539

DR-are involved in the presentation of antigen to T helper cells, whereas those of the MHC class I loci-HLA A, B, and C-are targets of cytotoxic T cells. Class II antigens have a limited expression on mature cells; they are largely confined to cells such as macrophages, dendritic cells, and B lymphocytes which are capable of presenting antigen. By contrast, class I antigens are widely expressed, being present on the surface of virtually all nucleated cells. MHC antigens show such extreme polymorphism that unrelated individuals have a minuscule chance of having identical tissue types. Class I and class II antigens are not expressed on the syncytiotrophoblast membrane or on the underlying cytotrophoblasts that surround the conceptus; lack of expression of these antigens occurs at the transcriptional level.4 In addition, class II antigens are not expressed on the cytotrophoblasts in direct contact with the decidua, but the presence of class I antigens is disputed. Although standard tissue typing antisera do not react with these cells, the monoclonal antibody

W6/32

(which

recognises non-polymorphic molecules) and antibodies to &bgr;-2-microglobulin (the non-polymorphic light chain of class I antigens) give positive staining reactions.5,6 Several explanations for these observations—eg, coating with maternal antibody7 low-density antigenic expression,8 and expression of truncated molecules9--have been advanced, but the answer has

determinants

on

class I

become clear. The HLA class I gene family contains many more members than can be accounted for by HLA A, B, and C genes and the products of several other loci have lately been described,10 among which are molecules designated HLA E, F, and G. Transfection of the HLA G gene into a lymphoblastoid cell line which does not express classic 44 kD class I antigens resulted in the surface expression of a 37-39 kD molecule that was associated with &bgr;-2-microglobulin and bound to W6/32.11 Immunoprecipitation of methioninelabelled HLA G transfected cells with W6/32 followed by two-dimensional gel electrophoresis showed a characteristic autoradiographic spot array. Identical patterns were obtained with this method when methionine-labelled pooled, first trimester villous cytotrophoblasts or certain choriocarcinoma cell lines were used.12 This work confirms the earlier suggestion by Ellis et al13 that villous cytotrophoblasts express surface HLA G. These researchers isolated and sequenced an HLA class I cDNA clone from a library derived from the chorion carcinoma cell line BeWo. This nucleotide sequence showed a high degree of homology with the published sequence of HLA G (then designated HLA 60). They then used the polymerase chain reaction to show the presence of similar sequences in cDNA from normal extravillous trophoblasts. Expression of HLA G seems to be limited to cytotrophoblasts.12 Interestingly, although the

now

aminoacid sequence of the molecule is invariant there is nucleotide polymorphism,13 which suggests that there is selection pressure against antigenic polymorphism. Thus it is likely that class I molecules are necessarily present on nucleated cells and serve a function beyond immunological recognition. So, why is a baby not rejected as foreign by his mother? The main reason seems to be that a special non-immunogenic form of the MHC class I molecule is expressed at the feto-matemal interface. RE. Transplantation immunity and the maternal fetal relation. N Engl J Med 1964; 270: 667-72. 2. Boyd JD, Hamilton WJ. The human placenta. Cambridge: Heffers, 1970. 3. Beer A. Immunology of reproduction. In: Samter M, Talmage DW, Frank MM, Austen KF, Claman HN, eds. Immunological disease. Boston: Little, Brown, 1988: 329-60. 4. Kawata M, Parnes JR, Herzenberg LA. Transcriptional control of HLA A, B, C antigens in human placental cytotrophoblasts isolated using trophoblast and HLA specific monoclonal antibodies and the fluorescence activated cell sorter. J Exp Med 1984; 160: 633-51. 5. Bamstable CJ, Bodmer WF, Brown G, et al. Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell 1978; 14: 9-20. 6. Ellis SA, Sargent IL, Redman CWG, McMichael AJ. Evidence for a novel HLA antigen found on human extravillous trophoblast and a choriocarcinoma cell line. Immunology 1986; 59: 595-601. 7. Bonneau M, Latour M, Revillard JP, Robert M, Traeger J. Blocking antibodies eluted from human placenta. Transpl Proc 1973; 5: 589-92. 8. Redman CWG. HLA DR antigen on human trophoblast: a review. Am J Reprod Immunol 1983; 3: 175-77. 9. Kress M, Cosman D, Khoury G, Jay G. Secretion of transplantation related antigen. Cell 1983; 34: 189-96. 10. Koller BH, Geraghty DE, DeMars R, Duvick L, Rich SS, Orr HT. Chromosomal organisation of the MHC class I gene family. J Exp Med 1989; 169: 469-80. 11. Shimizu Y, Geraghty DE, Koller BH, Orr HT, DeMars R. Transfer and expression of three cloned human non-HLA-A, B, C class I MHC genes in mutant lymphoblastoid lines. Proc Natl Acad Sci USA 1988; 85: 227-31. 12. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ, DeMars R. A class I antigen, HLA-G, expressed in human trophoblasts. Science 1990; 248: 220-23. 13. Ellis SA, Palmer MS, McMichael AJ. Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA class I molecule. J Immunol 1990; 144: 731-35.

