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oN~r~ Photomz~...I,hm No. : Tot:a2 Pan,,: GROXPP I~AZG~- ISSUER: DI~ON- /<,-I) K , S.TG (I) : I~.'1((I) : ~ (2) : :LMG (1) : I~.S~,T (3) : :~nl:r., I'.. a D.D. ausc. FS (I) : AJK (1) : CJP d., S (I) : DGF (i) : Library (!) :FiLc (Z) : ~S (i) (~) (4) (~) Prepaz~ mastic: ~./ ~lllI2e: ~,~-?., / "P-~"( ];)~t eI ];~,t e Sere% ! Rec~. ,tC~.G~Co ~O AuCho= I ! O.Co. ~'( CheclmLng n-t:e Zsmze~ ' V"/);. / n-¢e 2. 4- ~e ~fl~:~e~]possible. d~-u-~8 £o~ ~J~:L~4r should be v~t~en :L~ :LuM. ~I1 8ubseq~e~ ob~Li~ the ~p IAm~tr'J m~ IiIme'! tp~. CAz~zA~'~Aon shou/.4 be d,~'¢~c~t,n~ 1~ "~he Xss'a~z. Aftm_~ l~:L~tx~, one bout1 co1~ muJrC be re~A-~ed ~o ~J~e ~8mAe: ~c~ fina~ &~ an4 ~he Xss~e: wall e=m~e ~ o~e cow As me-- t~ +.he P/anage:. -.A' J BAT Co LTD - MINNESOTA TOBACCO LITIGATION

AI"TE~'!'I~ ION m This work was produced on tha Hagnetic Card typewr[tex. It is £mperaLive l~haL ~111 correcLions, alterations, etc., are mndc cZ~arly. E.G. pcnci.11cd co,,:,,.~s, £ull-stop~ could be cnsily missed. A cross in thc margin, in linc with Lhe cor~ection would drn~ ou~ attention to the i,as,_.r~ion. Nould you plc,lsc be Lind c2~r.ugh to wC.~.L- 1..~rr.c in.scrLio|is on ~I sepnr~,i.c s:heer of pnpe..r_r, and ,_~t: c-t ot~ portionc o£ typed worl:. Th~s i~ becat,se Lhc .~l:l~;:~ct~ic C~irds d].f£t:A" £rom Pnp,~r Tnp.~s. rx~ C~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION -...?

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A REVIEW OF THE GENETZCS AND CONSEQUENCES OF a |-ANTITR2?S IN DEFICIENCY REPORT NO. ED. 1394 UNCLASSIFIED 27.7.1976 AUTHOR: R.R. Baker ISSUED BY: K.D. Kilburn DZSTEIBUTZON: Dr. S.J. Green Dr. I.W. HuKhes Dr. R.A. Sanford R.M. Gibb, Esq. R.S. Wade, Esq. E.G. Nicholls, Esq. HeZT R. Sottorf Dr. ¥. Seehofer A.J. Krusz'ynski, Esq. Dr. C.J.P. de S£queira Dr. D.G. Felton Library Copy No. 1 |! t! 2 " " 3, 4 " " 6, 7, 8 " " 9, 10 " " 11 " " 12 " " 13 " " 14 " " 15 " " 16, 17 COPY NO: BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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SECTZON CONTENTS PAGE SUM~UY~Y INTRODUCTION i. Types of Emphysema 2. al-antitrypsin 3. Genetic Transmission of al-antiCrypsin defic~en=y 3. I Early Studies 3.2 Electrophoresls of Human Serum 3.3 The Pi Sysnem, and its GeneClcs 4. al-antitrypsin Deficiency, Pulmonary Emphysema, and CiEareCte Smok/nK 5. al-antitrypein Deficiency and Diseases of the Liver REFERENCES TABLE FIGURE 1 2 2 4 6 6 7 9 ].5 19 22 28 Z9 BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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~I~146D Group Research & Development Centre, Brlc£sh-American Tobacco Co. Ltd., SOUTHAMPTON. 27th July, 1976 A REVT'~W OF THE GENETICS AND CONSE(~UENCES OF o~-ANTITEYPSIN DEFICIENCY (Report No. RD.I394 Unclassified} SU~RY The susceptibility Co pulmonary emphysema can be inherited. Thus certain people are pre-d£sposed to emphysema, and for such subjects cisaretCe smoking couId be an ~mportanc factor in promoting the coudition. During the last twelve years many scud£es have shown Chic • de~Lcimzcy in the blood serum levml of the enzyme al-antitrypslu is s~rongly associated with the occurrence of e~physmma, and ~t is this deficiency which is inherited. The genetic transmissiou of al-antitrypsin deficiency Ls more complex than simple Mendelian genetics, and results in a range of observed levels and types of al-anCitrypsin, which d~ffez in different ethnic &~oups. The present report rev£ews the available literature on =l-ancitrypsin deficiency, including its relationship to cigarette smoking. 0 BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-2- INTEODUCTION The occurrence of emphysema of the lung has been associated with cigarette smoking (I, 2), although there is evidence that the susceptibility to emphysema is inherited (1). During about: the last twelve years stronE evidence has accumulated that a deficiency in the blood serum level of the enzyme al-antltrypsin (el-AT) pre-disposes people to emphysema, and thac it is this deficiency which can be inherited. Consequently, for individuals wiuh such a pre-d£sposition, smoking could be hazardous (2). It is obviously, in the interests of the tobacco industry to • ppreclate the large vol~me of research that is belnE carried out on of-AT, on the consequences of its deficiency, and on the genetics of its transm/ssion. In the lest two years, for example, at least 59 papers have been published on =I-AT. The present report reviews the available llt•rature on =l-AT. 1. Types of Emph_ysema Zn emphysema the lungs have lost much of chelr recoil capacity, and the force available to expel air ouc of the lungs is reduced (3). In addition, the airways inside the lung, having lost the support of the lung tissue, are narrower than normal at any given lung volume and offer a greater resistance to air flow. The volume of the lungs is often greatly increased, and a greater volume of air remains ~ them which cannot be exhaled. Emphysema is caused by desuructlon of alveolar tissue, which leads to emphysematous lungs having much enlarged air cavities, fewer alveolar walls than normal, within which the network • of capillary blood vessels in which the exchange of respiratory gases occurs is reduced. Emphysama can affect both Lungs, or part of • lung, BAT Co LTD - MINNESOTA TOBACCO LITIGATION r",o

