The Alpha1-antitrypsin Gene and Its Mutations

The Alpha1-antitrypsin Gene and Its Mutations· Clinical Consequences and Strategies for Therapy Ronald G. Crystal, M.D.; Mark L. Brantly, M.D.;t Richard C. Hubbard, M.D.; David T. Curiel, M.D.; David] States, M.D.; and Mark D. Holmes, M.D.

(a1AT) deficiency is an autosomal hereditary disorder characterized by low serum and lung levels ofa1AT, a high risk for the development of emphysema in the third to fourth decades, and a lesser risk for the development of liver disease, particularly in childhood. 1-7 This disorder is an extraordinary example of the power of modern genetics. It was first recognized in 1963, when Laurell and Eriksson5 discovered the disease "aI-antitrypsin deficiency" by noting a marked reduction of the "a1globulin" band in approximately 1 ofSOO serum protein electrophoresis patterns of a random Swedish population. Within 25 years, the clinical manifestations have been fully described, the responsible gene has been cloned, the molecular bases of the major deficiency states are defined, and techniques have been developed for accurate prenatal diagnosis. Although the liver disease still remains an enigma, the pathogenesis of why a defiCiency of alAT causes emphysema is well understood and a specific therapy is available to prevent the progression of the emphysema in these individuals. ~phal-antitrypsin

FORM AND FUNCfION OF aI-ANTITRYPSIN MOLECULE

Alphal-AT is an inhibitor of serine proteases, including neutrophil elastase, trypsin, chymotrypsin, cathepsin G, plasmin, thrombin, tissue kallikrein, factor Xa, and plasminogen. 8,9 For this reason, it is sometimes referred to by the more general terms "a1-antiprotease" or "a.-protease inhibitor."9 In vivo, however, the only real substrate for alAT is neutrophil elastase, a powerful protease capable of cleaving most protein components of the extracellular matrix, a variety of proteins of the coagulation and complement cascades, and E coli cell wall components.9-11 In this regard, the association rate constant of normal alAT with human neutrophil elastase is 107 M-1s-l, a value 25-fold greater than for the interaction of alAT with any other protease. 8,9,ll The interaction of alAT and neutrophil *From the Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda (Chest 1989; 95:196-208) tParker B. Francis Fellow in Pulmonary Research. Reprint requests: Dr. Crystal, Bldg 10, 8m 6003, National Institutes of Health, Bethesda 20892

198

elastase is rapid and tight; dissociation is negligible and under normal circumstances binding is a suicide interaction for both molecules. The mature alAT protein is comprised of a single chain of 394 amino acids with three, N asparaginyllinked complex type carbohydrate side chains, giving it a total molecular weight of 52 kDa 12,13 (Fig 1). Crystallographic analysis of alAT demonstrates a globular, elongated structure with 30 percent of the molecule in the form of a-helices and 40 percent as ~ sheets. 14 The carbohydrate side chains attached to residues Asn 46 , Asn83 , and Asn 247 are all on the outside surface of one-half of the elongated structure. On the opposite side of the molecule is an exposed loop with the reactive inhibitory site for serine proteases (MeflSSSer359) at the tip of the loop. The overall structure of the molecule stresses the active site loop such that the MeflSS-Ser359 tip fits closely into the reactive pocket of neutrophil elastase where it is held with great affinity. In normal humans, 34 mg of alAT are produced daily per kg body weight. 15 The alAT gene is expressed in hepatocytes and, to a lesser extent, mononuclear phagocytes. 16-l8 In normal individuals, alAT serum levels are 20 to 48 J-LM. 19 With a molecular weight of 52 kDa, the mature protein is capable of diffusing into most organs, but the molecular mass of the alAT results in lower concentrations of alAT within the organ. For example, the alAT level on the epithelial surface of the lower respiratory tract of normal individuals is 2 to 5 f.LM, approximately 10 percent that of plasma. 19-21 In the normal lower respiratory tract, alAT provides >95 percent of the protective screen against the omnivorous proteolytic capacity of neutrophil elastase. lO,21 This enzyme is a 220 residue,+ single chain protein with two carbohydrate side chains and a molecular weight of 29 kDa 23 (Fig 1). It is coded for by a 4 kb single copy gene located on chromosome 11 at qI4. 24 Neutrophil elastase is one of the few human enzymes capable of cleaving insoluble, cross-linked tThe published literature describes mature neutrophil elastase as a 218 amino acid polypeptide (22) but recent sequencing studies li'avis, U. have shown it to be comprised of 220 residues Georgia, personal communication).

a.

The Alpha,·antitrypsin Gene and its Mutations (Crystal at 81)

a1-antltrypsln

Neutrophil elastase

FIGURE 1. Structure of aI-antitrypsin and its natural substrate, neutrophil elastase. Alpha,-antitrypsin is a single chain 394 residue glycoprotein with three asparaginyl-linked (Asn"·S3 ·..,) complex carbohydrate side chains (shaded areas). The active inhibitory site centered at Met"""-SerO"" is localized on a stressed loop (-Ile356-ProW,-Met"""-SerO""-Ile300-Pro36I_) protruding from the molecule (striped region). Neutrophil elastase is a single chain 220 residue glycoprotein with two asparaginyl-linked (Asn..·..·) side chains (shaded areas). The reactive center has a specificity pocket (Val''', Phe"°, Ala '81 , Val''', PheOO3 ; see reference 29 and Bode W personal communication) (stippled area) adjacent to the catalytic triad Ser '13-His41 -Asp" (striped region). The two molecules interact with great avidity; dissociation is rare and binding is a suicide interaction for both.

elastin, the rubber-like macromolecule that modulates the elastic recoil of tissues, including the alveolar walls of the lower respiratory tract. lO•25 Despite its name, neutrophil elastase is relatively nonspecific about its targets-it attacks many connective tissue proteins other than elastin, including the major components of the alveolar interstitium such as collagens type I and III and the protein portion of proteoglycans, as well as type IV collagen and laminin, the major protein components of basement membranes.'o The neutrophil elastase gene is expressed only in bone marrow myeloid cell precursors;26.27 the elastase is stored in the azurophilic granules of the mature neutrophil and is released when the neutrophil is activated or when it disintegrates at the end ofits lifespan. 10.28 Neutrophil elastase has a large number of arginine residues on its external surface giving it a highly positive charge; consequently, it rapidly interacts with negatively charged macromolecules such as those comprising connective tissue. 22.23 Like other serine proteases, the proteolytic capacity of neutrophil elastase depends on a catalytic triad His41-Asp88-Ser'73 centered in its

reactive site pocket. 29 When a substrate is presented to the reactive center, a transfer of a proton within the triad allows the Ser173 to become a highly reactive nucleophile capable of attacking the peptide bond within the substrate. Alpha,-AT inhibits neutrophil elastase by interacting tightly, and essentially irreversibly, with the neutrophil elastase active site pocket. 9 •23 Under normal circumstances, there are some neutrophils in the lower respiratory tract, and thus Q,AT plays a critical role in providing a protective shield for the alveolar walls against a chronic, low level burden of neutrophil elastase. 21 .25 •30 In Q,AT deficiency, the low plasma levels of Q,AT are reflected in low lung levels. ' 9-2' Consequently, there is insufficient antiprotease protection for the fragile alveolar walls, resulting in a slovv, unimpeded destruction of the lung parenchyma. 25 Over several decades, the loss of alveoli is sufficient to affect the demands for gas exchange, and the disease becomes clinically apparent as emphysema. Although Q,AT is normally an excellent inhibitor of neutrophil elastase, it has an "Achilles heel"-the CHEST I 95 I 1 I JANUARY, 1989

197

IA 18

Ie

5' __.......

