis (NFl) where the main signs are caf&au-lait spots, intertriginous freckling, iris hamartomas (Lisch nodules) and multiple skin neurofibromas (schwannomas) - and neurofibromatosis 2 (NF2) characterized by bilateral acoustic neuromas and peripheral schwannomas (see Martua & Eldridge 1988). Apart from NFl and NF2, other types of neurofibromatoses have been proposed (Riccardi 1982, Riccardi & Eichner 1986, McKusick 1990) but it is not known whether some of these less defined types may be variant forms of NFl or NF2. The observation of specific loss of genes on chromosome 22 in acoustic neuromas as well as in other NF2-associated tumors tentatively assigned the locus for NF2 to chromosome 22 (Seizinger et al. 1986, 1987). This assignment was confirmed by family studies demonstrating linkage to DNA markers on chromosome 22 (Rouleau et al. 1987, Wertelecki et al. 1988). The isolation of the NFl gene on chromosome 17 (Fountain et al. 1989, Menon et al. 1989, O'Connell et al. 1989, 1990, Viskochil et al. 1990) was greatly facilitated by the existence of constitutional
translocations which disrupted the NFl locus (Schmidt et al. 1987, Ledbetter et al. 1989). So far, translocations or visible deletions have not been reported in NF2 patients. However, one constitutional chromosome anomaly, a ring chromosome 22 - r(22) - has been reported in association with a type of neurofibromatosis which did not fully meet the diagnostic criteria of either NFl or NF2 (Duncan et al. 1987). We report here another constitutional chromosome rearrangement involving chromosome 22 in a male who developed neurofibromatosis, an intracranial tumor and testicular seminoma.
Materials and methods Case report The proband is a severely mentally retarded 25year-old male, born to healthy parents when the mother was 19 years old and the father 24 years old. Body weight was 3000 g, and body length 50 cm.The reports from the neonatal and early childhood period are scanty but include severe mental retardation without development of speech, general hypotonia, unsteady gait, congenital rightsided hydronephrosis, hydroureter and an unspeci171
Tommerup et al. schwannomas, and the diagnosis neurofibromatosis (von Recklinghausen) was tentatively assigned. A progressive bilateral deafness associated with balance problems was gradually noted. At the age of 22 years, he had an ophthalmological examination. Vision was hand movements bilaterally, but he was disinterested in Teller acuity cards. Fixation and following large objects was present. There was no nystagmus and no ptosis. OFC was 58 cm, i.e. he was not microcephalic. The distance between the inner canthi, 35 mm, and between the outer canthi, 95 mm was normal. The ears were large and flat, they were not low-set. The nose was prominent. There was a scar from nephrectomy in the left flank, but there were no mapleleaf-shaped depigmentations of the skin, no cafe-au-lait spots and no intertriginous freckling. He was able to walk without support, but ran into every obstacle. There were no signs of cardiological problems. Slit-lamp examinations disclosed no cataract or smaller lenticular opacities. There were no Lisch nodes in the irides. By direct and indirect ophthalmoscopy the discs appeared atrophic, there were normal macular and peripheral retinal features. Phacomata were not observed. Refraction was close to emmetropia.
fied cardiac defect. At the age of 3 years he underwent unilateral nephrectomy due to repeated infections of the urinary tract. Pictures of his face from that time show a prominent occiput and a facial dysmorphism with ridged metopic sutures, downward slanting palpebral fissures, left esotropia and epicanthus, large low-set ears, tented upper lip (Fig. I), a small umbilical hernia, bilateral calcaneovalgus deformity and puffy feet. A high-vaulted palate, enamel defects of the incisors, and bilaterally undescended testes positioned in the inguinal canals were also reported. At the age of 17 years, he was surgically treated for a unilateral testicular tumor (6 x 5 x 4.5 cm);at that time both testes were descended, but occasionally positioned in the inguinal canals. Histology of the tumor showed grade I seminoma; a separate 5-mm large tumor was also detected and penetration of tunica or infiltration into rete testis was not detected. Subsequently there have been no signs of relapse. Reexamination of the histological sections was not possible; the original histological report did not mention signs of testicular dysgenesis or dysplasia. At the age of 19 years, five cutaneous tumors (0.61.5 cm) were noted. Due to suspicion of metastases from the seminoma, three of the cutaneous tumors were removed, histologically classified as
b
a
Fig.
