pathophysiological basis of IDDM and SMS; the potential of such work lies in diagnostic tests and therapeutic interventions. Departments of Child

Health and Cellular and Molecular Sciences, St George’s Hospital Medical School, London SW17 0RE, UK


S, Landin M, Kristenson JK. Antibodies to a 64,000 MW human islet antigen precede the clinical onset of insulin-dependent diabetes. JClin Invest 1987; 79: 924-34 2. Solimena M, Folli F, Aparasi R, Pozza G, De Camilli P. Autoantibodies of GABAergic neurons and pancreatic &bgr;-cells in stiff-man syndrome. N Engl J Med 1990; 322: 1555-60. 3. Baekkeskov S, Aanstoot H-J, Christgau S, et al Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 1990; 347: 151-56. 4. Erlander MG, Tobin AJ. The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochem Res 1991; 16: 215-66. 5. Karlsen AE, Hagopian WA, Grubin CE, et al. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc Natl Acad Sci USA 1991, 88: 8337-43. 6. Persson H, Pelto-Huikko M, Metsis M, et al. Expression of the neurotransmitter synthesizing glutamic acid decarboxylase in male germ cells. Moll Cell Biol 1990; 10: 4701-11. 7. Cram DS, Barnett LD, Joseph JL, Harrison LC. Cloning and partial sequence of human glutamic acid decarboxylase cDNA from brain and pancreas islets. Biochem Biophys Res Comm 1991; 176: 1239-44. 8. Kobayashi Y, Kaufman DL, Tobin AJ. Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein. J Neurosci 1987; 7: 2766-72. 9. Wyborski RJ, Bond RW, Gottlieb DI. Characterisation of a cDNA coding for rat glutamic acid decarboxylase Mol Brain Res 1990; 8: 193-98. 10. Michelsen BK, Petersen JS, Boel E, Moldrup A, Dyrberg T, Madsen OD. Cloning, characterization and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 1. Baekkeskov

Aminoacid sequences, predicted from cDNA sequences, of human (this letter) and rat10 GAD, are aligned. For rat data -


identity with

human sequence.

syndrome" (SMS) is a rare neurological condition characterised by a progressive symmetrical increase in axial muscle tone exacerbated by sensory stimuli. It is believed to be due to interference in neuronal pathways using the inhibitory neurotransmitter y-aminobutyric acid (GABA). It is associated in about 60% of cases with antibodies to glutamic acid decarboxylase (GAD), which catalyses the rate-limiting step in the synthesis of GABA.2 GAD activity is widely distributed in the nervous system but is also found in other tissues, including, notably, pancreatic islets. About 20% of patients with SMS have IDDM; other autoimmune diseases are also common.2 The 64 kDa antigen recognised by IDDM autoantibodies is GAD.3 GAD is now generally accepted to be a doublet of polypeptides of molecular weight 60-70 kDa. Complementary DNA encoding brain GAD from cat, rat, mouse, and fruitfly has been cloned; the sequences are highly conserved and code for a protein of predicted molecular weight 66-67 kDa (GADlarge). 4There is evidence for two rat brain GAD genes and it has been suggested that the 65 kDa polypeptide is coded by the larger "67 kDa" gene corresponding to that already cloned from cat, rat, and fruitfly, whereas the 64 kDa polypeptide is coded by a smaller "65 kDa" gene (GADsmall). Clearly, given the importance of the aetiology ofautoimmunity to GAD in the two diverse clinical conditions of IDDM and SMS, the structures of both forms of human GAD need to be ascertained. Although there are data on the GADsmall islet polypeptide,s the sequence of only a limited region of human GAD large has been published;6.7 this has sequence homology with the cat and rat

GAD large. 8,9

We screened a human frontal cortex cDNA library in ÀZAPII using the feline GAD large clone (kindly provided by Dr A. Tobin, Los Angeles) as a probe. After two rounds of screening, four positive plaques were selected. The ’Bluescript’ phagemids containing the DNA inserts were rescued, and these clones were characterised further by restriction mapping and dideoxy sequencing. The longest clone obtained (2-9 kb, including the 1 ’3 kb 3’ untranslated region) was entirely sequenced. By comparison with published feline and rat GAD large sequences, this human clone contained the entire coding region except for the N-terminal sixty residues and demonstrated 85% identity to the rat sequence at the nucleotide level and 97% identity to both the rat and cat at the protein level (figure). There is one report of differential expression of GAD genes

between human pancreas and brain.7 However, these two forms differ less than do the two forms occurring in rat brain,4 and the "islet form" is more similar to rat brain GAD large than to human brain GADlarge. Furthermore, our sequence data agree not only

with the limited brain sequence of these authors4 but also with the recent report of the partial sequence of human GADlarge from an insulinoma.10 The most reasonable conclusion is that the two forms of GAD4.5 are expressed in both human brain and pancreatic &bgr;-cell, as recently shown for rat. 10 Now that the structures of both forms of human GAD are known, studies can be undertaken into the

1991; 88: 8754-58.