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-3- or some regions of both lungs. The affected lungs of can do not collapse, as fs usuaL, when the thorax is opened during autopsy, 7et the lunS itself is not stiff but soft. The air does not come ouC of the lungs for reasons which are not fully understood. There are two main types of emphysema: centrilobular (or cencrlaclnar) emphysema, involving the central portion of the secondary lobule of the lung*, and panlobular (or panacinar) emphysema, involving all lobules of the lung. However, medical opinion is divided as to whether these two types are distinct diseases which affect different parts of individual lung lobules, or whether the only difference is the lobule affected, with no differences in onset, nature and duration of symptoms etc. (A). Alternative classifications of emphysema on the basis of the distribution and form of the enlargements ~rLChin the lung have also been proposed (5). External factors such as exposure Co coal and other dusts, and other pollutants, including those present in ciKareCte smoke, are considered by ~hacchinson (2), for example, co be important factors in the development of both types of emphysema. There ia also an apparently distinct clinical Kroup o£ people with emphysema who have a deficiency of el-AT in their blood serum, their el-AT levels being 10-20Z of that of the majority of the population (2). Furthermore, in these patients there is a strong tendency for the disease to have occurred in other members of the family, and for the lower zones of the lungs co be the most severely affected, and the main lesions generally have the features of panlobular emphysema. In emphysematous subjects wlCh normal el-AT levels, the lesions are found in the upper zones of the lunK in over half the cases. Wlth el-AT *The riKht funk consists of three lobes, and the left lung consists of two. BAT Co LTD - MINNESOTA TOBACCO LITIGATION "-.a

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-4- deficient patients, the onset of emphysema is usually between aaes 30 and 40 years) compared to beL~een 60 and 70 years for patients ~h normal el-AT levels (6). The course of deterioration of the lung function is usually more rapid than in patients with normal el-AT levels. Apart from these d~fferences) the broad similarities between the two groups of panlents sugaest that the biochemical and patholoSlcal processes which are responsible for emphysema have muuh in common, whether =l-AT is presenu in normal quantities or non. There is also a predominance of males who develop e.~physela) both with deficient and normal uI-AT levels. 2. a 1-anti~r~sin Proteases (i.e. protein-degrading enzymes) play an important part in various biochemical processes. Cernain proteins can inhibit pronease activity by combinin~ with the enzyme and inactivating it. The capacity of human blood serum to inhibit the enzymatic activiey of sc~e proteases has been known for almost eighty years (6). About 9Or of the serum's antlprotease activity derives from a &lycoprotein which forms most of the blood's =I sl°bulin" This substance is called a1-antitrypsin because its activity is usually quantified by measurement of its capacity to inhibit trypsin. Much of the remainder of the serum's inhibitory activity is due to a2-macroalobulin. Both this and at-AT ere active aaainst a number of proteases, includin8 trypsin) chymotrypsin) and elas~ase (2, 7). The reactions with trypsin and =hymotryps£n are apparently stoichic~etric (7). =I-&T is produced by the liver (8) and found in blood, semen, cervical mucus and other body fluids (9). It is a Elycoprorein with a carbohydrate portion of about 12~ containing galactoae, mannose, aceeyl BAT Co LTD - MINNESOTA TOBACCO LITIGATION rka -,,,,,/

