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&agr;1-Antitrypsin Deficiency andPrenatal Diagnosis

IF the haemoglobinopathies have acted as the

prototype for the study of inherited disease, thenai-antitrypsin (ai-AT) deficiency has not lagged farbehind. Since 1963, when an association was firstdemonstrated (somewhat fortuitously) between

(tt-A T deficiency and pulmonary disease,1 an

enormous amount of clinical and genetic informationhas accumulated concerning this curious and highlypolymorphic protease inhibitor. Clinically thereis firm evidence for a strong association with

emphysema,2,3 neonatal hepatitis,4,5 cirrhosis,6,7 andhepatoma8and limited support for an aetiological linkwith various renal disorders,9,10 rheumatoid

arthritis,l1 and psoriasis. 12 On the genetic side

progress has been startling. Over fifty allelic variantshave been described and are known as the proteaseinhibitor or Pi system. The alleles have been labelledon the basis of their electrophoretic mobility, with theZ allele representing a very slow variant, which in thehomozygote (PiZZ) leads to severe Ki-AT deficiency.The single locus coding for oci-AT is on the long arm

1. Laurell CB, Eriksson S. The electrophoretic &agr;1-globulin pattern of serum in

&agr;1-anritrypsin deficiency Scand J Clin Lab Invest 1963; 15: 132-40.2. Tobin MJ, Hutchison DCS. An overview of the pulmonary features of &agr;1-antitrypsin

deficiency. Arch Intern Med 1982; 142: 1342-48.3 Janus ED, Phillips NT, Carrell RW. Smoking, lung function, and &agr;1-antitrypsin

deficiency Lancet 1985; i: 152-54.4 Sharp HL, Bridges RA, Krivit W, Freier EF. Cirrhosis associated with alpha

1-antitrypsin deficiency: a previously unrecognised inherited disorder. J Lab ClinMed 1969; 73: 934-39.

5 Porter CA, Mowat AP, Cook PJL, Haynes DWG, Shilkin KB, Williams R.&agr;1-antitrypsin deficiency and neonatal hepatitis. Br Med J 1972; iii: 435-39.

6 Larsson C. Natural history and life expectancy in severe alpha1-antitrypsin deficiency,PiZ Acta Med Scand 1978; 204: 345-51.

7 Cox DW, Smyth S. Risk for liver disease in adults with alpha1-antitrypsin deficiency.Am J Med 1983; 74: 221-27.

8 Eriksson S, Carlson J, Velez R. Risk of cirrhosis and primary liver cancer in

alpha1-antitrypsin deficiency. N Engl J Med 1986; 314: 736-399 Moroz SP, Cutz E, Balfe JW, Sass-Kortsak A. Membranoproliferative

glomerulonephritis in childhood cirrhosis associated with alpha1-antitrypsindeficiency Pediatrics 1976; 57: 232-38.

10 Miller F, Kuschner M Alpha1-antitrypsin deficiency, emphysema, necrotisingangiitis and glomerulonephritis. Am J Med 1969; 46: 615-23.

11 Sjöblom KG, Wollheim FA. Alpha-1-antitrypsin phenotypes and rheumatic diseases.Lancet 1977; ii: 41-42.

12 Heng MCY, Moy RL, Lieberman J. &agr;1-antitrypsin deficiency in severe psoriasis. Br JDerm 1985; 112: 129-33

of chromosome 1413 within a 10 kilobase segment ofDNA and consists of five exons separated by fourintrons.14 The Z allele results from a point mutationcausing a G to A transition in exon V, which in turnleads to substitution of lysine for glutamic acid atresidue 342. An abnormal &agr;1-A T is produced which isnot processed normally by the liver, and this in turnhas the dual effects of local accumulation, whichimpairs hepatic function, and reduced secretion

resulting in low levels of plasma anti-protease activity.Studies of the molecular basis of &agr;1-A T deficiency

have progressed to the extent that prenatal diagnosishas been achieved in several families by analysis ofmaterial obtained at amniocentesis or chorionic villus

biopsy. Until 1984, prenatal diagnosis of al-ATdeficiency could be accomplished only by Pi typing offetal blood obtained during the second trimester byfetoscopy,15 a potentially hazardous technique with asignificant risk of pregnancy loss. Then recombinantDNA technology made it possible to developsynthetic oligonucleotide probes specific to the M andZ alleles.14,16 Under suitable conditions these probeswill hybridise to the mutational sequence causing thedeficiency in exon V, which is known to be located in a2-4 kilobase HindIII plus XbaI restriction fragment.Hybridisation is impaired by a single base-pairmismatch between the nineteen residue syntheticprobe and the &agr;1-A T allele, so that DNA from anindividual with an MM genotype will not hybridise tothe Z oligonucleotide probe and vice versa.

