1472

avium, E faecium (identified with API20 Strep, API, Biomerieux), and S epidermidis (also isolated from intravascular catheter). The E avium was resistant to vancomycin (by Stokes’ E

method with 30 Jlg discs), but was sensitive to ampicillin. Both the E faecium and S epidermidis were sensitive to vancomycin. The patient was treated with amoxycillin 500 mg three times daily and vancomycin 1 g twice a day for 10 days and recovered. Faeces cultured 24 h after the start of treatment yielded vancomycinresistant E avium and vancomycin-sensitive E faecium. E avium from blood was sent to the Antibiotic Reference Laboratory, Central Public Health Laboratory, UK, where resistance to both vancomycin and teicoplanin (minimum inhibitory concentrations > 32 mg/1) was confirmed. The strain hybridised with a DNA probe specific for the VanA glycopeptideresistance gene and could transfer vancomycin resistance in vitro to a recipient strain of Efaecalis. The origin of this vancomycin-resistant E avium strain is unclear. Neither the patient from whom it was isolated nor any of the other patients or staff on the ward had been transferred from any hospital in which vancomycin-resistant enterococci have been reported. The VanA gene has been reported in other members of ilie gut flora that would normally be regarded as non-pathogenic.5 Undisclosed transfer of this gene may be a possible explanation for the otherwise unexplained appearance of VanA in the E avium reported here. Microbiologists and clinicians should be aware that transferable vancomycin resistance in enterococci is not restricted to specialised units with large vancomycin usage but can also arise in general hospitals where vancomycin is used infrequently. We thank Mr R. Pollard for permission

to

report this

Third Department of Internal Medicine and Departments of Dermatology and Parasitology, Asahikwa Medical College, Asahikawa 078, Japan

KATSUYUKI HIRAI NOBUO TAKEMORI NOBUYUKI YANAGAWA MASAYOSHI NAMIKI HAJIME IIZUKA KENJI MIYAMOTO

1. Shulman LE. Diffuse fasciitis with hypergammaglobulinemia and eosinophilia: a new syndrome?J Rheumatol 1974; 1 (suppl): 46. 2. Stanek G, Konrad K, Jung M, Ehringer H. Shulman syndrome, a scleroderma subtype caused by Borrelia burgdorferi? Lancet 1987; i: 1490. 3. Sepp N, Schmutzhard E, Fritsch P. Shulman syndrome associated with Borrelia burgdorferi and complicated by carpal tunnel syndrome. J Am Acad Dermatol 1988, 18: 1361-62. 4. Abele DC, Anders KH. The many faces and phases of borreliosis II. J Am Acad Dermatol 1990; 23: 401-10. 5. Miyamoto K, Nakao M, Uchikawa K, Fujita H. Prevalence of Lyme borreliosis spirochetes in ixodid ticks of Japan, with special reference to a new potential vector, Ixodes ovatus (Acari: Ixodidae).J Med Entomal 1992; 29: 216-20.

case.

Serum

Pathology Department, North Tyneside General Hospital, North Shields, Tyne & Wear NE29 8NH, UK; and Laboratory of Hospital Infection, Central Public Health Laboratory, London NW9

disappearance of eosinophilia. He is currently on 10 mg prednisolone every second day. Abele and Anders4 suggested that B burgdorferi is closely associated with diverse skin diseases including Shulman syndrome. Various tick species are candidate vectors for B burgdorferi. In Japan Ixodes persulcatus Schulze and I ovatus Neumann are the vectors of B burgdorferi5 and Hokkaido is a focus for both. The patient had a history of a tick bite and mountain excursions in Hokkaido, and he was seropositive for B burgdorferi. This case supports the view that Shulman syndrome may be another clinical expression of B burgdorferi infection.