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-5- hexosau~ne, sialic acid and fructose (6). The amino acid composition shows • high content of •8p•rtic acid (9.75X), glutamic acid (12.9Z) and leuclne (9.9Z). Cysteine is noc found, suggesting that at-AT does not contain disulphide bridges. Values for th• molecular v•LKht of =l-AT of between A5,000 and 54,000 have been quoted (7, 10-13). Xt has b••n isolated in pure form from human serum (13), and its physical properties suggest chat iC has a sinai• polypeptide chain. The amino acid sequence for the N-termL~al eight residues is: Glutamic acid ! "Aspartic acid I Pro i ine I Glutamine l Glycine l Asparagine + Aspartic acid [ Alanine I Al•r~ne =l-AT readily forms polymers and higher 8g@regates when exposed to denatur~K •gents. Its half-llfe in both normal and QI-AT deficient subjects has been estimated to b• approximately four to six days (1A, 15). A con~an£ent and widely used method for the determinatlon of the concentration of =I-AT in serum in the pr•senc• of other £nhibitors is radial /~n~nod~fusion (16). In this mJehod, • specific antistrum is incorporated /nto an as•rose Kel, samples axe appl£ed in wells, and the area of the circular inmunoprecipi~aut is proportional to the concentration of antisen present. With this method the concentration of •l-AT in BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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~6m _I normal serum lies between 2.0 and 2.5 g £ (6, 15, 17) and tbls range satisfactorily accounts for the observed tTypsin £nhib£tion acCiv£L'y of serum if the reaction between trypsin and el-AT is stolchlometric (18). About 85-95X of people have this level of =l-AT, the exact proportion depending on the racial origin of the population (I9). The remainder of the population have various lower levels of =I-AT. The =l-AT concentration in blood can also be elevated by up to 5OZ under some condi=ions, e.g. during pregnancy and £n women .king oral contraceptives (6). 3. Genetic Transmission of el-AT Deficiency 3.1 Early Studies Tt was first reported in 1963 that some individuals have very low levels of =l-AT in their serum, approximately 10-20Z of normal (20). Subsequent studies suggested that the deficiency is inherited (21-24). All the available studies showed that when one parent has a low level and the other a normal level of =I-AT, all nhelr children have intermed£ate concentrations. Lf one parent has an intermediate and the other a normal concentration, approximately half ~he children have a normal concentration, and the other half have an intermediate concentration. This r.annot be explained by Mendelian genetics with one recessive and one dominant gate, since only normal individuals (containing either t~o dominant genes, or one dominant and one recessive gate) and deficient individuals (containin8 two recessive Series) would be observed. It was postulated that there were t~o codominant Kenes, with each sane resulting in =l-AT appearing in serum independently of the other. One sane contrlbutee about 50Z of the total normal el-AT concentration, and the mmmm~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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other about 1OZ. The genetic transmission is then in simple Mendelian fashion. Thus individuals deficient in =l-AT have t~0o genes, each giving 10~ or a total of 20~ of the normal el-AT concentration. Normal individuals have two genes each concrlbuting 5OZ to glve a total of lOOK of the normal =l-AT level. Individuals with intermediate levels of =I-AT are heterozygotes for the gene that gives 5OZ and the Eene that gives lot, Eiving them a tocai level of 6OZ of the normal =~-AT concentration. Thus the theory of codoe~nant inheritance offered an explanation of the observe<l Or/modal distribution of =I-AT concentrations in sere ~n families where al-&T dtficiency was found. Within the framework of the above theory of inheritance, the homozyEous deficient subjects can be readily distinEuished by their =I-AT levels. However, overlap in =I-AT concenUration values does occur, and the serum level of =I-AT fluctuates in response to acute or chronic infection, cancer, pregnancy and estrogenic hormones. C=nsequenCly, .heterozygotes cannot always be d£stinEulshed from normal subjects. Further invascigatlons on serum =l-AT has shown the blochemical features of =l-AT levels are more complex, and that the transmission Is more involved than the simple P~endellan inheritance schee outlined above. 3.2 Elec~Tophores£s of Human 5e~ ElecCrophoresis of human serum has shown that several biochemical forms or variants of =I-AT exist, each with its own electrophoret£c mob£1ity. In 1965, Laurel1 (25) reported chat the =I-AT of homozyaotes for the deficiency Eene differed in elacnrophoretlc mobillty from chat of individuals with normal =I-AT levels (h~zyEoces for the normal Edna). It was not clear if these results indicated a structural -..,.,.j r.....;- BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-8- difference in the =x-AT from deficient and normal subjects, or if they were due ~o interaction with some unknown substance. Furthermore, the low resolution of the Lu~unoelectrophoresis technique used did not distinguish two components of •I-AT in the serum of heteroryEotes (with intermediate levels of =l-AT). However, dlsconcinuous starch 8el eleccropboresls of serum aC an acidic pH (4.95) made this dlscinccion possible (26). Under these conditions, the el-AT zone is between that of the orosmucoid and albumin zones, and the aI-&T of most subjects gives rise Co • complex pattern of up to eight zones (27,. 28). There are t~o major bands that are usually recognized after staining the gel with smldo black. Two or more minor bands are also present, but cannot always be seen after this staining procedure (6). In order to define these weaker bands more clearly, and quantify them, antigen-antibody crossed electrophoresis has been used (29, 30), In this technique, after starch Eel electrophoresis a longitudinal scrip of starch is cut out and placed in an •Earose S•I containinS antibody Co el-AT. A second elecCrophoresls is thee performed at right angles to the direction of the first. As the ~I-AT migrates into the gel it prec£pltates with the antibody, resultin$ in peaks wb/ch can be stained for ident£flcation. Such methods have led to the detection of several phenotypes* of =l-AT. In all cases the complex starch gel/antigen-antibody crossed *Bet•use it is not always posslble to tell from the appearance of an orKanismwhat 8aces are present, the followin~ terms are used: phenotype - genocype - a classification of the orlanlsmby ~ts appearance, a classification of the organlsm by the sen• it carr£es, Slv~ng rise to the appearance characteristic. In the present context, "appearance" refers no type of =I-&T present in the subject. BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-9- eleccrophoresis pattern o£ an individual appears to be the sum of two components, one £ro~ each parent. A @enecic model has been proposed, which is described in the next section, together ~r~th the ~nterpretation of the electropherograms. 3.3 .The Pi S~,stem~ and its Genetics For the system of inherited =I-AT phenotypas, the term "?i system" (31) ~s used (Pi standing £or protease inhibitor). The indlv~dual phenotypes can be explained by assuming thaC there are several codonL~nant genes which are all alleles, i.e. they ell occupy the same position (locus) of the chromosome (26, 28, 31-34). The allele products vary in several respects: the electrophoratlc mobility, the distrlbution o£ GI-AT within the eight zones in the electropherogrem, the total el-AT concentration in serum, or a combination oF t~o or three of these characteristics (28). So Far 19 alleles, most oF them vet-y rare, have been identified, vith 190 possible combinations, o£ which only 36 have been recosnised (35, 36). The most common, or normal, allele is called the Fi H allele, it has a medium electrophoretic mobility, and contributes 5OX oF the total normal ~I-&T concentration to the genoese. The Pi Z allele has the slowest elactrophoretic mobility, and contributes 1OZ of the total normal Q1-AT concentration to the 8enotype. The P£ M and Pi Z Series are the "normal" and "deF£c£ent" genes referred Co in Section 3.1 above. The terminology in nm-lng the alleles is based on the electrophoretlc mobility oF Chair allele products. The allele Pi F has Fast mobility, Pi M has medium ~obility, Pi S has slow mobili~y, and ?i Z has the slc~west mobility. The position of the letters in the alphabet indicates BAT Co LTD - MINNESOTA TOBACCO LITIGATION c.j"l _r,,,.. r',,o

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-I0- the relative mobility of the allele product (31). This nomenclature has been aKreed at the First International =l-AT Pi System Workshop, held in 197A (37). A simplified drawing of the major zones in the el-AT acid starch gel electropherogram of some of the Pi phenotypes is shown in Figure I. The relative mobilicies and protein contents of the zones are indicated. The principal of antigen-antlbody electrophoresls is also indicated in Figure 2 for the major =I-AT zones of the Fi MZ phenotype. The mechanism that leads to the multiplicity of bands on acid starch geI and crossed antigen-antibody electrophoresis is unknown. Since the genetic date obtained to date suKKests that =I-AT is determined by a single chromosome locus with multiple alleles, there should be no more than two different allelic types of el-AT in the serum of a given individual. If the various electrophoretic species represent different structural el-AT molecules controlled by different alleles, it would sugKest the existence of several Kenetlc loci. Musianl and Tomasi (13) have pointed out Chat it is possible that several closely linked loci exist in whlch crossing over and recombination events are very rare and have not, therefore, been observed in the genetic analyses so far performed. However, a more straightforward explanation of the multipllcity of the electrophoretic bands Can be put forward. This is silply that the different types of allelic at-AT undergo various asgresation, de-iBidation, or buffer interaction reactions on acid starch eel electrophoresis (15). The structural variations of the various types of allellc aI-AT probably lle in the carbohydrate part of ~he molecules. Although the glycoprotein nature of =I-AT has been confirmed in several studies, no BAT Co LTD - MINNESOTA TOBACCO LITIGATION <3o r',o