Oligonucleotide analysis of fetal DNA obtained atamniocentesis or chorionic villus biopsy can be carriedout in three to five days and will identify the fetalgenotype if parental samples are available for

comparison. Comparative samples are necessarybecause any clinically silent mutation in the shortsequence of DNA under study would almost certainlyresult in a mismatch, thereby giving a spurious result.This method of analysis requires a high level of skill.Other potential disadvantages of this approach arethat it will detect only point mutations, and largequantities of DNA, of the order of 10 µg, are needed.An alternative molecular approach, which avoids

many of these difficulties, involves the use ofrestriction fragment length polymorphisms (RFLPs),a technique now well-established for other single-gene disorders including Huntington’s chorea, cysticfibrosis, and phenylketonuria. Several RFLPs havebeen identified in the &agr;1-A T gene and its flankingsequencesl’ and these have been utilised for prenatal

13. Cox DW, Markovic VD, Teshima IE. Genes for immunoglobulin heavy chains andfor &agr;1-antitrypsin are localised to specific regions of chromosome 14q. Nature 1982,297: 428-30.

14. Kidd VJ, Wallace RB, Itakura K, Woo SLC. &agr;1-antitrypsin deficiency detection bydirect analysis of the mutation in the gene. Nature 1983; 304: 230-34

15. Jeppsson JO, Cordesius E, Gustavii B, et al. Prenatal diagnosis of alpha 1-antitrypsindeficiency by analysis of fetal blood obtained at fetoscopy. Pediatr Res 1981; 15:254-56

16. Kidd VJ, Golbus MS, Wallace RB, Itakura K, Woo SLC. Prenatal diagnosis of&agr;1-antitrypsin deficiency by direct analysis of the mutation site in the gene. N Engl JMed 1984; 310: 639-42.

17 Cox DW, Woo SLC, Mansfield T. DNA restriction fragments associated with&agr;1-antitrypsin indicate a single origin for deficiency allele PiZ Nature 1985, 316:79-81.

422

diagnosis.18-20 Of particular benefit is the stronglinkage disequilibrium that exists between the Z alleleand a unique haplotype identified by means of AvaIldigests and a 6-5 kilobase &agr;1-AT probe extendingfrom exon II to the 3’ flanking region of the gene. Forthe uninitiated this means that, for reasons unknownbut almost certainly of intriguing evolutionarysignificance, a unique Southern blot band pattern,designated 371, is seen consistently and exclusivelywhen DNA from PiZZ individuals is hybridised withthe 6-5 kilobase probe after digestion by the AvaIlrestriction enzyme.Cox and Mansfield20 in Toronto have now

reaffirmed that all 32 PiZZ patients studied showedthis unique 371 haplotype, which was not present inany of the 32 controls. This being so, it should now bepossible to offer direct prenatal diagnosis of PiZZ&agr;1-AT deficiency by analysis of fetal DNA withoutrecourse to DNA from parents or siblings. Wisely, theCanadian authors urge caution since it may emergethat the 371 haplotype can occur with non-Z alleles orthat the Z allele may be associated with other

haplotypes. At present, therefore, parents and/orsiblings should always be studied. Other DNA

polymorphisims have been identified, including onewith the MaeIII enzyme,21 which promises to be ofparticular value in chorionic villus biopsy diagnosis, inwhich meticulous attention must be paid to separationof the fragments. Other extremely rare deficiencyalleles such as Mmalton and a null allele have been

described,22 so that accurate Pi typing of parentalsamples is essential before prenatal diagnosis is

attempted. The situation is further complicated by theexistence of an S allele which could be misinterpretedif only the M and Z oligonucleotide probes were used.Thus thorough clinical, biochemical, and molecularevaluation is essential for every family presenting forprenatal diagnosis.Many may ponder on the ethics of prenatal

diagnosis of &agr;1-AT deficiency, since only a smallproportion of deficient individuals encounters serioushealth problems in early life, and the pulmonarydisease of middle age can be modified byenvironmental factors, chiefly avoidance of cigarettesmoking. Furthermore, treatment with synthetic orblood-derived &agr;1-AT is on the horizon.23 A largeprospective population survey conducted in Sweden,where the PiZZ incidence of approximately 1 in 1500is at its highest, indicated that 14 of 120 PiZZ infants

18. Cox DW, Mansfield T Prenatal diagnosis for alpha1-antitrypsin deficiency. Lancet1985; i. 230

19. Hejtmancik JF, Sifers RN, Ward PA, Hams S, Mansfield T, Cox DW. Prenataldiagnosis of &agr;1-antitrypsin deficiency by restriction fragment lengthpolymorphisms, and comparison with oligonucleotide probe analysis. Lancet 1986;ii: 767-70.