Glutamic acid decarboxylase expression in islets and brain SIR,-A 1990 Lancet editorial debated the identity of the 64 kDa first described in 1982 as the target of autoantibodies in type I diabetes.’ The antigen was subsequently identified as glutamic acid decarboxylase (GAD), which catalyses the formation of y-aminobutyric acid from L-glutamic acid.2 Autoantibodies directed against the same target are also found in "stiff man syndrome" (SMS).3 The causative role of these autoantibodies in the two conditions remains unclear, especially since SMS is very rare in patients with type I diabetes. GAD is expressed in rat, mouse, and cat brain and in rat islets of Langerhans,4-7 the target organs of these autoimmune diseases. However, sera from patients with type I diabetes known to contain high titre 64 kDa antibodies tend not to react with GAD in sections of rat cerebellar cortex.3 Possible explanations are that the sequences of GAD isoforms are different in brain and in islets or that there is preferential expression of a different isoform in each tissue. Two GAD isoforms of molecular weights 65 kDa and 67 kDa exist in rat brain, and GAD65 and GAD67 are coded for by separate genes.8 A portion of a human homologue of GAD 67 from testis9 and the complete sequence of a GAD65 transcript (GAD-2) from human islets of Langerhans have been reported.10 To clarify the nature of GAD isoforms in man and to study their sequences and relative expression in human pancreatic islets and brain we isolated mRNA, synthesised the cDNA, and amplified the cloned GAD 65 and GAD67 transcripts. Human islets were obtained by collagenase digestion of pancreas obtained from live organ donors; human brain RNA was obtained from Clontech (Palo Alto). Transcripts were amplified in two separate but overlapping portions, one from position 1 (ATG codon) to 1000 and the other from position 900 to the stop codon at about 1800. The primers were selected on the basis of available sequences (partial sequence of human GAD6/and full-length sequences of cat and ratS GAD67 and rat GAD 65’ The small fragment of each gene between 900 and 1000 was also amplified and used as a probe to identify the larger fragments. The amplified fragments were cloned in ’BluescriptIISK’ (Stratagene, San Diego) and sequenced. We found two GAD isoforms in human brain, showing a high


degree of similarity with rat GAD 65 and GAD67 sequences. Human islets of Langerhans also expressed both isoforms and the islet


sequences were identical to those in brain. The sequence we have obtained for GAD65 is identical to that reported as GAD-210 while GAD67 has 100% similarity with the partial human testis sequence. Northern blot analysis showed that both transcripts are present at comparable levels in the brain, whereas in the endocrine pancreas GAD65 is expressed at a much higher level than GAD67. These results suggest that GAD, an autoantigen in type I diabetes and SMS, is encoded by the same genes in both human brain and pancreatic islets and that the structure of the mRNAs in the two tissues is the same. GAD transcripts in the islet and in the brain thus differ only quantitatively. Differences in anti-GAD response in the two autoimmune conditions cannot therefore be ascribed to different GAD sequences, although post-translational modifications in the two tissues cannot be excluded. Now that the sequences of both GAD isoforms are known it should be possible to map the epitope(s) recognised by the antibodies and to study B and T lymphocyte reactions to GAD in these two different autoimmune diseases.

Division of Immunogenetics, Department of Pediatrics, Rangos Research Center, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA


Department of Immunology, King’s College School of Medicine and Dentistry,


London SE5 9PJ, UK

Department of Surgery,

King’s College Hospital,

Homocyst(e)inaemia and bone density in elderly women SIR,—When we reported (Aug 10, p 355) that low bone density associated with stroke mortality in elderly women we speculated that the association might be a result of Moderately hyperhomocyst(e)inaemia. high levels of homocyst(e)ine in blood have been associated with stroke,’-3 and the was