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-11- two studies have obtained similar values for the carbohydrate con~ent (7, 45), suggesting that the b~ochemlcal variations lie in this part of the molecule. Furthermore, Bell and Carrell (47) have shown that in dear gel elecnrophoresis at pH 8.6 the mobility of =l-AT from a Pi M~4 individual fell to that characteristic of Pi ZZ =l-AT when the sialic acid content of the =l-AT was partially rlmoved (by tined digestion wLth neursminidase). More significantly, electrophoresls in acid starch gel of the aial£c acid-deflcient Pi ~ el-AT gave an exact reproduction of the Pi ZZ pattern both in mobility and proportion of protein in the peaks. The different appearances of Pi I~4 and Pi ZZ uI-AT thus apparently have their origin in the partial loss of sialic acid in the Pi ZZ el-AT. The mechanism that leads to a lower concentration of serum =I-AT in genetically deficient individuals is unknown. The most likely explanation is that the structural mutation that leads to the different electrophoretic mobilities of the different genotypes also leads to different synthetic rates of =I-AT (6, 15). However, the possibility of different elimination rates of the structurally different =I-AT molecules from the different genotypas has not been excluded, although it has been shown that the half-life time of radioactively labelled el-AT in the circulating blood is about 4-6 days in Pi MM, MZ, and ZZ phenotypes (14, 48). The response of individuals who differ in their =I--AT phenotype to an injection of typhoid vaccine supports the hypothesis that. the Pi Z Eene governs the synthesis of =~-AT at a slower rate than the Pi M sane. Serum =l-AT has been measured for about three weeks in several people following an injection of typhoid r~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-12- vaccine. Pi IdH and Pi HZ phenotypes both temporarily double their initial el-AT levels within 3 or 4 days, while Pi ZZ phenocypes show virtually no thanes in their el-AT concentration (49). The Pi Z allele is the "deficiency" sane referred to in Section 3.1 above, contributing about IOZ of the total normal (Pi ~) el-AT level to the genotype. It has been detected in the followinK combinm~ions: FZ, IZ, MZ, SZ, end XZ (28), all of which produce phenotypes with ~ow =l-AT levels. In addition the Pi P, Pi S, and Pi W alleles are also associated wlnh lower el-AT levels (19, 35), contributing about 30~ of the no~al normal (Pi MM) =I-AT level to the genotype. The approximate at-AT concentration, expressed as a percentage of Ehe concentration for the Pi P24 Kenotype (2.12 ± 0.32 g £-I), associated with various geno~ypes (2, 6, 15, 27) is: Genot7pe MS SS MZ SZ ZZ a I-AT Concentration Eelative to that for Pi MM i00 75-85 52-63 50-60 30-40 10-20 The distributions of Pi phanotypes in various population samples of unrelated individuals are shown in Table 1. The population studies BAT Co LTD - MINNF_....qOTA TOBACCO LITIGATION r',,,,,o

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-13- listed have been selected for large numbers of subjects and/or h~gh frequency of variants. The frequencles of Pi phenoCypes observed in the studies in references 38, A1 and A2 are compat£ble with those predicted by a Hardy-Welnberg equilibrium, givlng support to the theory of codouL~nant inheritance of the Pi types (28). Further support for the cheor7 is obtained from studies on a large number of families with a high number of children, where it was found that the distr£bution of P£ phenocypes in such children was close to that expected for codominant inheritance (28). Further information has also omerEed from population and family studies (28). The frequencies of variants are about the same for both sexes, and several variants have been transmitted from father Co son. These facts exclude the possibilities of X- and Y- linked Pi genes. Thus moat people (75-2OOZ) are Pi }~/ (Table I). The Pi M allele appears to be the "normal" allele in eli tested populations, and is probably the orig£nal allele from which all ochers have been derived by mutation (19). The differins number of variants found in the various population samples is remarkable. Althouzh the size of the samples are not the same for all populations and often relac£vely small, there appears to be a tendency for Central European populations Co possess a larzer number of Pi phano~ypas than Chose from Asia, Africa, and the Sub-Arctic area. The 8ene frequencies of the Pi alleles can be determ~ed from the data in Table I. The Pi M a11ale has a frequency between 0.86 and 0.99 and is always the most common. In the study where 2830 Norwesian subjects were screened (38), the frequencies of the Pi S, P£ Z, and BAT Co LTD - MINNESOTA TOBACCO LITIGATION C~o CO

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-14- P£ ~ alleles were 0.023, 0.016 and 0.013 respectively. It is interesting that in the group of Spaniards studies (&l) the Pi S frequency was very high at 0.112, whereas the frequencies of the other alleles were similar to those in the Norwegian group. Among Lapps and Finns the Pi S allele is almost absent, and in the Monsoloid, Korean end Indian populations (Table 1) the Pi S allele was not found at all. The available dana is insufficient to speculane whether these dlfferences in Eerie frequencies in the various populations are due no selecnion or drift. More recently, evidence has accumulaned for the existence of a Eerie which produces no el-AT at a11. In 1973 Talamo et al. (43) reported the case of a 24 year old man in whose serum no Ul-AT could be detected us~ng several analytical methods, and in which no =l-aT bands were found by acid starch gel and crossed antlgen-antlbody electrophoresis. ~t was postulated that the man was a homozygote for a null gene that produced no ~I-AT, i.e. his genotype was Pi --. Studies of the man's family indicated that both his parents, his matel-n~l grandmother, two of his three sisters, and his two children had intermediate concentrations of uI-AT in their sere which had electrophoresis mobilities similar to that of Pi MH phenotypes, i.e. they were probably all Eenotype Pi M-. Further evidence has also been reported for the existence of Eenotypes Pi M-, Pi Z-, and Pi S- (40, 44-46). Because of the difficulty ~n ~nferr~ng the presence of the Pi - gene, it has been agreed internationally (37) r/mr individuals will only be called phenotype Pi ~ only if there is evidence from a famLily study of homozy$osity; the nomenclature for suspected homozygous phenotypes when the presence of the Pi - allele has not been ruled out ~rlll be Pi M. BAT Co LTD - MINNESOTA TOBACCO LITIGATION r'xo ',,.o