......

TAA (stop)

3'

AnAA (polyadenylatlon signal)

FIGl'RE 2. Overall structure of the normal ai-antitrypsin gene. There are 7 exons dispersed over 12.2 kb of chromosome 14 at q31-32.3. At the 5' region of the gene are three non-protein coding exons referred to as I At la, Ie, respectively; these exons and the regions flanking them control transciption of the gene (see text). These non-eoding exons are followed by 4 protein coding exons, referred to as exons II-V (see Brantly et al 41 for details). The start codon (ATG) for translation of the alAT mRNA and the signal peptide (stippled area) are in exon II and the stop codon (TAA) is in exon ~ followed by the polyadenylation signal (ATIAA). The carbohydrate (CHO) attachment sites (Asn 46, 83, 247) are coded for in exons II and III, and the active inhibitory site MetJ58 is coded for in exon V

MetJ58 at the active site can be easily oxidized. 31 .32 When this occurs, the association rate constant of the alAT for neutrophil elastase is reduced 2000-fold. 33-37 Both cigarette smoke and inflammatory cells in the lower respiratory tract can oxidize the Met358; this explains, at least in part, why cigarette smoking markedly accelerates the development of emphysema in individuals with alAT deficiency.38 ai-ANTITRYPSIN GENE AND

ITS

EXPRESSION

The 12 kb alAT gene is comprised of seven exons (Fig 2).39-41 Exons lA' I B , and Ie are "noncoding" -they contain sequences found in alAT mRNA transcripts, but do not code for the actual alAT protein. Exons 11V contain the sequence information that defines the protein itself. The alAT gene is normally expressed in two classes of cells: hepatocytes and mononuclear phagocytes. 16-18.40 Most of the information relating to control of alAT gene expression is related to hepatocyte expression. In the region just 5' to exon Ie there are typical cis (ie, nearby)-controlling regions. 39 •42-45 In addition, there is at least one, and likely more, hepatocyte cis regulatory elements capable of interacting with specific nuclear protein(s), presumably to control expression of the gene. 46.47 Less is known about regulation of alAT gene expression in mononuclear phagocytes; current evidence suggests the control of mononuclear phagocyte expression is in the regions flanking and including exons I A and I B .40·45 The protein coding region of a l AT begins in exon II 198

and ends in exon ~39 AlphalAT mRNA transcripts in mononuclear phagocytes are slightly larger than liver alAT mRNA transcripts (mononuclear phagocytes 1.8 kb, liver 1.4 kb).16-18.39.4o The carbohydrate attachment sites are coded in exons II and III, and the active site is coded by exon ~ Following transcription, the mRNA is translated on the rough endoplasmic reticuluum (RER), producing a precursor protein of 418 amino acids.12.13.39 The signal peptide of 24 residues is removed during secretion into the cisternae of the RER where the protein is glycosylated with high-mannose type carbohydrates as it folds into a three-dimensional configuration. 48-51 The glycosylated protein is then translocated to the Golgi where the carbohydrates are trimmed to the complex form and the mature protein is then secreted. ai-ANTITRYPSIN ALLELES

The Q1AT gene is very polymorphic, with at least 75 alleles known. 41 Conceptually, these alleles are conveniently categorized into four groups based on the status of the alAT protein in serum (Table 1) including: (1) "normal" (associated with normal serum levels of alAT with normal function); (2) "deficient" (associated with serum alAT levels <35 percent of normal); (3) "null" (no detectable alAT protein in serum); (4) "dysfunctional" (the alAT protein is present, but does not function normally). Except for the null alleles and a few rare alleles, the nomenclature for the alAT alleles is based on a letter code (A to Z), relating to the position of migration of The Alpha,-antitrypsin Gene and its Mutations (Crystal et al)

Table 1- Classification ofAl"ha1-Antitrypsin Variantl·

Category Nonnal

Allele M1(Ala213) M1(Val213) M2 M3 M4 Balbambn

Xmintcburcb Deficient

Null

Others§ Z S Mprodda Mheerlen Mmalton Mduarte Others~

Nullbellingbam NU~iteW1s

Nullmatta..... Nullhoog kong

Nu~

Dysfunctional

Othersl\ Pittsburgh

Estimated Allelic Frequencyt 0.20-0.23 0.44-0.49 0.14-0.19 0.10-0.11 0.01-0.05 Rare Rare All rare 0.01-0.02 0.02-0.04 Rare Rare Rare Rare All rare Rare Rare Rare Rare Rare All rare Rare

Serum Q1AT Level (%):1: 100 100 100 100 100 100 100 100 10-15 40-70 <10 <10 <10 <10 <10 0** 0 0 0 0 0 Normal

Function as an Inhibitor of Neutrophil Elastase# Normal Normal Normal Normal ?

? ?

? Decreased Normal Normal

? ? ?

?

Decreased

Risk for Diseasett None None None None None None None None Lung, liver None Lung Lung Lung, liver Lung, liver Lung Lung Lung Lung Lung Lung Lung Hemorrhagic diathesis

*See reference 41 for further details. t Allelic frequencies for USA Caucasians; ccrare" = allelic frequency
the Q1AT protein in isoelectric focusing (IEF) ofplasma between pH 4.2 and 4.9. 1,41,52,53 The common normal variants migrate in the middle and are thus referred to as the "M-family" alleles, while the deficiency variant described by Laurell and Eriksson5 migrates close to pH 4.5 and is referred to as Z.54 When two alleles have an identical IEF pattern, and the sequence difference is knOWD, the relevant residue is specifically indicated (the two most common Q1AT alleles, Ml[Val213 ] and Ml[Ala213 ], have IEF patterns of"Ml," but differ at residue 213 by the neutral amino acids Val and Ala).55 Some rare alleles are labeled by a letter indicating the IEF position together with the birth site of the allele (M procida).46 The null alleles are labeled "null" together with the place of origin (Nullbellingham).57 Since the two parental genes are codominantly expressed, the Q 1AT serum phenotype, referred to as the Pi (for "protease inhibitor") phenotype, is determined by the independent expression of the two

parental alleles. 1,41,52,53 For all Q1AT alleles known, the phenotype determines the serum level of Q1AT (inheritance of two deficient alleles results in lower serum Q1AT levels than does inheritance of one deficient allele and one normal allele).l,41,52,53 Extensive epidemiologic studies have carefully delineated the association of certain Q 1AT phenotypes and the risk for development of disease .1,13,58,59 Inheritance of any combination of normal alleles or one normal allele with a deficient or null allele bears no risk for disease. However, certain combinations of the deficiency alleles, null alleles, or mixtures of deficiency and null alleles bring a risk for development of emphysema, liver disease, or both (Table 1). In the context that serum levels of Q1AT dictate lung Q1AT levels, since Q1AT provides the critical protective screen of the lower respiratory tract, the phenotype defines the relative risk for the development of emphysema. For example, an individual with serum levels of 5 J.LM CHEST / 95 / 1 / JANUAR~