G. GOPAL RAO K. J. MORRIS R. C. GEORGE N. WOODFORD

Vancomycin-resistant enterococci. Communicable Dis Rep 1992; 2: 27. Uttley AHC, George RC, Naidoo J, et al. High-level vancomycin resistant enterococci causing hospital infections. Epidemiol Infect 1989; 103: 173-81. 3. Leclercq R, Derlot E, Duval J, Courvalin P. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med 1988; 319: 1. 2.

157-61. 4. Noble WC, Virani

Z, Cree RGA. Co-transfer of vancomycin and other resistance genes from enterococcus faecalis NCTC 12201 to staphylococcus aureus. FEMS Microbiol Lett 1992; 93: 195-98. 5. French G, Abdulla Y, Heathcock R, et al. Vancomycin resistance in south London. Lancet 1992; 339: 818-19.

Borrelia burgdorferi and Shulman syndrome SIR,-Shulman syndrome (eosinophilic fasciitis)l is a scleroderma-like illness with firm taut skin, flexion contractures of extremities, and limitation of joint movement. Histopathology reveals striking thickening of the fascia infiltrated by lymphocytes and plasma cells. Some investigators have implicated Borrelia burgdorferi as a cause, on the basis of a history of tick bites and positive B burgdorferi serology.2,3 A 49-year-old man noticed swelling of the extremities in February, 1992. This gradually worsened, resulting in tightening of the skin and flexion contractures of the fingers. He had a history of a tick bite at age 17 when on a mountain excursion in Hokkaido, and until recently he had made other trips to mountains occasionally. He was admitted to our hospital on July 14. He had eosinophilia (white blood cells 8300/nl, 33% eosinophils) and hypergammaglobulinaemia. Rheumatoid factor, scleroderma antibodies, and serological tests for syphilis were negative. Right forearm biopsy revealed increased collagen bundles in subcutaneous fat and remarkable thickening of the fascia with infiltration of lymphocytes and plasma cells. Visceral manifestations were absent. Shulman syndrome was diagnosed. Tests for B burgdorferi (IgG/IgM FASTLYME kit; 3M Diagnostic System) were done four times while he was in hospital and were abnormal (14-15%, normal < 10%). Isolation of B burgdorferi from the skin lesion was unsuccessful. He did not respond to doxycycline and amoxycillin. Prednisolone 40 mg per day was started on Aug 20 and proved effective; his symptoms rapidly improved with the

lysosomal enzyme abnormalities in galactosaemia

reported serum glycoprotein abnormalities in galactosaemia (decreased thyroxine binding globulin [TBG] and abnormal isoelectric focusing of sialotransferrins with a shift towards the cathode).l These features are also found in the carbohydrate-deficient glycoprotein (CDG) syndromes, a newly recognised group of genetic diseases with major SiR,—We have untreated classic

system involvement.2,3 Since there

nervous

are

also

serum

lysosomal enzyme abnormalities in CDG syndromes,2,4 we studied serum p-N-acetylhexosaminidase and ot-fucosidase in two newborn babies with galactose-1-phosphate uridyltransferase deficiency before and during treatment with a galactose-free diet. Isoelectric focusing was done with 12 ul serum on agarose gels in a pH gradient 5’0-8-0 with a Pharmacia Phast apparatus. Enzyme activities were measured with fluorogenic substrates. The isoelectric focusing pattern of p-hexosaminidase was abnormal in both patients before treatment (figure). There were several additional cathodal bands as well as an abnormal anodal

A B C D E F *

Isoelectric

focusing

of

&bgr;-hexosaminidase.