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-15- 4. =I-AT DeficlencTp Pulmonary Emphysemae and Cilarette Smoking The most striking finding in Pi ZZ phenotypes, whose serum of-AT concentration is only 10-20Z that of the normal Pi MM phenotype, is their high susceptibility to develop emphysema, at least fifteen times higher than the general population (e.g. 2, 3, 6, 15, 21, 22 and 45). Presumably the other phenoeypes that have such low levels of •I-AT •re •leo particularly susceptible to emphysema, but the numbers detected are very small: the one recorded case of • Pi -- phenotype and no aI-AT had severe emphysema at 24 years old (43), the one recorded case of a Pi Z- pbenotype with 5Z of the normal =l-AT level did not have emphysema at 41 years of age (40). Whether phenotypes with intermediate levels of at-AT (in particular the heterozygotes Pi MZ and MS, who together make up to about IOZ of a normal population) are also more liable to emphysema r~ins extremely controversial. Several studies have concluded that heterozygote Pi phenotypes MZ and MS are more susceptible to emphysema than the common Pi M phenotypes (34, 50-56), while other studies have concluded that they are not (22, 24, 40, 57-61). It has 81so been suggested that heterozygous Pi MZ and MS pbenotypes are mainly associated with type B chronic obstructive pulmonary disease (primarily bronchitic), whereas homozygote Pi ZZ phenotypes ere largely associated with type & chromic obstructive pulmonary diseases (primarily emphyse~atous) (62). Huch of the erguaant lies in the lack of • universally agreed diagnostlc crlteria for emphysema (2), in differences in the general desizn of ~he studies, and in differences in subject and/or patient selection. The debate will undoubtedly continue, but it is probably reasonable to m BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-16- conclude that heterozygous Pi phenotypes HZ and HS are not partlcularly prone to severe emphysema. However, it has been claLmed chat heterozygoces vho are also exposed to an externaI cause of emphysema, e.g. those who smoke or work in certain polluted environments, could be more susceptible Co emphysema than the col~on Pi 14 phe~otype (2, 94). It is hOE specifically known how el-aT deficiency predisposes an individual to mnphysema, although evidence for a generalised mechanism has accumulated in recent years. Clearly not all =l-AT deficient individuals acquire emphysema, nor is the disease present initially but develops during the lifet~ne of the person. In blood, the leucocytes release leucoproteases to protect the body against invading foreiKn materLal by degrading the foreign material. These power£ul enzymes can also act on the tissues of the body, and the ant£protease activity of serum protects the tissues by inactivating excess leucoproteases. Since about 90Z o£ the blood serum's antiprotease activity is due to =I-AT, which does directly ~thibic the action of leucoproteases (63), it has been suKsested (2Z, 63) that the release of laucoproteases could be responsible for the development of pulmonary emphysema in =I-AT deficient subjects. Zn indlviduals with normal of-AT levels, the proteases that leak into the in~erstlt/~1 spaces of the lun~ are rap£dty inactivated by the el-ATtend proteolysls of the alveolar tissue is prevented. When there is a de£1ciency in the level of ~I-AT" the available inhibltory activity is easily overwhelmed, and digestion of the lung tissue occurs with the pathological appearance of pulmonary emphysema. The leucoproteases are produced in the lyososo~al granules of the leucocytes, and consist of a potent elsstase* (64) and collagenase (65). It has been demonstrated • rThe lysosomal elastase has different properties from porcine pazzcreatlc elastase - lysosomal elastase is less susceptible to the action of serum inhibitor, and is more active at pH 7.0, than the latter. m~b ~J %O BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-17- that lysosomal extracts can damage the vascular basement membrane (66), which is significant since lesions in the pulmonary capillaries are one of the early features of emphysema (57, 67, 68). Furthermore, since the blood flow per unit voIuma of lung tissue is considerably greater in the lower zones of the luns than in the upper zones, the release of leucoproteases in a -AT deficient individuals could account for the predominance of the 1 disease in the lower zones of the lung in these individuals (57). The alveolar macrophages play a major part in the defence of the lung against inhaled toxic agents, by releasing lysosomal enzymes. A~ain, as in the case of the leucoproteases, the macrophage proteases can damage the lung itself. Extracts from the macrophage cells also contain an elastolytic enzyme which has only about IOZ of the activity of leucocyte elastase (69), but is much less zasdily inhibited by a]-A~ and a2-macroglobulin than is the leucocyte elaJtase (70). It has been suggested (70) that the macrophage elastase ~ay play a part /~ the degradation of lung elascin in emphysema, particularly in cases with normal al-~T levels. Emphysema has been produced artificially in dogs by exposing them to an aerosol containing leucocyte elastase (71), in mice by exposure to human alveolar macrophage extract (72), end in other an/mals by £nCratracheal appl£catlon of the enzyme pepsin* (73). Thus, although the production of emphysema in the animals required relatively large doses of the enzymes, there is direct evidence for the causal relation between the enzyme and emphysema (71-73). *This enzyme is not found in human serum, and has no elastolytic properties. r~ ~D c~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-18- Woodcock et el. (74) have published data suggesting that (:he level of el-AT in arterial blood is lower then that in venous blood in subjects suffering from chronic airways obstruction and pulmonary infections. They suggest that the immunological property of =l-A~ is altered as it passes through the lungs, perhaps by attachment of the el-AT to proteases, thereby causing an apparent venoarterial difference in concentration. However, their findings have been contradicted by ~acer studies (75, 76), where no significant venoarnerlal difference in el-AT levels or serum trypsin inhibitory c~apacity was observed in most patients with chronic airways obsuructlon. It is non possible no assess the significance of these controversial findings to the mechanism of emphysema. Exposure to certain substances hastens the onset of emphysema. Ni~osan dioxide is believed to be such a substance, and it has b~n shown chat nitrogen dioxide promotes the release of proteases wiChln the lung (e.g. 77). Chronic industrial cadmium poison£ns is known to be associated with imphyslma (2). Cadmium ions produce • substantial reduction in the oxygen uptake of alveolar macrophales and the inhibition of some enzyme systems (78), and reduce the concentration and trypsin inhlbitory capacity of =l-AT when added to blood plasma in vitro (79). Both nitrogen d£oxide and cadmium (80) are present in cigarette smoke. Cigarette smoke produces marked changes in the alveolar macrophages of both human and animal lungs. ~n cans, the degradaclve capaclcy of free living macrophages ob~alned from excised lungs to various lysosomal enzymes increased markedly after only four days exposure of the rats to cigarette smoke (81). A comparison of the alveolar macrophages obtained BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢...m ',,O