1989

199

(PiZZ) is at much higher risk for the development of emphysema than an individual with a level of 10 JLM (PiSZ). Furthermore, the phenotype dictates the kind of disease for which the individual is at risk. For example, homozygous inheritance of the Z deficiency allele is associated with an increased risk for both emphysema and liver disease,I,3,41,52,53 while homozygous inheritance of a null allele (Pi Nullbellingham) is associated with an even higher risk for emphysema, but no risk for liver disease. 57 ,60,61

The cCNormal" Category Four alleles (MI[Ala213 ], MI[Val213], M2, and M3) represent >95 percent of the known a l AT variants associated with normal serum a l AT levels (Table 1).41,55,62,63 Among Caucasians, the only racial group for which there are extensive data, MI(Val213) is the most common, with MI(Ala213), M2 and M3 less frequent, in descending order, respectively. Sequence analysis of the coding exons of normal M-family genes has shown that single base changes are responsible for the different variants (Fig 3). When inherited in a homozygous fashion or with each other in a heterozygous fashion, all of the normal M-family alleles are associated with serum a.AT levels of 20 to 48 JLM and all 41 In addition to inhibit neutrophil elastase similarl~ the four common M-family normal variants, at least 42

other "normal" alAT variants have been identified. 4l Except for M4 (allelic frequency approximately 0.01 to 0.05), all are rare, with frequencies of less than 1 percent. 41 ,64 None has been completely sequenced. As far as is known, they are all associated with normal a l AT serum levels and inhibitory function.

The cCDeficiency" Category At least 11 a l AT deficiency variants have been identified. The "classic" deficiency allele is Z; it was this variant that was inherited in a homozygous fashion in the original patients described by Laurell and Eriksson.:5 The other common deficiency allele is S. The Z Variant: The Z allele is commonly found among Caucasians of Northern European descent, representing 1 to 3 percent of all alAT alleles in this population. 1,4l,:52,53,62,63,65,66 It is rarely found among Blacks or Orientals. The serum levels of individuals homozygous for the Z gene are markedly reduced, typically 2 to 5 JLM .•,4.,52,53 The coding exons of the Z gene differ from the common normal MI(Ala213) gene by one base, resulting in the amino acid substitution Glu342 -.+ 67-70 (Fig 3). This single amino acid substitution interferes with the ability of the cell to secrete a.AT, resulting in low serum a.AT levels. 71 -76 In alAT synthesizing cells of Z homozygotes, alAT mRNA levels, a.AT mRNA trans-

M1(A..21~ t - - - - - - t III ~----t

Nul...... __ ~

TyrteD v.tlt.Stop TAC40GTG TAG

1

delete

5' shift

GI~. GAG

Z

Lys _--~. MG

Pro=-. Leu CCC

~.

CTC

M........,

leu353 Phe353 Stop376

Null............

TTl _. TAG - . Nullmetmwe 3' shift T insertion A•

1

4.---

s .....• - - - - - Gtu2e4.VaJ GM GTA

MINIIton

4.------

Phe52 TTC

T

delete

leu3t1 Stop334 - - - - CTC _ _... TAA

T

--.Nullhongkong

5'$hift

delete

FIGURE 3. Normal, deficiency, and null ai-antitrypsin alleles for which the gene sequence is known. Shown at the top, center are the 4 coding exons (II-V) of the normal Ml(Ala213) gene, In an evolutionary sense, this is the "oldest" human ai-antitrypsin gene (see references 41, 55, 67, 110 for discussion). Below this are the 4 coding exons of the normal Ml(valI13), M3, and M2 genes, respectively. Between each are the relevant codons and amino acid residues defining the sequence differences among these genes. The specific base mutations are underlined. The mutations defining the deficiency and null alleles are indicated, with the arrows emerging from the exon of the normal ai-antitrypsin allele that serves as the "base" sequence for the mutation, The relevant codons, amino acid residues and specific base mutations, additions, or deletions (underlined) are indicated,

200

The Alpha,-antitrypsin Gene and its Mutations (Crystal at al)

lation and core glycosylation of the newly synthesized a lAT in the RER are all normal. 17,18,71-74 However, following glycosylation, the Z-type alAT molecules aggregate, resulting in fewer alAT molecules translocated to the Golgi and thus available for secretion.73,74,77 Consistent with this concept, liver biopsies of PiZZ individuals show accumulation of alAT in the rough endoplasmic reticuluum of hepatocytes. 3,77,78 There are two consequences of the Glu 342 --+Lys mutation that help explain this phenomenon; likely both contribute to the aggregation process. First, analysis of the crystallographic structure of alAT demonstrates a salt-bridge from the normal Glu 342 residue to residue Lys 290 .14 It is hypothesized that the Glu342--+Lys substitution in the Z protein interrupts this salt-bridge, thus altering the three-dimensional configuration of the protein, causing the molecule to fold into its three-dimensional configuration at a slower rate than normal and consequently allowing nearby Z molecules in an "open" configuration to interact through their hydrophobic residues, resulting in aggregation. 14 Second, mutagenesis and transient expression studies utilizing the alAT eDNA suggest that the substitution of Glu at residue 342 per se also plays a central role in causing the aggregation, probably because the positive charge of the Lys 342 in the Z form of a lAT modifies interactions in the local milieu independent of the salt bridge with Lys 290. 75,76 Not only is the Z protein present in reduced amounts in serum, but it also does not function normally as an inhibitor of neutrophil elastase. This is manifested by an association rate constant of the Z molecule for neutrophil elastase that is lower than that ofthe normal M-family variants. 79 The relative impotence of the Zmolecule likely occurs because some of the complexes of the Z molecule with neutrophil elastase are unstable-the elastase cleaves the MeflSS-SerJ59 bond, and the elastase is released. The S Variant: the S allele is more common than Z, representing 2 to 4 percent of all Cl IAT alleles of Northern European Caucasians and up to 15 percent of individuals living in the Iberian peninsula. 1,41,52.53,62.63,66 PiSS individuals have serum a l AT levels of 13 to 19 J.LM .1,52,53,80,81 These levels are apparently sufficient to protect the lung, so PiSS individuals are not at risk for emphysema, .despite the relative deficiency state. 19,59,82 However, PiSZ heterozygotes have serum levels of 6 to 11 J.LM, levels that are borderline "protective," and some of these individuals do develop emphysema. 1,19,20,41,52,53,82,83 The coding exons of the S gene differ from the Ml(Val213) gene at a single base, resulting in the amino acid substitution Glu 264 --+Val (Fig 3).39,84,85 Alpha lAT synthesizing cells of S homozygotes have Cl IAT mRNA transcripts of normal length and levels, but the cells secrete approximately 40 percent of the amount of

CllAT secreted by comparable cells of M-homozygotes. 86 Unlike the Z protein, the newly synthesized S protein does not accumulate in the alAT synthesizing cells, and PiSS individuals are not at risk for the development of liver disease. 87 There is evidence that the serum deficiency occurs in S homozygotes because the newly synthesized protein is unstable and destroyed intracellularly shortly after synthesis. 88 The mechanism for this is unknown, but crystallographic analysis suggests the Glu264 --+Val mutation results in the loss ofa salt-bridge Glu264 - Lys387, possibly causing the instabilit)r.14 Once secreted, the S molecule has a normal life span in plasma89 and it is relatively effective as an inhibitor, with an association rate constant for neutrophil elastase of 7.1 x 1()6 M -IS -1.90 Other Deficiency Variant: Several rare Cl I AT alleles, including 1,91 Mheerlen,92 Mprocida ,S6 Mduarte ,93 M malton ,94 M like ,95 M rouen ,96, ~97 and Zaugsburg98 are associated with reduced serum a lAT levels. Together these alleles represent far less than 1 percent of all a lAT alleles. The mutations responsible for Mheerlen,99 Mprocida,56 and Mmatton100 are known (Fig 3), but the mechanisms causing the reduced a l AT levels of these rare deficiency alleles have not been completely defined, and the mutations responsible for the other rare deficiency alleles are unknown.