20. Cox DW, Mansfield T. Prenatal diagnosis of &agr;1-antitrypsin deficiency and estimates offetal nsk for disease. J Med Genet 1987, 24: 52-59.

21. Cox DW, Billingsley GD. Restriction enzyme MaeIII for prenatal diagnosis of&agr;1-antitrypsin deficiency Lancet 1986; ii: 741

22. Garver RI, Momex JF, Nukiwa T, et al. Alpha1-antitrypsin deficiency andemphysema caused by homozygous inheritance of non-expressing alpha1-antitrypsin genes. N Engl J Med 1986, 314: 762-66.

23. Editorial. Treatment for &agr;1-antitrypsin deficiency Lancet 1985; ii: 812-13.

had prolonged neonatal jaundice,24 but by the age offour years only 4 of these children had clinicalevidence of liver disease.25 This low risk of severe

hepatic involvement in early childhood would scarcelyjustify termination of pregnancy. However liverdisease in PiZZ children shows a strongly positiveintrafamilial corelation, so that if one sibling isseverely affected the risk to subsequent PiZZ siblingsmay be as high as 78 %,26 although the Canadiansurvey suggests that a figure of 40 % is more

appropriate.20 Prenatal diagnosis for these familieswould seem entirely justifiable.There is still much to learn about &agr;1-AT deficiency,

Why, for example, do only a few PiZZ homozygotesget serious hepatic problems and why do these tend torecur in siblings? Did the Z allele originate in a singlenorthern European individual and spread throughoutthe Caucasian population as the result of heterozygoteadvantage (or Viking marauders)? The answers tothese and many other equally intriguing questionsare eagerly awaited. Meanwhile, thanks to thecollaborative efforts of molecular scientists in North

America, parents who have had one severely affectedchild now have the option of reliable first trimesterprenatal diagnosis.

Epilepsy—La Belle Indifférence?EPILEPSY is a common condition with a remarkably

uniform prevalence in Western countries of 1 in 200individuals;1 2-5 % of the population have a fit at sometime. It represents the clinical expression of a largenumber of cerebral disorders affecting all age groups,2Specialist investigation is required and diagnosticfacilities include various electroencephalographictechniques and access to an expanding range ofcomplex (and expensive) scanning procedures. Man-agement consists primarily of anticonvulsant drugs,which are often given life-long. The realisation thatanticonvulsant polypharmacy confers no benefit overappropriate monotherapy4 has led to increasingreliance on therapeutic drug monitoring to tailor drugdosage for the individual patient. 5

Whilst the majority of patients can be renderedseizure-free by pharmacological means, chronicintractable epilepsy develops in a sizeable minority(approximately 20%6), particularly in those with an

24. Sveger T. Liver disease m alpha1-antitrypsin deficiency detected by screening of200 000 infants. N Engl J Med 1976; 294: 1316-21.

25. Sveger T, Thelin T. Four-year old children with &agr;1-antitrypsin deficiency Acta

Paediatr Scand 1981; 70: 171-77.26. Psacharopoulos HT, Mowat AP, Cook PJL, Carlile PA, Portmann B, Rodeck CH

Outcome of liver disease associated with &agr;1-antitrypsin deficiency (PiZ) Arch DisChild 1983; 58: 882-87.

1. Zielinsky JJ. Epidemiology. In: Laidlaw J, Richens A, eds. Textbook of epilepsy. Drugs

ed. Edinburgh: Churchill Livmgstone, 1982: 16-33.2. Delgaco-Escueto AV, Treiman DM, Walsh GD. The treatable epilepsies N Engl J

Med 1983; 308: 1508-13.3. Davidson DLW. Anticonvulsant drugs. Br Med J 1983; 286: 2043-45.4. Reynolds EH, Shorvon SD. Single drug or combination therapy for epilepsy. Drugs

1981; 21: 374-82.5. Brodie MJ. Therapeunc drug monitoring. Practitioner. 1986; 230: 1003-096. Reynolds EH, Elwes RDC, Shorvon SD. Why does epilepsy become intractable?

Lancet 1983; ii: 952-54.