172
I. The proband at the age of a) 3 years and b) 25 years.
Ring chromosome 22 and neurofibromatosis
Fig. 2. CT-scan of the brain. Arrows on the tumor.
The finding of optic atrophy associated with a chromosome 22 abnormality prompted us to examine whether he had an acoustic: neurinoma. Brain stem-evoked response audiometry suggested bilateral audiological affection. CT scanning of the skull revealed a large (4 cm)unilateral tumor in the left cerebellopontine area a.nd a slight hydrocephalus (Fig. 2). Surgical intervention was decided against; judged by the CT scan it could be a unilateral acoustic neuroma or a cystic process. Both parents were healthy, admittedly without cafi-au-iait spots or any other signs of neurofibromatosis. This included normal slit-lamp examinations.
digested with the appropriate restriction endonucleases (Boehringer Mannheim) under the recommended conditions, separated on 0.7% agarose gels, and transferred to Hybond N + filters. The following 32P-labeIledprobes (Feinberg & Vogelstein 1983) were used: a 1.7 kb arylsulfatase A cDNA probe (HT14ICP8) (Stein et al. 1989), pMS3-18 (D22S1) (Barker et al. 1984) and W23C (D22S28) (Rouleau et al. 1989). X-ray films were exposed for 3-5 days. As controls for dosage analysis, filters were rehybridized either with a 464 bp zeta-globin cDNA probe (probe 4p7-7) from chromosome 16 (Cohen-Solal et al. 1982) or with CRI-P137 (D7S65) (Barker et al. 1987). Autoradiograms were scanned with a Shimadzu CS-9000 dual-wavelength flying spot scanner.
Results Cytogenetic studies
A ring G chromosome, with an extra small marker chromosome in most of the cells, was diagnosed in the proband in 1970. Also, in 1970, a mosaic karyotype, mos46,XY /46,XY, -G, + r(G) was reported in the peripheral lymphocytes of the phenotypically normal father, which would indicate a paternal origin of the ring chromosome. However, in 1989, a repeat chromosome analysis of the father and the mother revealed completely normal karyo-
Cytogenetic studies
Due to the severe mental retardation, chromosome analyses had been performed at ages 3 and 5 years. At the age of 25 years, peripheral PHA-stimulated lymphocytes were studied from both the proband and the parents. The following staining methods were applied: plain Giemsa-staining and later on Q-and C-banding as well as AgNOR-staining (see Andersen et al. 1990). An Epstein-Barr-virus-transformed cell line established from peripheral blood lymphocytes and a skin fibroblastic cell line from the proband established from the periphery of an excised cutaneous schwannoma were also karyotyped. h z situ hybridization with a biotin-labelled chromosome 14/22 specific alphoid repeat probe C1 22 (Kslvri et al. 1991) to peripheral blood chromosomes was performed essentially as described in Andersen et al. (1990). DNA studies
DNA was extracted from peripheral blood and from two samples of a cutaneous neurofibroma,
1 3 :
C t
* '
Fig. 3. Sequential Q- and AgNOR-staining of chromosomes 22 in the family showing the paternal origin of the normal chromosome 22 in the proband (arrow). b) Sequential Q-and C-banding. c) I n siru hybridization with 14/22-alphoid repeat with signals on both the normal chromosome, the ring and the small marker chromosome.