The "Null" Category A "null" Cl IAT gene is an Cl I AT gene that does not code for Cl IAT protein identifiable in serum and thus individuals inheriting a null gene from both parents have Cl I AT serum levels of zero (Table 1). The null alleles are very rare; their frequency among Caucasians of Northern European descent is estimated to be
The "Dysfunctional" Category "AlphaIATpittsburgh," the only known example of a dysfunctional Cl I AT variant, was found in an individual with a bleeding disorder.l()5 This form of Cl I AT was CHEST I 95 I 1 I JANUAR'f, 1989

201

partially sequenced and shown to have a single anlino acid substitution at the (llAT active inhibitory site (Met358--'Arg). Interestingly, this gives the active site of the (llAT nlolecule a remarkable homology with antithrombin III, the natural inhibitor of thrombin, and the affected individual died from hemorrhage following traunla. Evaluation ofthe (llATpittsburgh protein has delllonstrated it is an inhibitor of thronlbin and a poor inhibitor of neutrophil elastase. 106-109 Identification of (lIAT Phenotypes

Classically, identification of (lIAT phenotypes capitalizes on 1\\'0 facts:1. 41. 52 -54 (1) nlost (lIAT alleles code for (lIAT proteins that differ fronl one another by charge, and thus can be identified by IEF analysis of serunl; and (2) the (lIAT allele dictates the serum (lIAT level. Thus, the (llAT phenotype is traditionally deternlined by analyzing serUlll for the (llAT IEF pattern, by using a nleasureluent of serum (lIAT levels, and if necessary, by family studies. 54 However, as more (llAT genes are sequenced, it has become clear that this approach does not identify all alleles, (IEF analysis of serum cannot distinguish the COlllnlon normal alleles Ml[Val 213 ] and Ml[Ala213 ]).55 Furthernlore, when fanlily analysis is not possible, it is difficult to identify "null't heterozygotes (distinguish PiZ Nullgranite falls fronl PiZZ54 •102). To circumvent these problems, several approaches have been used to identify (llAT alleles in gellonlic DNA. First, for a few alleles, restriction endonucleases have been identified that cut DNA at the specific site of the polylllorphislll. Capitalizing on this, Southern blotting with exon-specific DNA probes can be combined with the endonucleases BstEII to identify the restriction fragment length polynlorphism in exon III that distinguishes the M1(Va}213) and M1(Ala213) gene, 55 an RsaI PolYlllorphisnl in exon II that distinguishes the M2 variant frolll the COlunlon M1 variants,l1O and a PvuII polynlorphislll in exon II identifies the Mprocida deficiency gene fronl all other known (llAT genes. 56 Second, analysis of genolllic DNA with various restriction endonucleases and intron-specific probes has demonstrated that all Z alleles seem to be within a comnlon 12 kb segment, thus defining a specific Z haplotype. lll This approach has been used for prenatal screening. 112 Third, oligonucleotide probes have been used to detect the (lIAT deficiency genes. 83. 113 To accomplish this, the exons coding for the (llAT protein are conveniently cut fronl genonlic DNA into separate fragments \\rith the endonucleases BgI1 or with a cOlllbination of PstI and Sstl, ~nd the exon with a specific single base change is detected in agarose gels using 32P-Iabeled oligonucleotide probes. 83 Alternatively, the polymerase chain reaction can be used to amplify individual segments of the (llAT coding exons, permitting easier 202

evaluation using labeled nucleotide probes. 114,115 Finally, using allele-specific primers combined with the polymerase chain reaction, individual (lIAT alleles are amplified, depending on the specificity of the probe. 116 This method is the fastest and simplest of all of these techniques, permitting accurate identification of known single base (llAT mutations within one day. CLINICAL MANIFESTATIONS OF (lIAT DEFICIENCY Emphysema is the most common clinical manifestation of (lIAT deficienc)'. Greater than 95 percent of these individuals are homozygous for the Z-type (lIAT gene, but any individual with Q 1AT levels of
the pathogenesis of the lung destruction in a lAT deficiency results from insufficient amounts of a lAT available to protect the alveoli from attack by neutrophil elastase, strategies to prevent the emphysema associated with alAT deficiency have focused on augmenting the antineutrophil elastase screen ofthe lower respiratory tract. 19,20 In contrast, as the pathogenesis of liver disease is not understood, no rational therapeutic strategies have been devised, except for liver transplantation, with its attendant mortality and morbidit~ 124-126

General Concept of Augmentation Therapy with alAT Purified from Plasma In 1979, we approached the problem of preventing emphysema associated with a lAT deficiency with the same strategy used to treat hemophilia-the affected individual is "deficient," so why not augment a lAT levels by administering intermittent alAT infusions? Using human alAT partially purified from pooled plasma, Gadek et al l27 demonstrated that once weekly intravenous infusions of approximately 4 g of a lAT would maintain reasonable alAT serum levels and that the infused alAT would diffuse into the lung. When sufficient amounts of purified a lAT became available to mount larger scale clinical trials, Wewers et al l9 treated 21 individuals with severe alAT deficiency once weekly with 60 mglkg of alAT intravenously for six months. This study convincingly demonstrated that: (1) a lAT serum levels could be chronically maintained at sufficient levels to put the individual out of the "at risk" group for emphysema; (2) the infused a lAT diffused into the lower respiratory tract and the alveolar epithelial lining fluid levels of alAT were in the protective range; (3) the a lAT that had diffused into the lung was functional, and capable of inhibiting neutrophil elastase; and (4) this therapy was safethere was no evidence of immune reactions or viral disease, including hepatitis or human immunodeficiency virus infection. What the study did not show (and could not by its design), was that augmentation therapy halted the progression of the disease. This is understandable, since the emphysema is slowly progressive (it takes decades to develop). Furthermore, insufficient numbers of patients have been identified who would be suitable candidates for a large-scale clinical trial, and even if they could be identifed, the slowly progressive nature of the disease would necessitate an extremely costly trial involving comparison of large numbers of treated and untreated individuals for many years. 19,l28 On the basis of these considerations, and with the evidence that augmentation therapy with purified human plasma Q 1AT safely reverses the biochemical abnormalities at the site of the target organ, the USA Food and Drug Administration li-

censed aI-antitrypsin augmentation therapy in December 1987 for general use.