Lane A=control (newborn baby), B = patient with CDG syndrome, C and D infants with classic galactosaemia before treatment, E = D after 1 week of galactose-free diet, F=control (infant). Normally in newborn babies (A), band pattern is heavier than in infants (F). =

1473

band. Activity of this enzyme was increased in both patients (189 and 102 IU/1, normal 10-50) and similar to values in patients with CDG syndrome (40-112 IU/1, n = 10). A repeat analysis after 1 week of a galactose-free diet showed a normal isoenzyme pattern (data of patient 2 in figure) and normal activity. a-fucosidase had a normal activity but a similarly disturbed isoenzyme pattern with abnormal cathodal bands that normalised during treatment. These data are similar to those found in CDG syndromes (figure).4 Taken together with our findings of decreased serum TBG and abnormal sialotransferrin pattern, these results are a further argument for grouping classic galactosaemia as a CDG syndrome, albeit a secondary and treatable one. In serum glycoproteins of patients with CDG syndrome there is a partial deficiency of the terminal trisaccharide sialic acid, galactose, and Nacetylglucosamine.5 Sialic acid bears a negative electric charge and the deficiency of this carbohydrate residue explains the cathodal shift of sialotransferrins and other glycoproteins. We postulate that in classic galactosaemia the accumulated galactose-1-phosphate interferes with the carbohydrate processing at the Golgi galactosyltransferase step and leads to a partial deficiency of the terminal disaccharide (sialic acid and galactose) of glycoproteins such as transferrin, TBG, and the lysosomal enzymes. Defective galactosylation of proteins in cultured skin fibroblasts and deficiency of glycolipids containing galactose or Nacetylgalactosamine in brain and lymphocytes with classical galactosaemia patients has been reported.6,7 Classical galactosaemia and CDG syndromes are both multisystemic disorders affecting the nervous system, liver, gastrointestinal tract, kidneys, and gonads. We suggest that these glycoprotein abnormalities play a major part in the pathogenesis of classic galactosaemia that thus may be regarded as a secondary "Golgi disorder". Department of Paediatrics, University of Leuven, 3000 Leuven, Belgium

JAAK JAEKEN

Department of Paediatrics, University of Ghent,

Jos KINT

Department of Molecular Cell Biology and Genetics, University of Limburg, Maastricht, Netherlands

LEO SPAAPEN

Vulsma T, Theunissen PMVM, van der Meer SB, Jaeken J. Galactosaemia, a carbohydrate-deficient glycoprotein syndrome. Society for the Study of Inborn Errors of Metabolism, 30th Annual Symposium, Leuven, Sept 8-11, 1992 (abstr). 2. Jaeken J, Vanderschueren-Lodeweyckx M, Casaer P, et al. Familial psychomotor retardation with markedly fluctuating serum prolactin, FSH and GH levels, partial TBG deficiency, increased serum arylsulphatase A and increased CSF protein: a new syndrome? Pediatr Res 1980; 14: 179. 3. Jaeken J, Stibler H, Hagberg B. The carbohydrate-deficient glycoprotein syndrome. Acta Paediatr Scand 1991; suppl: 375. 4. Jaeken J, Kint J. Abnormal serum lysosomal isoenzymes in leukodystrophy with sialic add deficient proteins. Society for the Study of Inborn Errors of Metabolism, 25th Annual Symposium, Sheffield, Sept 22-25, 1987 (abstr). 5. Stibler H, Jaeken J. Carbohydrate deficient serum transferrin in a new systemic hereditary syndrome. Arch Dis Child 1990; 65: 107-11. 6. Omstein KS, McGuire EJ, Berry GT, Roth S, Segal S. Abnormal galactosylation of complex carbohydrates in cultured fibroblasts from patients with galactose-1phosphate uridyltransferase deficiency. Pediatr Res 1992; 31: 508-11. 7. Petry K, Greinix HT, Nudelman E, et al. Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes. Biochem Med Metab Biol 1991; 46: 93-104.

1. Spaapen LJM,

Nested PCR of PAH cDNA and sequence analysis of V388M.