173
Tommerup et al. types (100 cells), and sequential Q- and AgNorstudies indicated that the normal chromosome 22 originated from the father (Fig. 3a), suggesting a maternal origin of the aberrant chromosomes. Unfortunately, the early slides from 1970 have been lost. The cytogenetic findings in the proband are summarized in Table 1. The majority ( ~ 7 0 % of ) the metaphases in PHA-stimulated lymphocytes, both at ages 3 and 25 years, and in the lymphoblastoid cell line, contained both the ring chromosome and the small marker chromosome; the large majority of the remaining cells had only lost the small marker. Other types (complete monosomy 23, two rings) were rare. Thus, the mosaic pattern in PHAstimulated peripheral lymphocytes was remarkably stable over time. In contrast, the fibroblastic cell line was more unstable, with 8% of the cells being completely monosomic for all of chromosome 22, and 30%)of the cells being monosomic for that part of chromosome 22 contained in the ring chromosome. Only 14% of the fibroblasts contained both the ring and the marker. Both the ring chromosome and the small marker contained C-band positive material (Fig. 3b), and both were intensely labelled following in situ hybridization with the chromosome 14122 alphoid repeat probe, along with the normal chromosome 22 and both chromosomes 14 (Fig. 3c). DNA studies
The family was not informative for the polymorphisms (TuqI: 5 kb, 3 kb; BamHI: 19 kb, 7 kb) which can be detected with the ARSA cDNA probe. However, the dosage analysis indicated that the proband was hemizygous (Fig. 4). Thus, the mother was heterozygous for both the BumHI (data not shown) and the TuqI polymorphism, whereas the father was double homozygous (BumHI: 19 kb; Tuql: 5 kb) (Fig. 4). Only the BumHI: 19 kb and TuqI: 5 kb alleles were detected in the proband. As exemplified by Fig. 4, the density of
kb
C1 F
Ratio
1
1
M
B T1 T2 C2 C3
1 0.5 0.5 0.5
1
1
Fig. 4. Southern blot o f TaqI digested DNA hybridized with probe (ARSA cDNA) and rehybridized with dosage controf probe (zeta-globin). Ratio: The mean ratio of the densities of ARSA:zeta-globin hybridization signals in the normal controls was set at 1. Lanes: CI-C3, control DNA; F, bther; M, mother; 9, proband, peripheral blood; TI -T2, proband, tumor samples I &2.
the TbqI 5 kb allele in the proband was very similar to the density of the 5kb allele in the heterozygotic mother, but of considerably less density than in the homozygotic father, supporting that the proband is actually hemizygotic at the ARSA locus. Furthermore, there was no difference in density between the constitutional tissue (peripheral blood) and the two parts of the schwannoma. The proband was heterozygous at the D22SI locus (BgcII) (Fig. 5a) and had retained this heterozygosity in both schwannoma samples, indicating that this locus had neither been lost during the constitutional chromosome rearrangement nor during tumor development. All three individuals were homozygous at the D22S28 locus (with TuqI, BgnI, S a d ) (Fig. 5b). In the proband, dosage analysis of DNA from peripheral blood did not suggest loss at this locus, either. A reduced intensity of the hybridization signal in one of the tumor samples was not observed in the other sample (Fig. 5b). Discussion
Table 1. Cytogenetic findings in the proband
No. of cells with kafyotype Age (y)
45,xl,-22 +r(22) +r(22)'2 +r(22)+mar
+mar
Total
0
18
50 50
Peripheral blood
3 5 25
5 15
0 0
13 35
14
2
33
0 0
0
12
2
36
0
50
4
24
0
7
15
50
0 0 1
Lymphoblastoid cell line
25 Skin Fibroblasts
25
174
Based on the cytogenetid in situ hybridization data, the most likely assumption is that not only the recognizable ring chromosome, but also the small supernumerary chromosome (which may also be a ring) were derived from the missing chromosome 22, either from an original larger ring chromosome which had undergone secondary rearrangements, or by a primary rearrangement with breakpoint within the centromere itself. The demonstration that the short arm Q-band heteromorphism on the normal chromosome 22 originated from the father suggests that the aberrant chromo-
Ring chromosome 22 and neurofibromatosis
a kb
C1
F
M
BTl'l2C2
D22S1
b C1
F
M
B T1 'x2 C2 D22S28
5.4-
Ratio
1
1
1
I
1.1 0.6
1
Fig. 5. Southern blot of u ) g h l digested DNA hybridized with D22SI-probe. b) TuyI digested DNA hybridized with D22S28-
probe and rehybridized with dosage control D7S65-probe (only constilnt band is shown). Ratio: The mean ratio of the densities of D22S28:D7S65 (constant band) hybridization signals in the normal controls was set at 1. Lanes as in Fig. 4.