Treatment of a l AT Deficient Individuals Augmentation therapy for alAT deficiency is straightforward. The following is the routine approach used by the Pulmonary Branch, NHLBI. First, the diagnosis of a lAT deficiency is made by analysis of serum for a lAT levels. While the measurement itself is simple (usually by radial immunodiffusion, but enzyme-linked immunoassay or nephelometry can be used), there is some confusion relating to standard values, and the values considered to be "deficient." The problem stems from the reference standards in use. 19 Unfortunately, the reference standard supplied with the most commonly used commercial radial immunodiffusion kit (Calbiochem-Behring) leads to an approximately 35 percent overestimation of the a lAT levels. Using this "clinical" standard, the normal range for serum a l AT levels is 150 to 350 mwdl, "a IAT deficiency" is defined as levels <80 mwdl, and Z homozygotes are invariably<50 mWd1.1,19,20,41 In contrast, using a true laboratory standard, the normal values are 107 to 249 mwdl (20 to 48 JLM), with a lAT deficiency defined as<57 mwdi «10 JLM), and Z homozygotes invariably<36 mwdl «5 JLM). To resolve this confusion, an international standard is being developed and should be available to laboratories within the next six months. Until then, it is critical to know the normal and deficient ranges for the laboratory doing the analysis. Second, the a lAT phenotype is determined using isoelectric focusing of serum. This information is helpful because it confirms the a lAT serum level data and it clearly defines the specific type of a lAT deficiency. The latter is important because it defines the extent of risk for lung and/or liver disease (Table 1). AlphalAT phenotyping is carried out by several academic and commercial laboratories and a reference laboratory has been established in conjunction with a phase IV study ofa lAT augmentation therapy. Because the individuals heterozygous for a null allele will appear to be homozygous for the other parental allele, definitive phenotyping cannot be carried out without family analysis. 1,41,52-54 However, for routine clinical purposes, it is usually sufficient to determine the phenotype only on the individual being considered for augmentation therapy. Third, a clinical determination is made as to whether or not the individual has evidence of lung destruction-emphysema. This is important because epidemiologic studies suggest a small proportion of a lAT deficient individuals never develop clinically relevant emphysema. 38,59,82,1l7 In this regard, since chronic augmentation therapy is not a trivial undertaking, we reserve its use for a lAT deficient individuals who have CHEST I 95 I 1 I JANUAR~

1989

203

A. ---.

20

140

.

~ CI 120 E

16

~

> 100 .!

...

80

E

60

~

~

~

12 8

~

I

40 4

20 2

1

3

4

5

6

7

8

10

9

11

12

Duration of therapy (months)

B.

6

. ~

:E ~ ......,

> J!

..cs

I-

C

&&. .J W

5 4

3 2

1 Pre

6

4

2

8

10

12

Duration of therapy (months)

I

•

~

6

5

..!I .~ '-:I

4

~~ !l =3 c

2

:c~

c.

-Su 3

•

II. .J W

1

Pre

2

4

6

8

Duration of therapy (months) 204

10

12

FIGURE 4. Serum alAT levels, lung epitheliallining fluid (ELF) alAT levels, and lung epithelial lining fluid anti-neutrophil elastase capacities associated with long-term intravenous augmentation Shown are therapy of alAT deficienc~ data for three PiZZ individuals receiving 60 mglkg alAT at weekly intervals for 12 months. A: Serum alAT levels prior to initiation of therapy and at day 7 just prior to each next infusion. Left ordinate, serum alAT level based on a commercial standard (mg/dl); right ordinate, serum alAT level based on a true laboratory standard (.... M~ Note that these nadir levels are chronically maintained above the threshold protective level (80 mg/dl commercial standard, 10 ....M true standard). B: Average ELF alAT levels at various times during the 12 month treatment period. All samples were collected within a 24-hour period just prior to an infusion of a1Al: The normal range of ELF alAT levels is 2.4 to 5.6 ....M, and the threshold protective level is 1.3 .... M. C: Average ELF anti-neutrophil elastase capacity during long-term augmentation therap~ ELF samples assayed are the same as those in B. The normal range of anti-neutrophil elastase capacity is 2.2 to 4.4 ....M. See references 19, 20 for details of assays, normal values, and discussion of threshold protective levels.

The Alpha,-antitrypsin Gene and its Mutations (Crystal et 8/)

clinical evidence of emphysema as determined by a combination of history, physical and chest x-ray examinations, lung function tests and ventilation-perfusion scintigraphic scanning. Fourth, prior to the first administration of therapy, we perform baseline studies including serum electrolytes and complete serum chemistry panels, complete blood count, blood clotting parameters (prothrombin, partial thromboplastin and thrombin times, total fibrinogen), hepatitis B surface antigen and core antibody and anti-human immunodeficiency virus antibody determinations, urinalysis, 24-hour creatinine clearance, electrocardiogram, chest x-ray film (posterior-anterior and lateral), and routine pulmonary physiologic studies (vital capacity, total lung capacity [by helium dilution, or body plethsmography], forced expiratory volume in 1 s [FEV 1], FEV/forced vital capacity, and diffusing capacity [single breath for carbon monoxide D. Although there has been no case of hepatitis B in association with this therapy, until more experience has been gained, active and passive immunization against hepatitis B virus is initiated as long as there is no prior history of hepatitis or of documented antibodies against the hepatitis B virus. Fifth, therapy is started. The dosage is 60 mglkg of active (XIAT given intravenously at 140 mg/min, once weekly (see reference 19 for complete details). With this approach, serum and lung levels of (XIAT remain above the level necessary to protect the alveolar structures (Fig 4). In this regard, the serum (XIAT levels from week to week are remarkably constant and lung (XIAT levels and anti-neutrophil elastase capacities invariably remain above the threshold protective level. Thus, it is not necessary to monitor serum (XIAT levels and function or lung (XIAT levels and anti-neutrophil elastase capacities by bronchoalveolar lavage. Finally, we re-evaluate the patients on therapy at six-month intervals with the same blood, urine, electrocardiogram, x-ray, and lung function studies performed prior to initiation of therap~ Future Approaches Ongoing studies have been directed at two alternatives to once-weekly augmentation therapy: monthly intravenous infusions and aerosol. Both are in the development stage, but both are promising. The monthly infusion approach is based on the knowledge of the pharmacokinetics of (XIAT demonstrating a half-life in serum of 4.5 days following intravenous infusion. 19 In this regard, we hypothesized that once monthly infusions with 250 mglkg (approximately four times the amount administered once weekly) should provide the same protection as once weekly with 60 mglkg. Studies of Hubbard et al l29 evaluating nine individuals over a one-year period have demonstrated this to be true. Furthermore, the once-

monthly infusions are well toJerated and no safety problems have developed. The only caveat to this approach is that, to prevent transient hypertension (due to the oncotic pressure of the infused protein), the rate of infusion must be maintained at s50 mgt min, requiring the recipient to have an intravenous infusion for 4 to 6 h. Aerosol therapy is based on the concept that since the lower respiratory tract is the only site needing protection in (XIAT deficienc~ direct delivery by aerosol would provide the most efficient route of administration. In this regard, using an aerosol generator capable of developing (XIAT droplets sufficiently small (S3 JJ.m diameter) to reach the lower respiratory tract, using twice daily administration of 100 mg of (XIAT for one week, we have been able to raise alveolar epithelial lining fluid (XIAT levels in Z homozygotes to the normal range .130 There was a concomitant increase in the alveolar anti-neutrophil elastase capacit~ Importantly, aerosol therapy appears to be safe; there has been no allergic reaction and no change in lung function. While the detailed pharmacokinetics of this approach have yet to be worked out, these promising results suggest this is a feasible and rational approach to therapy in (XIAT deficienc~ REFERENCES 1 Gadek JE, Crystal RG. aI-antitrypsin deficiency. In: Stanbury JB, Wyngaarden JB, Fredrickson OS, Goldstein JL, Brown MS, eds. Metabolic basis of inherited disease. New York: McGraw Hill, 1982; 1450-67 2 Morse JO. Alpha1-antitrypsin deficiency. N Engl J Med 1978; 299: 1045-48; 1099-05

