Clin Genet 1994: 46: 295-298 Printed in Denmark . All righis reserved

Copyright Q Munksgaard 1994

CLINICAL GENETICS ISSN 0009-9163

X; 1 translocation in a female Menkes patient: characterization by fluorescence in situ hvbridization

Beck J, Enders H, Schliephacke M, Buchwald-Saal M, Turner Z. X; 1 translocation in a female Menkes patient: characterization by fluor- escence in situ hybridization Clin Genet 1994: 46: 295-298. 0 Munksgaard, 1994

Menkes disease is an X-linked recessive disorder of copper metabolism, characterized by progressive neurological degeneration, abnormal hair and c0nnectiv.e tissue manifestations. We present a female Menkes patient, with classical Menkes features, carrying a de noyo balanced translo- cation 46,X,t(X; l)(ql3;q 12). The breakpoint on the X chromosome was narrowed down to Xq13.3 within a 1 Mb YAC contig containing the Menk- es gene, using fluorescence in situ hybridization. The translocated X chromosome was of paternal origin and non-randomly active leading to the expression of the disease. This was additional evidence for paternal origin of de novo chromosome rearrangements, including all the X; auto- soma1 translocations examined so far.

Menkes disease, first described in 1962 (Menkes et al. 1962), is an X-linked recessive disorder of cop- per metabolism, leading to death in early child- hood. It is mainly characterized by progressive neurological degeneration, mental retardation, pe- culiar hair and connective tissue manifestations. Biochemical findings include low concentrations of serum copper and ceruloplasmin. Increased ac- cumulation of copper in cultured fibroblasts forms the basis of the definitive biochemical diagnosis (for review, see Horn et al. 1992). An inadequate supply of copper to the known copper-dependent enzymes explains most of the somatic features found in Menkes disease. The Menkes locus has been localized to Xq13.3 (Turner et al. 1992b) and the gene has been isolated by means of positional cloning (Chelly et al. 1993, Vulpe et al. 1993, Mer- cer et al. 1993). The protein is predicted to be a

James Beck', Herbert Enders2, Martin Schl[ephacke2, Monika Buchwald-Saall and Zeynep liimer3 'Department of Developmental Medicine, Children's Hospital, 'Department of Clinical Genetics, Institute of Human Genetics and Anthropology, University of Tiibingen, Germany and The John F. Kennedy Institute, Glostrup, Denmark

Key words: chromosomal translocation - fluorescence in sifu hybridization (FISH) - Menkes disease - yeast artificial chromosomes (YACs)

Dr. 2. Turner, The Danish Center for the Human Genome Project, The John F. Kennedy Institute, GI. Landevej 7, 2600 Glostrup, Denmark

Received 1 September 1993, revised version received 16 May, accepted for publication 18 May 1994

metal-transporting ATPase in agreement with the suggested underlying pathology.

In Menkes disease no visible deletions were de- tected in a cytogenetic survey of 181 unrelated pa- tients (Tommerup et al. 1993). On the other hand, in 16% of 200 patients, the disease was found to be associated with microdeletions (Chelly et al. 1993; and unpublished results). Two Menkes pa- tients have been reported carrying chromosome re- arrangements without any visible deletions in the Xq13.3 region. One of them was a male patient with a unique intrachromosomal rearrangement, where the long arm segment Xq13.3-q21.2 was in- serted into the short arm (Turner et al. 1992b). The other patient was a girl with a de n o w balanced X;2 translocation (Kapur et al. 1987), with a breakpoint at Xq13.3 (Verga et al. 1991, Turner et al. 1992a), and the disease was expressed because

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of non-random inactivation of the normal X chromosome. Females with the X-linked recessive Menkes disease are very rare indeed; only five pre- vious cases have been described (Matsubara et al. 1978, Iwakawa et al. 1979, Barto et al. 1983, Favier et al. 1983, Gerdes et al. 1990).

In this study we describe a female Menkes pa- tient with an X;1 translocation, and fine localize the breakpoint using fluorescence in situ hybridiza- tion.

Materlals and methods Case report

The proposita (Fig. 1) was born spontaneously after 38 weeks of gestation as the third child of healthy, non-related parents. There was no family history of neurologic diseases. Pregnancy and de- livery were normal. Birth weight was 2750 g, length 49 cm, and head circumference 32 cm. Apgar scores were lO/l, 106, 10/10.

The child had normal dark hair in the neonatal period. At the age of 6 weeks the hair became coarse, sparse and ivory colored, and pili torti was observed.