Upper= PCR lane 1 =size marker, 2=first PCR with lymphoblast cDNA, 3=second PCR with lymphoblast cDNA, 4=first PCR with liver cDNA, and 5=second PCR with lymphoblast poly(A+) RNA without reverse transcription. PCR product was not detected after first PCR with lymphoblast cDNA. Negative control with lymphoblast poly (A+) RNA without reverse transcription proves no contamination of cDNA. Primer sequences for first PCR: 5’dAGGGAAACCTGCCTGTACGT, 3’dCTCCATCAACAGATTCACAGC, and for second PCR: 5’dAAAAGCCAGAGACCTCACTC, 3’dACAGACCACATTCTGTCCATG. can be detected by nested polymerase chain reaction (PCR).2-4 We used this method to analyse PAH mRNA from lymphoblasts of a Japanese phenylketonuria patient and identified a novel mutation. Poly (A + ) RNA was isolated from a patient-derived EpsteinBarr-virus-immortalised lymphoblastoid cell line. Single-stranded cDNA was synthesised by reverse transcription with oligo(dT) primer and subjected to nested PCR with two sets of primers to amplify the entire coding region of PAH cDNA. A DNA fragment of the predicted size (1416 bp) was obtained (figure). Sequencing analysis of the amplified DNA revealed two point mutations: a single base substitution of G to A at base 1384 that alters valine at codon 388 in exon 11 to methionine (figure), and another substitution of G to C at base 1460 that alters arginine at codon 413 in exon 12 to proline. The valine-to-arginine mutation (V388M) has not been reported, whereas the arginine-to-proline mutation (R413P) has been described.6 Analysis of genomic DNA of the patient as well as the parents showed that this patient is indeed a compound heterozygote of V388M and R413P, whereas the father is a heterozygote of V338M and the mother is a heterozygote of R413P. Our study demonstrates that PAH mRNA in a phenylketonuria patient can be analysed with lymphoblasts and there is no need for risking liver biopsy. The method is not only rapid and reliable but also advantageous for the analysis of compound heterozygotes, because each allele can be examined separately. Various types of mutations responsible for the disease have been reported by genomic analysis of the PAH gene. Despite extensive studies for nearly a decade, less than 50% of the mutations have been identified in most populations.’ The analysis of ectopically transcribed PAH mRNA would facilitate the discovery of unknown mutations.

Department of Biochemical Genetics, Tohoku University School of Medicine, Aoba-ku, Sendai 980, Japan

KAZUTOSHI TAKAHASHI SHIGEO KURE YOICHI MATSUBARA KUNIAKI NARISAWA

Novel phenylketonuria mutation detected by

analysis of ectopically transcribed phenylalanine hydroxylase mRNA from lymphoblast is one of the most common inherited of aminoacid metabolism and is caused by altered activity of phenylalanine hydroxylase (PAH). To date molecular analysis of phenylketonuria has focused on extensive sequencing of 13 exons in the PAH gene’ because it is believed that PAH mRNA is expressed only in liver, and biopsy or necropsy liver specimens are required for the analysis of mRNA. Sequencing of each exon is laborious. Extremely low concentrations of mRNA synthesised by "ectopic" or "illegitimate" transcription in non-expressing tissues

SIR,-Phenylketonuria

inborn

errors

RC, Woo SLC. Phenylketonuria and the phenylalanine hydroxylase gene. Mol Biol Med 1991; 8: 3-18. 2. Sarkar G, Sommer SS. Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 1989; 244: 331-34. 3. Chelly J, Concordet JP, Kaplan JC, Kahn A. Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci USA 1989; 86: 2617-21. 4. Roberts RG, Bently DR, Barby TFM, Manners E, Bobrow M. Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA. Lancet 1990; 336: 1523-26. 5. Chomczynski P, Sacchi N. Single-step method of RNA isolation by add guanidium thiocyanate-phenol-chloroform extraction Anal Biochem 1987; 162: 156-59. 6. Wang T, Okano Y, Eisensmith R, et al. Molecular genetics of PKU in onentals. Am J Hum Genet 1989; 45: A228. 7. Konecki DS, Lichter-Konecki U. The phenylketonuria locus: current knowledge about alleles and mutations of the phenylalanine hydroxylase gene in various populations. Hum Genet 1991; 87: 377-88. 1. Eisensmith