3 Sharp HL, Bridges RA, Krivit ~ Freier, EF. Cirrhosis associated with alpha-I-antitrypsin deficiency: A previously unrecognized inherited disorder. J Lab Clin Med 1969; 73:93439 4 Kueppers F, Black LF. aI-antitrypsin and its deficiency. Am Rev Respir Dis 1974; 110:176-94 5 Laurell C-B, Eriksson S. The electrophoretic aI-globulin pattern of serum in aI-antitrypsin deficiency. Scand J Clin Lab Invest 1963; 15:132-40 6 Eriksson S. Pulmonary emphysema and alpha-I-antitrypsin deficiency. Acta Med Scand 1964; 175:197-205 7 Eriksson S. Studies in aI-antitrypsin deficiency. Acta Med Scand 1965; 177(suppl 432): 421-28 8 Carrell ~ Boswell DR. Serpins. The superfamily of plasma serine proteinase inhibitors. In: Barrett AJ, Salvesen G, eds. Proteinase inhibitors. Amsterdam: Elsevier, 1986; 403-20 9 Travis J, Salvesen GS. Human plasma proteinase inhibitors. Ann Rev Biochem 1983; 52:655-709 10 Bieth JG. Elastases: Catalytic and biological properties. In: Mechan R, ed. Regulation of matrix accumulation. New York: Academic Press, 1986; 217-320 11 Beatty K, Bieth J, Travis J. Kinetics of association of serine proteinases with native and oxidized a-I-proteinase inhibitor and a-l-antichymotrypsin. J Bioi Chern 1980; 255:3931-34 12 Carrell ~ Jeppsson J-0, Laurell C-B, Brennan SO, Owen MC, Vaughan L, et ale Structure and variation of human alantitrypsin. Nature (London) 1982; 298:329-34 13 Carrell ~ Owen MC. aI-antitrypsin: Structure, variation and disease. Essays Med Biochem 1979; 4:83-119 CHEST I 95 I 1 I JANUAR~

1989

205

14 Loebermann H, Tokuoka R, Deisenhofer J, Huber R. Human aI-proteinase inhibitor: Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J Mol Bioi 1984; 177:531-56 15 Jones EA, Vergalla J, Steer CJ, Bradley-Moore PR, Vierling JM. Metabolism of intact and desialylated aI-antitrypsin. Clin Sci Mol Med 1978; 55: 139-48 16 Rogers J, Kalsheker N, Wallis S, Speer A, Coutelle CH, Woods D, et al. The isolation of a clone of aI-antitrypsin and the detection ofa I-antitrypsin in mRNA from liver and leukocytes. Biochem Biophy Res Commun 1983; 116:375-82 17 Perlmutter DH, Cole FS, Kilbridge ~ Rossing TH, Colten HR. Expression of the a I-proteinase inhibitor gene in human monocytes and macrophages. Proc Natl Acad Sci USA 1985; 82:795-99 18 Mornex J-F, Chytil-Weir A, Martinet Y, Courtney M, LeCocq J-~ Crystal RG. Expression of the alpha-I-antitrypsin gene in mononuclear phagocytes of normal and alpha-l-antitrypsindeficient individuals. J Clin Invest 1986; 77:1952-61 19 Wewers MD, Casolaro MA, Sellers SE, Swayze SC, McPhaul KM, Wittes JT, et al. Replacement therapy for alpha.-antitrypsin deficiency associated with emphysema. N Engl J Moo 1987; 316: 1055-62 20 Hubbard RC, Crystal RG. aI-antitrypsin augmentation therapy for aI-antitrypsin deficiency. Am J Med 1988; 84(Suppl 6A):52-62 21 Gadek JE, Fells GA, Zimmerman RL, Rennard SI, Crystal RG. Antielastases of the human alveolar structures: Implications for the protease-antiprotease theory of emphysema. J Clin Invest 1981; 68:889-98 22 Sinha S, Watorek W, Karr S, Giles J, Bode W, Travis J. Primary structure of human neutrophil elastase. Proc Natl Acad Sci USA 1987; 84:2228-32 23 Baugh RJ, Travis J. Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry 1976; 15:836-41 24 Takahashi H, Nukiwa T, Yoshimura K, Quick CD, States DJ, Holmes MD, et al. Structure of the human neutrophil elastase gene. J BioI Chern (in press) 25 Janoff A. Elastases and emphysema: Current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis 1985; 132:417-33 26 Takahashi H, Nukiwa T, Basset ~ Crystal RG. Myelomonocytic cell lineage expression of the neutrophil elastase gene. J BioI Chern 1988; 263:254347 27 States DJ, Fouret ~ du Bois RM, Bemaudin J-F, Takahashi H, Yoshimura K, et ale Restricted expression of neutrophil elastase gene expression during granulocytic differentiation in bone marro~ Fed Proc (in press) 28 Janoff A. Elastase in tissue injury. Ann Rev Moo 1985; 36:20716 29 Bode W, Wei A, Huber R, Meyer E, Travis J, Neumann S. X-ray crystal structure of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor. EMBO J 1986; 5:2453-58 30 Hunninghake Gw, Crystal RG. Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers. Am Rev Respir Dis 1983; 128:833-38 31 Johnson D, Travis J. Structural evidence for methionine at the reactive site of human a-I-proteinase inhibitor. J BioI Chern 1978;253:7142-44 32 Johnson D, Travis J. The oxidative inactivation of human a-lproteinase inhibitor: Further evidence for methionine at the reactive center. J Bioi Chern 1979; 254:4022-26 33 Gadek JE, Fells GA, Crystal RG. Cigarette smoking induces functional antiprotease deficiency in the lower respiratory tract of humans. Science 1979; 206:1315-16 34 Carp H, Miller F, Hoidal lR, Janoff A. Potential mechanism of 208

35 36

37

38

39

40

41 42

43

44

45

46

47

48 49

emphysema: aI-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capaci~ Proc Natl Acad Sci USA 1981; 79:2041-45 Blue M-L, Janoff A. Possible mechanisms of emphysema in ciagarette smokers. Am Rev Respir Dis 1978; 117:317-24 Kilburn K, McKenzie W Leukocyte recruitment to airways by cigarette smoke and particle phase in contrast to cytotoxicity of vapor. Science 1975; 189:634-37 Hubbard RC, Ogushi F, Fells GA, Cantin AM, Courtney M, Crystal RG. Oxidants spontaneously released by alveolar macrophages of cigarette smokers can inactivate the active site of aI-antitrypsin rendering it ineffective as an inhibitor of neutrophil elastase. J Clin Invest 1987; 80:1289-95 Larsson C. Natural history and life expectancy in severe alpha 1antitrypsin deficiency, Pi Z. Acta Moo Scand 1978; 204:345-51 Long GL, Chandra T, Woo SLC, Davie EW, Kurachi K. Complete sequence of the cDNA for human aI-antitrypsin and the gene for the S variant. Biochemistry 1984; 23:4828-37 Perlino E, Cortese R, Ciliberto G. The human aI-antitrypsin gene is transcribed from two different promoters in macrophages and hepatocytes. EMBO J 1987; 6:2767-71 Brantly M, Nukiwa T, Crystal RG. Molecular basis of alantitrypsin deficiency. Am J Med 1988; 84(SuppI 6A): 13-31 Ciliberto G, Dente L, Cortese R. Cell-specific expression of a transfected human aI-antitrypsin gene. Cell 1985; 41:531-40 Sifers RN, Carlson JA, Clift SM, DeMayo FJ, Bullock DW, Woo SLC. TIssue specific expression of the human alpha-lantitrypsin gene in transgenic mice. Nucl Acids Res 1987; 15:1459-75 Kelsey G D, Povey S, Bygrave AE, Lovell-Badge RH. Speciesand tissue-specific expression of human aI-antitrypsin in transgenic mice. Genes Dev 1987; 1:161-71 De Simone ~ Ciliberto G, Hardon E, Paonessa G, Palla F: Lundberg L, et ale Cis- and trans-acting elements responsible for the cell-specific expression of the human aI-antitrypsin gene. EMBO J 1987; 6:2759-66 Courtois G, Morgan JG, Campbell LA, Fourel G, Crabtree GR. Interaction of a liver-specific nuclear factor with the fibrogen and aI-antitrypsin promoters. Science 1987; 238:68892 Grayson DR, Costa RH, Xanthopoulos KG, Darnell JE. One factor recognizes the liver-specific enhancers in aI-antitrypsin and transthyretin genes. Science 1988; 239:786-88 Hirschberg CB, Snider MD. Topography of glycosylation in the rough endoplasmic reticulum and golgi apparatus. Ann Rev Biochem 1987; 56:63-87 Elbein AD. Inhibitors of the biosynthesis and processing ofNlinked oligosaccharide chains. Ann Rev Biochem 1987; 56:497-