Somatic parameters developed normally during the first 11 months. The child had brachycephaly. Her mouth was small and her palate was high. Fat pads appeared in the thighs with constriction rings, and the extremities were relatively short. The skin appeared very dry although the patient sweat- ed frequently. She suffered from recurrent diarrhea and infections. From birth, she had reduced muscle tone and at the age of 11 months was un- able to sit or stand. No appropriate reaction to acoustic signals was seen. Fixation and following with the eyes was possible only for a short while. Ophthalmological examination and hearing tests were normal. She had had no episodes of con-

Fig. I. The girl with the Menkes disease at the age of 11 months.

vulsions. The electroencephalogram was somewhat dysrhythmic but there were no epileptic discharges. CT-scan disclosed marked atrophy of the brain, es- pecially in the infratentorial region.

Serum copper was 0.19 mg/l (range: 0.9-1.73 mg/l), serum ceruloplasmin was 80 mg/l (range: 150-500 mg/l) and serum lactate was 3.8 mmol/l (range:<2.0 mmoV1). Treatment with substitution of copper histidinate (Nadal et al. 1988) was started at the age of 12 months, despite question- able results of clinical trials previously performed in the late stage of the disease.

Cytogenetic analysis and X-inactivation studies

Chromosomes were prepared from peripheral lymphocytes and skin fibroblasts by standard tech- niques and analyzed with GTG-banding. A late replicating, inactive X-chromosome was visualized by high resolution RBG-banding of lymphocytes.

X-inactivation studies on the DNA level were carried out by polymerase chain reaction (PCR), using two primers flanking two methylation sensi- tive Hpal sites and a highly polymorphic CAG re- peat region within the Androgen-Receptor (AR) gene (Allen et al. 1992).

64Cu uptake was measured in cultured skin fibroblasts according to Tonnesen & Horn (1989).

Fluorescence in situ hybridization

In situ suppression hybridization and detection were carried out according to Kievitis et al. (1990) with some modifications. Metaphase and prometa- phase chromosomes were prepared from lympho- cytes by standard techniques. Ten nanograms of X-chromosome-specific alpha-satellite probe (BamX7) and 300 ng DNA from each of the two overlapping YACs, 4542 (500 kb) and 4548 (750 kb) were labeled with biotin-1 1-dUTP (Sigma) by nick translation. Repetitive sequences were sup- pressed for 2 h at 37°C and hybridization accom- plished at 36°C for 3 days. Detection was carried out at room temperature with FITC-conjugated avidin, and the signal was amplified with one addi- tinal layer of biotinylated goat anti-avidin, fol- lowed by an avidin-FITC layer. Chromosomes were counterstained with propidium iodide and preparations were visualized on a Leitz Diaplan epifluorescence microscope. Photographs were taken with Kodak Ektachrome 64T.

Results

The clinical examination of the patient indicated Menkes disease, supported by the findings of low serum copper (0.19 mg/l) and ceruloplasmin (80

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mg/l). Abnormally high 64Cu-incorporation in the cultured skin fibroblasts (86.7 ng 64Cu/mg protein/ 20 h, the control range for males 9.0-21.1 ng) con- firmed the diagnosis.

Cytogenetic analysis revealed a balanced X; 1 translocation, where the breakpoint on the X chromosome was at Xq13, the locus designated for Menkes disease. The karyotype of the patient is thus, 46,X,t(X;l) (q13;q12) (Fig. 2). Both parents had normal chromosomes indicating a de novo oc- currence of the translocation. To localize the breakpoint on the X chromosome more accurately, we analyzed the metaphase and prometaphase chromosomes with fluorescence in situ hybridiza- tion using two YAC probes: YAC 4542 and 4548. These two overlapping YACs form a 1 Mb contig, which was previously localized to Xq13.3 and shown to contain the gene responsible for the Menkes disease (Turner et al. 1992a, Chelly et al. 1993). Both YACs gave strong hybridization sig- nals at Xq13.3 on the normal X chromosomes. YAC 4542 gave hybridization signals on both of the translocation chromosomes (Fig. 3), while YAC 4548 gave signals only on the derivative X chromosome. This result indicated that YAC 4542 extended over the breakpoint, and the break on the X chromosome had occurred distal to YAC 4548 and proximal to the PGKl (Phosphoglycerol Kin- ase) locus.

The normal X chromosome was late replicating in all lymphocytes examined. The non-random X- inactivation pattern was also reflected by the skew- ed methylation of the HpaII sites within the An- drogen Receptor gene. The presence of highly poly- morphic CAG repeats in the same region allowed us to determine the paternal origin of the translo- cated chromosome.