1. Billingham

CYSTIC FIBROSIS: CLOSING THE GAP Since the gene defective in cystic fibrosis (CF) was cloned last year/ various research groups world wide have been busy isolating mutations within the gene (cystic fibrosis transrriembrane conductance regulator, CFTR), looking for clues to the function of the protein. Some of this work has now been published.2,3 From the nucleotide sequence, a protein of 1480 aminoacids has been predicted1 with two hydrophobic membrane-spanning domains and two hydrophilic nucleotide-binding folds. This structure resembles that of multidrug resistance proteins and of several other proteins involved in the transport of molecules across cell membranes.1,4 There is also a large domain, unique to CFTR, that may be a target for phosphorylation by protein kinases. The most common CFTR mutation in populations of North European origin is &Dgr; F5081,5-a deletion of three base-pairs in the first nucleotide binding fold. Another fourteen mutations have been identified in this region; nine cause single aminoacid substitutions, three are non-sense mutations, one disrupts a splice acceptor site, and there is another three-base-pair deletion adjacent to &Dgr; F508.3,6 A nomenclature has been developed based on the single letter

540

aminoacid code and the codon number (eg, G551D is the replacement of glycine with aspartic acid at position 551; R553X is the introduction of a termination codon in place of

3.

4.

arginine at position 553). Putative structures for the nucleotide binding folds of CFTR and related genes (known as ABC genes for ATP-binding cassette-ie, a domain found in various proteins that binds and hydrolyses ATP) have been predicted by computer modelling.4 The deletion and substitutions observed in CFTR occur in highly conserved regions of the nucleotide binding fold. Although they are unlikely to affect ATP binding and hydrolysis directly, the aminoacid substitutions observed may affect changes in protein conformation which normally take place after nucleotide binding. Frameshift mutations in exons 13 and 19 (a two base-pair insertion and a single base deletion, respectively) introduce premature termination codons.6,7 Another series of mutations, reported in and around the first transmembrane segment,2are all associated with milder disease phenotypes. Kerem et a15 have suggested that patients with pancreatic insufficiency and a generally severe phenotype will be homozygous, or compound heterozygotes, for mutations that have a severe effect on CFTR function. Patients with a milder phenotype, including those who have no pancreatic deficiency, will carry an allele that disrupts gene function to a lesser extent. Several patients have been reported who are homozygous, or compound heterozygous, for non-sense or splice mutations.6 The generally milder phenotype in these patients suggests that loss of CFTR function is not as deleterious as a breakdown in its regulation. Although a defect in the transport of chloride ions appears to be fundamental in CF, it is unlikely that CFTR functions as a chloride channel. CFTR is homologous to a family of unidirectional active transport proteins; by contrast, ion channels permit bidirectional ion flow and do not normally require ATP hydrolysis. Moreover, all CF patients seem to have a defect in a single gene whereas different tissues appear to have chloride channels encoded by different genes. A protein as large as CFTR might be expected to transport molecules larger than ions; Ringe and Petsko8 have suggested leukotriene LTC4 and prostaglandin D2 as candidates for this ligand. Since more than 50 mutations have been found within CFTR in the past eight months, any plans to screen entire populations for CF heterozygotes for all mutations are probably over-optimistic. In some populations it is possible to identify 85% of CF mutations by analysis of only two exons (10 and 11). In the UK, population screening for heterozygotes has been thought desirable, and pilot schemes are being initiated.9 This approach contrasts with the confused picture emerging from the USA,10 where a fear of litigation seems to be slowing progress in this area of primary health care, although it has lately been reported that a National Institutes of Health working group has supported the initiation of pilot programmes.ll If the combination of mutation analysis with clinical data and structural modelling leads to a greater understanding of the function of CFTR, the gap between gene and treatment should be progressively shortened. 1. Riordan JR, Rommens JM, Kerem B-S, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-73. 2. Dean M, Amos J, Hsu JMC, et al. Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 1990; 61: 863-70.