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-19- £rom healthy smokers and non-smokers by pulmonar7 lavage (82-84) has shown that smokers have many more macrophages than uon-smokerss and the smokers' macrophages had a greater size and number of lysosomal bodies than those o£ the non-smokers. Thus the results of the above studies suKKest that ciKarette smoke produces elutnges which are believed to precede emphysema, and that it contains substances which are independently associated with emphysm-=. In fact, patients with emphysema vho have never smoked are rare, and in Pi phenotypes ZZ the available evidence suKKests that smoklns hastens the onset of the disease (2). Tt ~uld appear thac smokln8 &cts as a "trigger" ~or emphysema, parc£cularly in Yi ZZ phenotypes vho are pre-disposed to the disease. 5. a~-A~ De£icienc7 and Diseases of the Liver T, 1969, the qu£Ce u~expecced observaC£on yes reported o£ a n~er o£ cases o£ el-AT def£c4ency among • SToup o£ ch£1dren ~r~Ch e£rrhosis of the liver (85). TI~s £indi~K hms been authenticated in ocher studies, £rom vh£ch ks is apparent chat Pi ZZ phenotTpes are pce-dlsposed Co neonatal hepaCltls end ci~rhosls o£ the liver in childhood (36, 86-88), as yell as llver disease in adult llfe, both cirrhosls and hepatoma (36, 86). As with the incidence o£ emphysema of the lunK, it is not clear whether hetarozygotes ~-~th intermediate levels of al-AT (Pi HZ and HS) have am iJ~creased pre-dlspositlon to neonatal hepatitis and ci.Trhosis relsCive Co people ~th noxlnal el-AT levels: some studies report that they do (89, 90), others that they do not (87, 88). An important observation Chat has emerged in the last four yeats Is that the livers of vlrcually &11 indlvlduals carrying the Pi Z gene <~ r~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-20- have a detectable m~croscopic abnormaZity - the presence of deposits within parenchymal liver cells (15, 36, 86, 88). These are observed in individuals wlUh or without liver disease, and in several phenotypes containing the Fi Z lena, but never in phenotypes without the Pi Z gene. These deposits are globules wlnhin the cynoplesm of a variable number of hepanocynes, roughly spherical or oval in shape and 0.5-40 wm in diameter. There is ample evidence from seudles reviewed in references 36 and 86 that the slobules are almost cer~alnly accumulations of =I-AT - i.e. that the material in the globules represents the missing serum = I-AT. It is not known why these inclusions occur. ~r may be that ~I--AT synthesised under nhe control of the Pi Z gene can only be released from the liver cell at a reduced rate because of its structural characteristics. Alternatlvely, a complex of Pi Z el-AT with a protease is formed bun cannot leave the rough endoplasmic reticulum (an intracellular structure believed no be • site of synthesis of the c~rbohydrate portion of glycoprotelns). The synthesis of a slycoprotein like =I-AT includes two steps: synthesis of the polypeptide chain followed by addition of carbohydrates to the chain (91). Since =I-AT from Pi ZZ phenotypes has less sialic acid than Ul-AT from Pi phenotypes (47), it would appear that, in the presence of the Pi Z Kene, carbohydrates are defectlvely added to the polypeptide chain £n the synthesis of aI-ATo Modification of the carbohydrate moiety could lead to the intracellular accumulation of Q1-AT; a small diffusion of el-AT out of the cell would lead to a low concentrat£on of the protein in the serum. BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-21- There is no obvious relaclonshlp between accunnalacion of aI-AT within the hepatocytes and hepatic damage. Moreover, it is not understood vhy the decreased aI-AT lerum concentration may lead to llver disease, although Gans (92) has suggested a mechanism sim/lar to ~he emphysema mechanism, viz proteases from leucocytes o~ Kupffer =ells in the liver could be responsible for hepatocellular ~mmge when the seru~ =I-&T concanuration is low. Others have proposed chat an aggressive agent, such as uhe hepanltls-B (Australia) antigen could be responsible for cellular damage in hepatocytes "sensitised" by the serum =l-AT deficiency (g3). The tanrer hyponhesis has little support, since the hepatitis-B annigen has not been found in Ehe serum of aI-~T deficient panients with neonatal hepatitis (86, 88). BAT Co LTD - MINNESOTA TOBACCO LITIGATION O

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-22- REFZ~CES 1. Royal College o£ Physicians Report "Smoking and Health Now", Chapter 5, P£~nan Medical and Scientific Publishlng Co. Ltd., London, 1971. 2. D.C.S. Hutchinson, Brit. J. Dis. Chest, 1973, 67, 171. 3. F. Prleto, New Scientist, 1975, 69, 504. British Medical Journal, LeadinE Article, 15 June 1974, No. 5919, 571. 5. A.G. Heppleston, British Medical Journal, 27 July 1974, 253. 6. F. Kueppers, Humangenenik, 1971, Ii, 177. 7. A. R/mon, Y. Shamash, and B. Shapiro, J. blol. Chem., 1966, 261, 5102. 8. H.E. Schultze and J.¥. Heremans, "Molecular BioloEy of Human Serum Proteins", Vol. 1, p. 365, Elsevler, Amsterdam, 1966. 9. R.M. Fineman, K.K. Kidd, A.M. Johnson, and W.R. BreE, Nature, 1976, 260, 320. I0. H.F. Bundy and J.W. Mehl, J. b~ol. Chin., 1959, 236, 1124. 11. M. SchSnenberser, Z. Na~urforicho, 1955, IO___~b, 474. 12. Y. Shlmlsh end A. Riwon, Biochim. biophys. Acts, 1966, 12__~I, 35. 13. F. Mueiau/ and T.B. Tomasl Jnr., Biochemisnry, 1976, 15, 798. 1A. F. Kueppere and R.J. Feller, Clin. chim. Acts, 1969, 2A, 401. 15. F. Keuppers, Environ. Res., 1973, 6, 603. 16. M. Mancinl, A.O. Carbonara, and J.F. Heremans, Immunochemietry, 1965, 2, 235. 17. F. Kueppers, RumanEenetlk, 1967, 5, 54. 18. F. Kueppers, New England J. Med., 1968, 279, 16A. 19. C. Kellermann and H. Walter, HumanEenetik, 1970, IO, 145. 20. C.-B. Laurel1 and S. Eriksson~ Scan. J. clin. Lab. Invest., 1963, 15, 132. BAT Co LTD - MINNESOTA TOBACCO LITIGATION w "--4