homozygous form of the disorder (homocystinuria) causes premature stroke, myocardial infarction, and severe osteoporosis.4 To test the hypothesis we investigated homocyst(e)ine concentrations in 23 women aged 65-80 randomly selected from participants in our study of osteoporotic fractures whose bone density was in the highest decile for age, and in 23 age-matched (within 1 year) participants whose bone density was in the lowest decile. Bone density was measured at the distal radius by single photon absorptiometry. Serum taken when the bone density measurements were made was frozen at - 20°C for up to 2 weeks and then at -190°C for up to 3 years. Homocyst(e)ine was assayed blindly by high-pressure liquid chromatography with electrochemical detection. ("Homocyst(e)ine", expressed as homocysteine, refers to the sum of homocysteine and the homocysteinyl moieties of the disulphides homocystine and cysteine-homocysteine, whether free or bound to proteins.) Homocyst(e)ine values ranged from 4-95 to 17.49 nmol/ml and the mean (SD) was 8-66 (3-05) in the 23 women in the highest decile of bone density, and 8-31 ( 1 62) nmol/ml in those in the lowest decile (p=0’63 by paired t-test, 95% confidence interval for difference 1 16 to + 1 86). These data, from a small number of women, suggest that hyperhomocyst(e)inemia is not a common cause of low bone density in elderly American women but do not eliminate the possibility that hyperhomocyst(e)inemia may be involved in the association between low bone density and stroke mortality. -


Department of Immunology, King’s College School of Medicine and Dentistry,


Division of Immunogenetics, Rangos Research Center, Pittsburgh



1. Editorial The 64K question in diabetes. Lancet

1990; 336: 597-98. 2. Baekkeskov S, Aanstoot H-J, Christgau S, et al. Identification of the 64K autoantigen in insulin dependent diabetes as the GABA-synthesising enzyme glutamic acid decarboxylase. Nature 1990; 347: 151-56. 3. Solimena M, Folli F, Dennis-Donmi S, et al. Autoantibodies to GABA-ergic neurons and pancreatic beta cells in Stiff Man Syndrome. N Engl J Med 1990, 322: 1555-60. 4. Julien J-F, Samama P, Mallet J. Rat brain glutamic acid decarboxylase sequence deduced from a cloned cDNA. J Neurochem 1990; 54: 703-05. 5. Katarova Z, Szabo G, Mugnaini E, Greenspan RJ. Molecular identification of the 62 kD form of glutamic acid decarboxylase in the mouse. Eur J Neurosci 1990; 2: 190-202. 6. Kobayashi Y, Kaufman DL, Tobin AJ. Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein. J Neurosci

1987; 7: 2768-72. 7. Okada Y, Taniguchi H, Shimada C.

High concentration of GABA and high glutamate decarboxylase activity in rat pancreatic islets and human insulinoma. Science 1976;

194: 620-22. 8. Erlander MG, Tillakaratne NJK, Feldblum S, Patel N, Tobin AJ. Two genes encode distinct glutamate decarboxylases. Neuron 1991; 7: 91-100. 9. Persson H, Pelto-Huikko M, Metsis M, et al Expression of the neurotransmittersynthesising enzyme glutamic acid decarboxylase in male germ cells. Mol Cell Biol

Division of Clinical Epidemiology, University of California at San Francisco, San Francisco, California 94105, USA


Primate Center,

Oregon Regional Beaverton, Oregon


MR, Beamer N, Sexton G, Nordt F, de Garmo P. Elevated concentration as a possible independent risk factor for stroke. Stroke 1990; 21: 572-76. 2. Brattstrom L, Lindgren A, Israelsson B, Malinow MR, Norrving B, Upson B. Hyperhomocysteinaemia in stroke: prevalence, cause, and relationships to type of stroke and stroke risk factors. Eur J Clin Invest (m press). 3. Clarke R, Kaly L, Robinson K, et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991; 324: 1149-55. 4. Mudd SH, Levy HL, Skovby F. Disorders of transulfuration. In: Scriver CS, Beaudet AL, Sly WL, Valle D, eds. The metabolic basis of inherited disease, 5th ed. New York: McGraw-Hill, 1983: 522-59. 5. Malinow MR, Kang SS, Taylor LM, et al. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation 1989; 79: 1180-88. 1. Coull BM, Malinow

plasma homocyst(e)ine


1990; 10: 4701-11. 10. Karlsen AE, Hagopian WA, Grubin CE, et al. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10. Proc Natl Acad Sci USA 1991; 58: 8337-41.

Upper airway obstruction during nasal intermittent positive-pressure hyperventilation in sleep.-In this article by Dr P. Delguste and colleagues (Nov 23, p 1295), the figure on p 1296 showing continuous polygraphic tracing during sleep was reproduced poorly in some copies of the London edition, and is reprinted below.

Glutamic acid decarboxylase expression in islets and brain.

1469 pathophysiological basis of IDDM and SMS; the potential of such work lies in diagnostic tests and therapeutic interventions. Departments of Chil...
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