534

50 pfeffer SR, Rothman JE. Biosynthetic protein transport and sorting by the endoplasmaic reticulum and golgi. Ann Rev Biochem 1987; 56:829-52 51 Lodish 8F, Kong N. Glucose removal from N-linked oligosaccharides is required for efficient maturation ofcertain secretory glycoproteins from the rough endoplasmic reticulum to the golgi complex. J Cell Bio 1984; 98: 1720-29 52 Kueppers F. Inherited differences in alpha-I-antitrypsin. In: Litwin SD, ed. Genetic determinants of pulmonary disease. New York: Marcel Dekker, 1978; 23-74 53 Fagerhol MK, Cox DW The Pi polymorphism: Genetic, biochemical, and clinical aspects of human aI-antitrypsin. Adv Hum Genet 1981; 11:1-62 54 Cox D~ Johnson AM, Fagerhol MK. Report of nomenclature meeting for aI-antitrypsin: INSERM, Rouen/Bois-Guillaume1978. Hum Genet 1980; 53:429-33 55 Nukiwa'I: Brantly M, Ogushi F, Fells G, Satoh K, Stier L, et The Alpha,·antitrypsin Gene and its Mutations (Crystal et al)

56

57

58 59

60

61

62

63 64 65

66 67

68 69

70 71

72

73

74

75

76

ale Characterization of the MI (Ala2I3) type of aI-antitrypsin, a newly recognized common "normal" aI-antitrypsin haplotype. Biochemistry 1987; 26:5259-67 Takahasi H, Nukiwa'I: Satoh K, Ogushi F, Brantly M, Fells G, Stier L, et ale Characterization of the gene and protein of the aI-antitrypsin "deficiency" allele Mprodda. J BioI Chern (in press) Satoh K, Nukiwa J: Brantly M, Garver RI Jr, Courtney M;, Hofker M, et ale Emphysema associated with complete absence of aI-antitrypsin in serum and the homozygous inheritance of stop codon in an aI-antitrypsin coding exon. Am J Hum Genet 1988; 42:77-83 Fagerhol MK, Hauge HE. Serum Pi types in patients with pulmonary diseases. Acta AllergolI969; 24:107-14 Hutchison DCS. Natural history of alpha-I-protease inhibitor deficienc~ Am J Med 1988; 84(6A): 3-12 Garver RI Jr, Mornex J-F, Nukiwa T, Brantly M, Courtney M, LeCocq J-~ et ale Alpha-I-antitrypsin deficiency and emphysema caused by homozygous inheritance of non-expressing alpha-I-antitrypsin genes. N Eng} J Med 1986; 314:762-66 Feldmann G, Martin J-~ Sesboue R, Ropartz C, Perelman R, Nathanson M, et al. The ultrastructure ofhepatocytes in alphaI-antitrypsin deficiency with the genotype Pi--. Gut 1975; 16:796-99 Kueppers F, Christopherson MJ. Alpha-I-antitrypsin: Further genetic heterogeneity revealed by isoelectric focusing. Am J Hum Genet 1978; 30:359-65 Dykes DD, Miller SA, Poleslcy HF. Distribution of alantitrypsin variants in a US white population. Hum Hered 1984; 34:308-10 Constans J, Viau M, Gouaillard C. Pi M4: An additional Pi M subtype. Hum Genet 1980; 55:119-21 Pierce JA, Eramo B, Dew TA. Antitrypsin phenotypes in St. Louis. JAMA 1975; 231:609-12 Evans HE, Bognacki NS, Perrott LM, Glass L. Prevalence of alpha-I-antitrypsin Pi types among newborn infants ofdifferent ethnic backgrounds. J Pediatr 1977; 90:621-24 Nukiwa T, Satoh K, Brantly ML, Ogushi F, Fells GA, Courtney M, et al. Identification of a second mutation in the proteincoding sequence of the Z-Type alpha-I-antitrypsin gene. J BioI Chern 1986; 34:15989-994 Chan SK, Rees DC. Molecular basis for the aI-protease inhibitor defiCiency. Nature (London) 1975; 255:240-41 Yoshida A, Lieberman J, Gaidulis L, Ewing C. Molecular abnormality of human alpha-I-antitrypsin variant (Pi-ZZ) associated with plasma activity deficiency. Proc Natl Acad Sci USA 1976; 73: 1324-28 Jeppsson J-O. Amino acid substitution Glu--+Lys in ai-antitrypsin PiZ. FEBS Lett 1976; 65: 195-97 Errington OM, Bathurst IC, Janus EO, Carrell RW In vitro synthesis of M and Z forms of human ai-antitrypsin. FEBS Lett 1982; 148:83-6 Bathurst IC, Travis J, George PM, Carrell RW Structural and functional characterization of the abnormal Z ai-antitrypsin isolated from human liver. FEBS Lett 1984; 177:179-83 Foreman RC, Judah JO, Colman A. Xenopus oocytes can synthesize but do not secrete the Z variant of human alantitrypsin. FEBS Lett 1984; 168:84-8 Verbanac KM, Heath EC. Synthesis, processing, and secretion ofM and Z variant aI-antitrypsin. J BioI Chern 1986; 261:997989 McCracken AA, Kruse KB, Brown JL. Secretion of mutant alpha-I-proteinase inhibitors. J Cell Biochem 1988; Suppl12B: 286 Brantly M, Nukiwa T, Stier L, Benavente A, Courtney M, Crystal RG. In vitro demonstration of the molecular basis of the secretory defect associated with expression of the Z alantitrypsin gene. Am Rev Respir Ois 1987; 135:A291