somes originated from the mother, especially since uniparental paternal disomy can be ruled out by the finding of an allele originating from the mother at the D22S1 locus (Fig. 5a). The only consistent feature of patients with r(22) chromosomes is mental retardation, most often of a severe or profound type (see Schinzel 1984). There is no consistent dysmorphism pattern. Both cardiac and renal malformations have been described in other cases. Several adult patients have been described. Thus, the clinical findings in the present case are not inconsistent with the r(22) diagnosis. The clinical features of the present case do not completely meet the diagnostic criteria of either NF1 or NF2 (see Martuza & Eldridge 1988), but the lack of a family history favors an association between the de nova rearrangement of chromosome 22 and the development of neurofibromatosis. The clinical symptoms of neurofibromatosis in the other described case with r(22) (Duncan et al. 1987) did not meet these criteria either, Since the gene for NF2 has been mapped to chromosome 22, this locus is a likely candidate locus, and the lack of cafe-au-lait spots, intertriginous freckling and iris hamartomas in both cases with r(22), together with the presence of relatively few peripheral
neurofibromas and a tumor mass in the cerebellopontine area in the present case is more in line with NF2 than with NFI. The demonstration of a maternal origin of the constitutional rearrangement in the present case may also be of relevance with respect to the development of N F since the NF2 locus is suspected to be imprinted (see Hall 1990). The function and nature of the NF2 gene are not known, but the loss of genes on chromosome 22 in acoustic neuroma and other tumors associated with NF2 suggests that it is a tumor suppressor gene (Seizinger et al. 1986, 1987). The loci D22S1 and D22S28 have been shown to flank the NF2 locus (Rouleau et a]. 1990). In the present study, DNA analysis of peripheral blood indicated that no loss of heterozygosity had occurred at D22S1 during ring formation, and dosage analysis of D22S28 did not support constitutional loss at this locus, either. Since dosage analysis of D22S28 in tumor DNA gave ambiguous results, we cannot safely draw any conclusions with respect to whether this locus has been subject to allele loss during tumor formation or not. The present study does not exclude the involvement of another N F locus situated somewhere else on chromosome 22. However, apart from the other case with r(22) (Duncan et al. 1987), there is no other evidence of a sepirate N F locus on chromosome 22. if the NF2 locus is associated with the development of N F in the two r(22) cases, the data support either 1) the presence of a tiny constitutional deletion or truncation of the NF2 locus without involvement of the two flanking loci D22S1 and D22S28 in the present case, or 2) that the NF2 locus is present in intact form, but may be subject to loss in association with somatic instability. With respect to the latter possibility, one common factor in the case described by Duncan et al. (1987) and the present case is that they both involve ring chromosomes. Mitotic instability due to breakage/reunion cycles is a well-known feature of ring-formed chromosomes, which may lead to both partial deletions and duplications, as well as complete monosomy (McClintock 1938, Kistenmacher & Punnett 1970). In the present case this is amply illustrated by the different kind of mosaicism found in lymphocytes and fibroblasts, and by the loss, in the majority of fibroblasts, of either one or the other of the aberrant chromosomes. In a recent review of ring chromosome carriers with reported tumors, we have found evidence suggesting that somatic instability may predispose ring carriers to development of tumors associated with those tumor suppressor loci located on the specific chromosome (Tommerup & Lothe, in press). If so, 175
Tommerup et al. the development of a testicular seminoma in the present case may not be fortuitous or merely associated with undescended testes, but may suggest that a gene on chromosome 22 could be involved in testicular tumorigenesis. Since mutations associated with this type of somatic instability would be expected to occur at later stages of life than inherited germline mutations, in a much more erratic fashion and involve only single cells and their progenitors, this hypothesis would predict that the associated clinical symptoms would be less clear than in cases with inherited germline mutations. In this context it is possible that some of the heterogeneity of neurofibromatoses may be caused by somatic mosaicism of mutations involving either the NF1 or NF2 locus. Unlike segmental N F which is a likely candidate for somatic mutation of the N F l gene (Nicolls 1969, Miller & Sparkes 1977), the type of somatic mutations associated with ring chromosomes would not be confined to specific body segments. The potential for clinical variation could be even further augmented by the parental origin of the mutation, by the involvement of different cell types and by the age when the somatic mutation may occur. Acknowledgements Biotin-l I-dUTP was kindly supplied by Dr. N. Gregersen. The zeta-globin cDNA was kindly donated by Dr. Corinne Boehm, Mrs. A. Sand, B. Jespersen and Mr. P. Holst provided expert technical help. This study was supported by The Danish Cancer Society, The Norwegian Cancer Society. The Carlsberg Foundation and The Danish Center for Human Genome Research.
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