Fig. 2. Partial karyogram of the patient representing the bal- anced translocation 46,X,t(X;l) (q13;q12). G-banded (right side) and BrdU-R-banded chromosomes (left side). A) normal chromosome I, B) translocated chromosome (l;X), C) normal chromosome X, D) translocated chromosome (X; I ) .

Fig. 3. In situ suppression hybridization of the metaphase chromosomes of the female Menkes patient with X;l translo- cation using YAC 4542. Hybridization signals of YAC 4542 are observed at Xq13.3 both on the normal (large arrow) and the derivative X chromosome (small arrow), and on the long arm of derivative chromosome 1 (small arrow), indicating that the break occurred within this YAC sequence. x: signal of the X- chromosome specific alpha-satellite probe (A generous gift of Torben Cruse).

Discussion The typical clinical features and biochemical par- ameters in the female patient presented here estab- lished the diagnosis of the X-linked recessive Menkes disease. Cytogenetic analysis showed that she had a balanced X;1 translocation and, with in situ hybridization, the breakpoint on the X chromosome was localized to Xq13.3 within a 1 Mb YAC contig containing the Menkes gene (Turner et al. 1992a, Chelly et al. 1993).

In Menkes disease no visible deletions were ob- served in a cytogenetic screening of 200 unrelated affected males (Tommerup et al. 1993; and unpub- lished results). Lack of cytogenetically detectable deletions also applies to the three chromosome re- arrangements associated with Menkes disease, in- cluding the present case. One of these patients was a male with a unique intrachromosomal rearrange- ment involving the Xq13.3 region (Rimer et al. 1992b). The female Menkes patient described by Kapur et al. (1987) carried a balanced X;2 translo- cation localized to Xq13 (Verga et al. 1991). The breakpoints of these patients were localized within the overlapping region of the YACs, 4542 and 4548 (Turner et al. 1992a). The X chromosome break- point of the present case is localized distal to these breakpoints.

The mother of the male patient with the intra- chromosomal rearrangement was a carrier of the mutation, in whom the abnormal X chromosome

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was non-randomly inactive. On the other hand, in both translocation females the normal X chromo- some was non-randomly inactivated, leading to the expression of the disease.

Females with the X-linked recessive Menkes dis- ease are rare and only five previous cases have been described. The case described by Barton et al. (1983) showed 45W46XX mosaicism, and two cases were cytogenetically normal (Favier et al. 1983, Gerdes et al. 1990). In these cases the disease was probably expressed due to monosomy of the abnormal X chromosome in most of the cells in the 45W46XX mosaicism or due to odd X inacti- vation. Cytogenetical analysis was not performed in the other two cases (Matsubara et al. 1978, Iwakawa et al. 1979).

In both Menkes females carrying the X-auto- some translocation, and in the mother of the male patient with the intrachromosomal insertion, the structural rearrangement occurred de novo and was of paternal origin. Most of the de nuvo chromo- somal rearrangements, including all the X;autoso- ma1 translocations examined so far (Bodrug et al. 1990, Robinson et al. 1990, Bodrug et al. 1991) have been found to be of paternal origin. This finding, together with the general lack of trans- mission of X-autosome translocations through meiosis in the male, led Bodrug et al. (1991) to suggest that the translocations causing Duchenne and Becker muscular dystrophy in females result from post-meiotic non-homologous recombination in spermiogenesis. This hypothesis may also apply to Menkes disease in females. Molecular analysis of these patients may provide more information concerning the mechanism of the translocations.

Acknowledgements We thank Mrs. H. Tigges and Mrs. H. Janda for excellent tech- nical assistance; A. I? Monaco, MD, PhD, for the YAC clones; N. Horn, PhD, and T. Ternnesen, PhD for 64Cu uptake studies.

References Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW. Methylation of Hpal l and Hhai sites near the polymor- phic CAG repeat in the human androgen-receptor gene corre- lates with X chromosome inactivation. Am J Hum Genet 1992: 5 1 : 1229-1 239.

Barton NW, Dambrosia JM, Barranger JA. Menkes’ kinky-hair syndrome: report of a case in a female infant. Neurology 1983: 33 (Suppl 2): 154.

Bodrug SE, Roberson JR, Weiss L, Ray PN, Worton RG, Van- Dyke DL. Prenatal identification of a girl with a t(X;4) (p21;q35) translocation: molecular characterisation, paternal origin, and association with muscular dystrophy. J Med Genet 1990: 27: 426-432.