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-23- 21. S. Er£ksson, Acl~a reed. Sc~nd., 1964, 175, 197. 22. S. Er£ksson, Acta med. Scand., 1965, 177, Suppl. 432. 23. F. Kueppers, W.A. Brlscoe, and A.G. Beam, Science, 1964, 14__6, 1678. 24. R.C. Tal~mo, J.D. Allen, M.G. Kahan, and K.F. Austmn, New England J. Med., 1968, 278, 345. 25. C.-B. Laure11, Stand. J. olin. Lab. ~ves~., 1965, 17, 27Z. 26. M.K. FaKerhol and M. Braend, Science, 1965, 14__~9, 986. 27. M.K. Fagerhol, Stand. J. clln. Lab. Invest., 1969, 23, 97. 28. M.K. Fagerol, in "Pulmonary Emphysema and Proceolysls", Ed&ted by C. Miccman, Academic Press, New York, 1972, p. 123. 29. C.-B. Laurell, Analyt. Biochem., 1965, IO, 358. 30. F. Kueppers and A.G. Beam, Science, 1966, 154, 407. 31. M.K. Fagerhol and C.-B. Laurel1, Clin. chim. Acta, 1967, 16, 199. 32. M.K. F&serhol and C.-B. Laurell, Prosr. Med. Genet., 1970, 7, 96. 33. M.K. F&serhol and T. Gedde-Dahl Jr., Hza,an Heredity, 1969, 19, 354. 34. F. Kueppers, Biochem. Genet., 1969,--3, 283. 35. P.J. L Cook, PostEraduate Medical Journal, 1974, 50, 362. 36. P.W. Brunt, Gut, 1974, 15, 573. 37. M.K. Faserhol, A.M. Johnson, and R.C. Tale-so, American Rev. Rasp. Dis., 1975, 112, 148. 38. M.K. Paserhol, Ac~a PeChol. M/c~obiol. Seand., 1967, 70, 421. 39. M.K. FaHerhol, A.W. Ez-lksson and E. Moun, Human Heredity, 1969, I__99, 360. 40. R.B. Cole, N.C. Nevln, G. Bluadell, J.D. Merrett, J.R. McDonald, and W.P. Johnston, Thorax, 1976, 31, 149. 41. M.K. FaEerhol and O.W. Tenfjord, Acta Pethol. Microbiol. Stand., 1968, 72, 601. BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-24- V 42. K. Omol;o and S. lbLrada, Jap. J. Buman Genet:£c$, 1970, 14, 293. 43. R.C. Talamo, C.E. Lar~gley, C.E. Reed, and S. Hakino, Science, 1973, 181, 70. 44. G. Blunde11, R.B. Cole, N.C. Nevin, and B. Bradley, The Lancet, 17 AuKusC 1974, 404. 45. W.D. Williams and L.¥. Fajardo, Am. J. Clin. Patho1., 1974, 61, 311. 46. C. Altay, H.K. Fagerhol and N. Erdosan, New England J. Hed., 4 October 1973, 289, 754. 47. O.F. Bell and R.W. Carrel1, Nature, 1973, 243, 410. 48. S. Hakino and C. Reed, J. Lab. Clin. Hed., 1970, 75, 742. 49. F. Kueppers, Humangenetik, 1968, 6, 207. 50. J. Lfeberman, New Englend J. Hed., 1969, 281, 279. 51. J. Lieberman, C. PL~tCman, and A. $. Schneider, J. &me=. Hod. &ss., 1969, 210, 2055. 52. C. Mier~nan and 3. L£eberman, Clln. Kes., 1972, 20, 242. 53. E. Fallat, F. r,,eppers, M. Powe11, E. Lilker, J.A. Nadel, and 3.F. Hurray, Clin. ]has., 1969, 17, 413. 54. D. OsCrov and R.H. Cherniack, Amer. Rev. KIsp. Dis., 1972, 106, 377. 55. E. Remnick, N.L. Lapp, and W.K.C. Horgan, J. &met. Hed. aasoc., 1971, 215, 1101. 56. R.E. Kanner, H.R. Klauber, end S. Watauabe, Amer. J. )fed., 1973, 54, 706. 57. M.H. Welch, H.E. Reiuecke, 3.F. ~mmersten, and C.A. Guenter, Ann. Zncern. Had., 1969, 71, 533. 58. H.H. Welch, R.R. Richardson, W.R. Whitcomb, R.F. Hanm~rsten, and C.K. Gushier, J. nucl. Med., 1969, 10, 687. BAT Co LTD - MINNESOTA TOBACCO LITIGATION N

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-25- 59. L.S. Geisler, G.W. Bactunanu, ¥. Launen, D. Nolte, H. Wentzel and H.-D. Rest, Deutsche Med. Wocheusch., 1972, 97, 329. 60. M.C. Jones and G.O. Thou~s, Thorax, 1971, 26, 652. 61. D.C.S° Hutchinson, C.E. Barter, P.J.L. Cook, J.W. Laws, N.A. ldartelll, and P. Hugh-Jones, Quart. J. Mad., 1972, 41, 301. 62. G.A. Falk and W.A. Br£scoe, Ann. intern. Meal., 1970, 72, &27. 63. F. Kueppers and A.G. Beam, Prec. Soc. exp. Biol. Med., 1966, 1..~.~2~, 1207. 64. A. Jano££ and ..I. Schere~, J. exp. MQd., 1968, 12B, 1137. 65. G.S. La--&rus, R.S. Broom J.R. D&~iels, and M.E. Pull~aer, ScienGe, 1968, 159, 1483. 66. A. 3anoff and J.D. Zeliss, Science, 1968, 16I, 702. 67. H.B. Martin and K.S. Boa~nan, Amer. Key. Resp. Dis., 1965, 91, 206. 68. J.R. G£11erp~e and W.S. Tyler, Amer. Rev. Resp. D£s., 1967, 95, 484. 69. A. Jano£f, E. ItosenberK, and M. Galdstou, Prec. Soc. exp. Biol. Hed., 1971, 136, 1054. 70. R. RosenberS, ~. Sandhaus and &. Janoff, Amer. Eev. Resp. Dis., 1972, 106, 114. 71. P. F~hnbel, B. Mass, T. Xkeda, and G. Veinb&uma, in "Pulmonaz-y Emphysema and Pzoteolysis", F~ited by C. l~Lccman, &cadenL~C Press, New York, 1972, p. 411. 72. A. Janoff, Proc. ]Fed=. Ame.=. Soc. Exp. B£ol., 1972, 31, 254. 73. P. Gross, M.A. Babyak, E. Tolker, and H. Kaschak, J. occup. Hed., 1964, 6, 481. 74. A.J. Woodcock, W. Green, and A. Crockett, British Medical Journal, 1972, (2), 134. BAT Co LTD - MINNESOTA TOBACCO LITIGATION r',,o m

