Letter to the Editor

DNA deletion in patients with von Reclinghausen neurofibromatosis Received 27 November 1991, revised 11 February, accepted for publication 21 February 1992

To the Editor: Von Recklinghausen neurofibromatosis (NFl) is a common (1 /3000 births) autosomal dominant disorder, although spontaneous mutations are responsible for half the cases. According to the diagnostic criteria devised by an NIH Consensus Panel (Stumph et al. 1987), affected subjects have to have at least two of the following signs of NFI:(I) at least five cafe-au-lait spots 0.5 cm or greater in diameter if prepubertal and at least six cafe-au-lait spots 1.5 cm or greater in diameter if older; (2) at least two cutaneous or subcutaneous lumps suggestive of neurofibromas; (3) biopsy-proved neurofibroma; (4) axillary or inguinal freckling; (5) Lisch nodules of the iris; (6) characteristic bone abnormality, such as pseudoarthrosis of the tibia or fibula; and (7) an affected parent or sibling, diagnosed by the aforementioned criteria. A diagnosis is usually established by the finding of multiple cafeau-lait spots and cutaneous neurofibromas. However, neurofibromas do not often appear until adolescence. Therefore, the diagnosis in childhood is occasionally difficult. The gene for NF1 has been cloned and partially sequenced (Cawthon et al. 1990). Several deletions and translocations that interrupt the gene were found in N F l patients (Viskochil et al. 1990). Therefore it is likely that the NF1 mutation results in loss of activity of one allele, but there are still few reports on loss of heterozygosity of marker

alleles in the NF1 gene. Here, we report DNA deletions recently found in NFI patients. Genomic DNAs were prepared from leukocytes of four patients that fit the criteria of NF1, two patients with Noonan-Neurofibromatosis syndrome (NFNS) and ten normal individuals. The patients were less than 12 years old and their chromosome karyotypes were normal. A subcloned DNA, 17LlA, that was closely assigned to the translocation breakpoint at 17q11.2 of a NF1 patient (Fountain et al. 1989) was used as a probe for Southern hybridization (Fig. 1). A cDNA clone of human prealbumin, pPA1, was utilized as an internal standard. To standardize the conditions for the hybridizations to the genomic DNAs, the two probes were made approximately equal in length (0.7-0.8 kb). Autoradiographic densities were analyzed by densitometry. The gene doses associated with 17LlA alleles were determined (Table 1). The density reduction by half of the alleles was found in three NFl patients. Two copy densities were detected in one NF1, two NFNS patients and ten normal individuals. The results suggest that three of four NF1 patients had a DNA deletion involving the 17LlA locus. Therefore, it is most likely that the deletions in these patients expanded to involve the N F l gene, causing the disorder. NF1 in the fourth patient might be due to some other genetic defect. The two

Table 1. Densitometric data on 4 NF1 and 2 NFNS patients Lane

Ci1

2 3 4 5 6 C 2

Density ratios' Copy numbers

c,

4

6.1 kb(pPA1)

1 2 3

4.4kb(17LlA)

4 5

The hybridization was performed with 'P-labelled 17LlA and pPAl (internal standard). Polymorphic 17L1A allele is indicated by an asterisk.

1 1

0.69

2

0.36

1

0.72

2 2b

-

6

c, Fig. 1. Southern hybridization of PstI-digests of NFI patients (lanes I+, Noonan-neurofibromatosissyndrome (NFNS) patients (lanes 5 and 6), and normal individuals (lanes C, and Ch.

2

0.64 0.35 0.30

0.60 _

_

_

2 _

_

~

~

~

Densities were measured from several separate hybridbations. The ratios were calculated as the density of the 17L1A allele devided by that of pPA1 allele. Copy number for lane 6 was judged by the Presence of two polymorphic alleles.

53

Letter to the Editor

NFNS patients may not be associated with large DNA deletions. The probe employed in this study will be useful for an early diagnosis of NF1, particularly in sporadic cases. Prenatal diagnosis in familial cases will also be possible with this probe. Acknowledgements We thank to D r J. Hamabe for his advice and the members of Shimane Institute of Health Science for technical assistance. This work was supported by Grants-in-Aid from Saitama Prefecture, Japan.

T. Kameil*2 Y. Fukushima2 A. Shibata2 Y. Hayashi2 N. Tachibana3 I. Takeda4 N. Niikawa' F. S. Collins6 K. Takahashi' S. Masumura' References Cawthon RM, Weiss R, Xu G, Viskochil D, Culver M. Stevens J, Robertson M, Dunn D,Gesteland R, OConnell P, White R. A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations. Cell 1990: 62: 193-201.

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Fountain JW, Wallace MR, Bruce MA, Seizinger BR, Menon AG, Gusella JF, Michels W, Schmidt MA, Dewald GW, Collins FS. Physical mapping of a translocation breakpoint in neurofibromatosis. Science 1989: 244: 1085-1087. Stumph DA, Alksne JF, Annegars SS, Brown PM, Connealy PM, Housman D, kppert M, Miller JP, Moss ML, Pileggi AJ, Rapin I, Strohman RC, Swanson LW,Zimmerman A. Neurofibromatosis. NIH Consensus Development Conference Statement 1987: 6 (12). Viskochil D, Buchberg AM, Xu G, Cawthon RM, Stevens J, Wolff RK, Culver M, Carey JC, Copeland NG, Jenkins NA, White R, OConnell P. Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus. Cell 1990: 62: 187-192. Correspondence: 'Department of Physiology Shimane Medical University Izumo 693, Japan 2Division of Medical Genetics; and Division of Hematology and Oncology, and Division of Dermatology Saitama Children's Medical Center Iwatsuki 339, Japan 'Division of Pediatrics Aomori Prefectural Central Hospital Aomori 030, Japan 'Department of Clinical Pathotogy Shimane Prefectural Central Hospital lzumo 693, Japan *Department of Human Genetics Nagasaki University School of Medicine Nagasaki 852, Japan 6Department of Internal Medicine University of Michigan Ann Arbor, MI 48109-0618, USA