Bodrug SE, Holden JAJ, Ray PN, Worton RG. Molecular analysis of X-autosome translocations in females with Duch- enne muscular dystrophy. EMBO J 1991: 10: 3931-3939.

Chelly J, Tdmer Z , Tennesen T, Petterson A, Ishikawa-Brush Y, Tommerup N, Horn N, Monaco AP Isolation of a candi- date gene for Menkes disease that encodes a potential heavy metal binding protein. Nature Genet 1993: 3: 14-19.

Favier A, Boujet C, Joannard. Possibility of a Menkes-like dis- order of copper metabolism in a girl. J Inher Metab Dis 1983: 6 (Suppl 2): 89.

Gerdes AM, Ternnesen T, Horn N, Grisar T, Marg W, MBller A, Reinsch R, Barton Nw, Guiraud P, Joannard A, Richard MJ, Gtittler F. Clinical expression of Menkes syndrome in females. Clin Genet 1990: 38: 452-459.

Horn N, Ternnesen T, Tlimer Z. Menkes disease: an X-linked neurological disorder of the copper metabolism. Brain Path-

Iwakawa Y, Niwa T, Tomita M. Menkes kinky hair syndrome; report on an autopsy case and his family sibling with similar clinical manifestations. Brain Develop (Tokyo) 1979: 11: 260- 266.

Kapur S, Higgins JV; Delp K, Rogers B. Menkes syndrome in a girl with X-autosome translocation. Am J Med Genet 1987: 26: 503-510.

Kievitis T, Dauwerse JG, Wiegant J, Breuning MH, Cornelisse CJ, van Ommen G-JB, Pearson PL. Rapid subchromosomal localization of cosmids by non-radioactive in situ hybridiza- tion. Cytogenet Cell Genet 1990: 53: 134-136.

Matsubara 0, Takaoka H, Iwakawa Y, Okeda R. An autopsy case of Menkes kinky hair disease. Acta Pathol Japn 1978: 28: 585-594.

Menkes JH, Alter M, Steigleder GK, Weakley DR, Sung JH. A sex linked recessive disorder with retardation of growth, peculiar hair and focal cerebral and cerebellar degeneration. Pediatrics 1962: 29: 769-779.

Mercer JFB, Livingston J, Hall B, Paynter JA, Begy C, Chand- rasekharappa S, Lockhart P, Grimes A, Bhave M, Siemieniak D, Glover TW. Isolation of a partial candidate gene for Menkes disease by positional cloning. Nature Genetics 1993:

Nadal D, Baerlocher K. Menkes’ disease: long-term treatment with copper and D-penicillamine. Eur J Pediatr 1988: 147: 62 1-625.

Robinson DO, Boyd Y, Cockburn D, Collinson MN, Craig I, Jacobs PA. The parental origin of de novo X-autosome trans- locations in females with Duchenne muscular dystrophy re- vealed by M27P methylation analysis. Genet Res Camb 1990: 56: 135-140.

Tommerup N, Tdmer Z, Ternnesen T, Horn N. A cytogenetic survey in Menkes disease: implications for the detection of chromosomal rearrangements in X linked disorders. J Med Genet 1993: 30: 314-315.

Ternnesen T, Horn N. Prenatal and postnatal diagnosis of Menkes disease, an inherited disorder of copper metabolism. J Inher Metab Dis 1989: 12 (Suppl 1): 207-214.

Tamer Z, Chelly J, Tommerup N, Ishikawa-Brush Y, Ternnesen T, Monaco AP, Horn N. Characterization of a 1.0 Mb YAC contig spanning two chromosome breakpoints related to Menkes disease. Hum Mol Genet 1992a: 1: 483-489.

mmer Z, Tommerup T, Tsnnesen T, Kreuder J, Horn N. Map- ping of the Menkes locus to Xq13.3 distal to the X-inacti- vation center by an intrachromosomal insertion of the seg- ment Xq13.3-ql2.2. Hum Genet 1992b: 88: 668-672.

Verga V, Hall BK, Wang S, Johnson S, Higgins JV, Glover TW. Localization of the translocation breakpoint in a female with Menkes syndrome to Xq13.2-ql3.3 proximal to PGKI. Am J Hum Genet 1991: 48: 1133-1 138.

Vulpe C, Levinson B, Whitney S, Packman S, Gitschier J. Iso- lation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nature Gen- etics 1993: 3: 7-13.

ology 1992: 2: 351-362.

3: 20-25.

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