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-26- 75. J. Lieberman, British Medical Journal, 13 July 1974, 93. 76. J.S. Milledge, British Medlcal Journal, 17 August 1974, 471. 77. K.D. Lunan and G. Freeman, in "Pulmonary Emphysema and Proteolysls", Edited by C. Mittman, Academic Press, New York, 1972, p. 463. 78. M.G. Mustafa, C.E. Cross, R.J. Munn, and J.A. Hardie, J. Lab. Clin. Meal., 1971, 77, 563. 79. P. Chowdhury and D.B. Louria, Science, 1976, 191, 480. 80. R.A. Nadkarnl, Chem~sCry and Industry, 1974, 693. 81. B-A.T. Report No. RD.1576 Rastri=ted, Z0.5.76. 82. S.A. Pratt, T.N. Finley, M.H. Smith, end A.J. Ladman, Arian. Rec., 1969, 163, 497. 83. S.A. Pratt, M.H. Smith, A.J. Ladman, and T.N. Finley, Lab. Invest., 1971, 24, 331. 84. J.O. Harris, E.W. Swenson, and J.E. Johnson, J. C1in. Invest., 1970, 49, 2086. 85. H.L. Sharp, R.A. ~ridges, W. Krivlt, and E.F. Freier, J. Lab. Clin. Med., i969, 73, 934. 86. 6. Feldman, J. Bignon, and P. Chahin£an, Digestion, 1974, IO, 162. 87. T. Mor~n, G. Feldman, J.-P. Beuhsmou, J.-P. Martin, B. Eueff, and C. Ropartz, The Lancet, 1 February 1975, 250. 88. ~. Aagenaes, k. Matlary, K. Elgjo, E. Munthe, and M. Fagerhul, Acta Paediat Stand., 1972, 61, 632. 89. J.L. Campra, J.P. Craig, R.L. Peters, and T.B. Reynolds, Ann. intern. Med., 1973, 78, 233. 90. B. Brand, G.H. BezahleE, and R. Gould, Gastroentero~ofy, 1974, 66, 264. BAT Co LTD - MINNESOTA TOBACCO LITIGATION R

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-27- 91. R.G. Sp£ro, New ]England J. Hed., 1969, 281, 991, 1043. 92. H. Gans, in "Pulmonary F~mphysema and Proteolysis", Edited by C. M£¢Cman, Academic Press, New York, 1972, p. 115. 93. C.A. Porter, A.P. Howat, F.J.L. Cook, D.W.G. Haynes, K.B. Shilkin, and R. Williams, British Medical Journal, 1972, (2), 435. 94. J. Liebermann, Path. Biol., 1975, 23, 517. 95. P.J.L. Cook, Ann. Human Genetics, 1975, 38, 275. BAT Co LTD - MINNESOTA TOBACCO LITIGATION

N o r- | Z Z © J © Z Pop, tat[on Horve8ians Finns YLnuiah Lapps Worvqtan Lapps lcttmders British Iorthe~ Irish Gernanm Hunsar[ans Greeks Spaniards U.S.A, Ilhlte Iranians Indians (kshenlus) Indians (U.K. tmisrants) Koreans (Female) Japanese Japanese Atnus He|rose (Honnbique) Nesroes (U.K. Lmiarants) Honsololds Arabs (U.L imiarauts) L If L [;Ol DISTRIBS.,Ofl (Z OF TOTAL) OF fl PilENQTYPES IH UNRELATL,, SUBJECTS Humber of Subjects 2830 223 ,is 3O2 94 4549 1995 516 182 ~00 378 251 271 430 394 90 965 238 27& &22 68 85 ri Phenotype HH H8 HE FH IH 8S $2 72, FF FS IS Others 89.7.5 4.10 2.96 2.5h 0.21 0.16 0,14 0.07 0.04 0.06 0.04 0.04 99.11 0.89 99.14 0.54 0.21 98.34 1.65 75.53 2.13 20.21 1.05 88.85 8.05 1.55 0.55 0,00 0.22 0,04 86.50 7.97 3.85 0.40 0.50 O. lO 0.2.5 78.68 3.29 0.58 14.34 0.19 83.55 1.74 0.58 9.88 0..58 0,58 92.50 0..50 1.75 2.50 i.25 75.60 18,76 1.85 0.53 0.25 1..59 0.53j l 91.19 3.07 4.60 00.44 2.58 2,21 10.33 0.73 0.35 0.69 2.03 99.06 97.21 98.89 96.79 0.52 2.49 95.00 0.42 3.78 97,08 0,30 1.80 96.10 0.24 1.18 0.24 100.00 95.17 3.53 1.05 0.02 0.05 0.58 1.35 0.97 1.15 1.74 0.58 0.75 0.38 0.30 1.11 1.11 1.11 0.23 1.10 O, io 0.59 0.25 0,5S 0,75 1.O5 O.38 0.76 0.10 O. 70 Reference 38 39 39 39 19 95 tO 19 19 19 41 t+2 19 19 95 19 42 12 19 9.5 41 95

INDEX HAS INDICATED GAP IN BATES RANGE HERE

FIG. I R,D. 13'=)4 UNCLASSIFIED -I- \ ACID STARCH GEL ELECTROPHORETIC PATTEP~HS OF .SOME 'PHENOTYPF.S ~'5C HEM'A'Tt~ ONLY THE TWO MA3"0R ZONES IN EACH ALLELE PRODUCT ARE INDICATED. TI-IE Ehq, EADTH OF THE ZONES CORKESPONOS To THE RELATIVE PPJ)TEIN C0efTENT. MM ZZ SS MZ I~S SZ I I ' ' r ' | l • I FIG.2 CROSSED, ANTIG.EN -ANTIBODY ELECTROPHOREStS OF THE MZ PHEHOTYPE (~SCHE.MATICJ STAGE I- ACID STARCH GEL ELECTROPHOKESIS IN DIRECTION L i STAGE Z - CR0~ED ,NNTIGEN - ANTI6OOY ELECTEOPHOEESIS IN DIRECTION Z.. z z I I J '-',.a r',,,j BAT Co LTD - MINNESOTA TOBACCO LITIGATION