Cutting GR, Kasch LM, Rosenstein, BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein. Nature 1990; 346: 366-69. Hyde SC, Emsley P, Hartshorn MJ, et al. Structural model of ATP-binding associated with cystic fibrosis, multidrug resistance and

bacterial transport. Nature 1990; 346: 362-65. B-S, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245: 1074-80. 6. Kerem B-S, Zielenski J, Markiewicz D, et al. Identification of mutations in regions corresponding to the 2 putative nucleotide (ATP)-binding folds in the cystic fibrosis gene. Proc Natl Acad Sci ( USA) (in press). 7. White MB, Amos J, Hsu JMC, Gerrard B, Finn P, Dean M. A frame-shift mutation in the cystic fibrosis gene. Nature 1990; 344: 665-67. 8. Ringe D, Petsko GA. A transport problem? Nature 1990; 346: 312-13. 9. Brock DJH. Population screening for cystic fibrosis. Am J Hum Genet 5. Kerem

1990; 47: 164-65. 10. Gilbert F. Is population screening for cystic fibrosis appropriate now? Am J Hum Genet 1990; 46: 394-95. 11. Roberts L. CF screening delayed for awhile, perhaps forever. Science 1990; 247: 1296-97.

CHILDREN WHO WALK LATE For parents, few milestones in their child’s development are exciting, memorable, and easily defined as the first steps. In Britain 97% of children achieve six steps unaided by 18 months of age,’ and it has been suggested that screening for children who walk late should form part of a preschool health surveillance programme.2 What would be achieved by such a policy? In one study of 404 late walkers3 a quarter were already known to paediatricians because of cerebral palsy, mental retardation, or other neurological, developmental, or orthopaedic disorders. Of 275 new referrals from screening, only 5% had neurological signsmainly cerebral palsy, although about a fifth had other evidence of delayed development. The frequency of neurological abnormalities in late walkers rises to 56% when the perinatal period has been abnormal.4 Theoretically, screening of boys not walking at 18 months with a blood test for creatine kinase will detect half the cases of Duchenne dystrophy in the population. In practice, detection rates are unacceptably low and population screening can not be recommended. Delayed motor skills in Duchenne dystrophy are often associated with generalised developmental delay and it is this combination which necessitates creatine kinase estimation in boys. Late walking is usually a benign normal variant but differentiation of "normal" from "abnormal" late walkers is not always easy. The typical child in the normal category has a family history of late walking and bottom shuffling, and the perinatal history is uneventful. Examination reveals a hypotonic bottom shuffler with hypermobile joints, hyperextended knees, and the sitting-on-air sign when the child is suspended under the arms. Such children do not have weakness and tendon reflexes are preserved. The picture may be less clear-50% of normal late walkers have no family history and 45 % are crawlers.3 The cerebral palsy diagnosed in late walkers at 18 months is usually a diplegia;1 in 8 such children is said to be a bottom shuffler.7 However, the increased resistance to passive dorsiflexion of the foot usually found in these cases contrasts with the hypotonia of most normal late walkers. Observation of a normal gait at 18 months is recommended as the last step in the screening programme for congenital dislocation of the hip.6 Apart from this, seeking out children who walk late does not fulfil recognised criteria for screening. Most of the children detected will be normal and the benefits of early diagnosis in the remainder are uncertain. By contrast, a child surveillance programme as