77 Bhan AK. Grand RJ. Co1ten HR. Ch€'ster AA. Li\'er in a.antitrypsin deficiency: morpholo~c ohs€'rvations and in vitro synthesis of at-antitrypsin. Pediat Res 1976; 10:35-40 C, d€'Croote J. Desmet 78 Callea F, Fevery H, Mal\si G, Lie\'en~ VJ. Alpha-I-antitrypsin (AAT) and its ~timulation in the liver of PiMZ phenotype individuals: a "r~('ruitm€'nt-secr~tory hlock" CR-SB") phenomenon. Liver 19R4~ 4:325-37 79 O~ushi F. Fells CA. Hubhard RC. Straus SD. Crystal RC. Ztype a I-antitrypsin is less rompet~nt than ~f I-type a I-antitrypsin as an inhihitor of neutrophil €'Iastase. J f:lin In\"€'st 1987; 80:1366-74 80 Goedde H~ Hirth L. Benkmann H-G, P€'lIi('€'r A, P€'lIicer T. Stahn M, et al. Population J:tenetic studies of s€'rum prot€'in polymorphisms in four spanish populations: Part II. Hum Hered 1973; 23: 135-46 81 Martin J-E S~shou~ R. CharlioTlet R, Ropartz C, P€'r€'ira T. Genetic variants of serum ai-antitrypsin (Pi types) in Portuguese. Hum H~red 1976; 26:310-14 82 Larsson C. Dirksen H, Sundstrom (;, Eriksson S. Lun~ function studi€'s in asymptomatic' indi\'idtlal~ \\;th nlod€'rat€'ly (Pi SZ) and s~verely (Pi Z) redu('€'d I€'\'els of a I-antitrypsin. Scand J Respir Dis 1976; 57:267-RO 83 Nukiwa T. Brantly M. Garver R. Paul L. Courtn€'y ~f. Lef:ocq J-~ et al. Evaluation of "at risk" alpha-I-antitrypsin ~€'notype SZ with synthetic oli~onuc1eotide Jiten~ prohes. J Clin Inv€'st 1986; 77:528-37 84 Yoshida A, Ewin~ C. Wess~ls M, Li€'h€'rman J. Gaiclulis L. Molecular ahnormality of Pi S variant of human alphal-antitrypsin. Am J Hum Cenet 1977; 29:23.'3-39 85 Owen MC, Llri~r M. Carr€'11 RW a1-antitrypsin: Stnlctural relationships of the sllh~titutions of th€' Sand Z variants. FE BS Lett 1978~ 88:234-36 86 Curiel D. Garver R, Chytil A. Stier L, Courtney ~f. Crystal R. a I-antitrypsin S-types deficiency results from ahnormal posttranscriptional processin~. Am Rev Respir Dis 1987; 135:A29I 87 Sharp HL, Alpha-I-antitrypsin: An i~or€'d prot€'in in und€'rstandin~ liver disease. Sem Liver Dis 1982; 2:314-28 L. Crystal RC. M€'chanism of th€' S88 Curiel D, Chytil A, Sti~r type a I-antitrypsin defici~ncy: retroviral ~ene transfer demonstrates the gene mutation caus€'s a pre~lycosylati()n proc€'ssing abnormality. Clin Res 1988; 36(3):591A 89 Jeppsson J-O, Laurell C-B, Nosslin B. Cox OW Catabolic rate of a I-antitrypsin of Pi types S. and M "_Itfln and of asialylated M-protein in man. Clin Sci ~1ol Med 1978; 5.5: 103-07 90 Ogushi F, Hubbard R, Fells G. Casolaro A. Curiel 0, Brantly M, et a1. Evaluation of th~ S-type of a I-antitrypsin as an in vivo and in vitro inhihitor of neutrophil €'Iastas€'. Am Rev Respir Dis 1987; 137:364-70 91 Arnaud ~ Colette C-C, Vittoz E Fud€'nher~ HH. Genetic polymorphism of serum alpha-l-proteal\e inhihitor (alpha-Iantitrypsin): Pi I, a deficient allele of the Pi system. J Lab Clin Med 1978; 92:177-84 92 Kramps JA, Brouwers J~ Maesen F, Dijkman JA. Pi Mt-orlf'n' a Pi M allele resultin~ in very lo\\~ a I-antitrypsin serum levels. Hum Genet 1981; 59:104-07 93 Cox OW A new defiCiency allele of ai-antitrypsin: Pi M mahfln • In: Peeters H, ed. Protides of hiolo~cal fluids. Toronto: Pergamon Press, 1975;375-78 94 Lieberman J, Gaidulis L, Klotz SD. A new deficient variant of aI-antitrypsin (M duatt.): Inability to detect the heterozy~ous state by antitrypsin phenotypin~. Am Rev Respir Dis 1976; 113:31-6 95 Kueppers F, Utz G, Simon B. Alpha-I-antitrypsin deficiency with M-like phenotype. J Med Genet 1977; 14:183-86 96 Martin J-~ Sesboue R, Charlionet R. Ropartz C. Does alphaI-antitrypsin PI null phenotype exist? Humangenetik 1975; CHEST I 95 I 1 I JANUAR~

1989

207

30:121-25 97 Fagerhol MK, Hauge HE. The Pi phenotype Mf! Discovery of a ninth allele belonging to the system of inherited variants of serum aI-antitrypsin. Vox Sang 1968; 15:396-400 98 Weidinger S, Cleve H, Patutschnick W Alpha-I-antitrypsin: Evidence for a fourth Pi M allele. Distribution of the Pi M subtypes in Southern Germany. Z Rechtsmed 1982; 88:203-11 99 Hofker MH, Nukiwa T, van Paassen HMB, Nelen M, Frants RR, Klasen EC, et aI. A Pro-+Leu substitution in codon 369 in the aI-antitrypsin deficiency variant Pi Ma-rieu' Am J Hum Genet 1987; 41:A220 100 Curiel D, Chytil A, Brantly M, Stier L, Okayama H, Courtney M, et aI. Use of retroviral gene transfer to characterize the molecular pathophysiologic mechanisms underlying a I-antitrypsin deficiency. Fed Proc (in press) 101 Laurell C-B, Sveger T. Mass screening of newborn swedish Am J Hum Genet 1975; infants for a. antitrypsin deficienc~ 27:213-17 102 Nukiwa T, Takahashi H, Brantly M, Courtney M, Crystal RG. aI-antitrypsin Nullpuite faU" a nonexpressing aI-antitrypsin gene associated with a frameshift to stop mutation in a coding exon. J Bioi Chern 1987; 262:11999-12004 103 Curiel D, Brantly M, Curiel E, Stier L. Crystal R. a1Antitrypsin defiCiency caused by aI-antitrypsin null mattawa: An insertion mutation rendering the aI-antitrypsin gene incapable of producing aI-antitrypsin. Am Rev Respir Dis 1988; 137:A210 104 Sifers RN, Brashears-Macatee S, Kidd VJ, Muensch H, Woo SLC. A frameshift mutation results in a truncated a.-antitrypsin that is retained within the rough endoplasmic reticulum. J Bioi Chern 1988; 263:7330-335 lOS Owen MC, Brennan SO, Lewis JH, Carrell RW Mutation of antitrypsin to antithrombin: aI-antitrypsin Pittsburgh (358 Met-+Arg), a fatal bleeding disorder. N Engl J Med 1983; 309:694-98 106 Scott CF, Carrell ~ Glaser CB, Kueppers F, Lewis JH, Coleman RW Alpha-1-antitrypsin-Pittsburgh: A potent inhibitor of human plasma factor XIa, kallikrein, and factor XII. J Clin Invest 1986; 77:631-34 107 Courtney M, Jallet S, Tessier L-H, Benavente A, Crystal RG, LeCocq J-P: Synthesis in E. coli of aI-antitrypsin variants of therapeutic potential for emphysema and thrombosis. Nature (London) 1985; 313:149-51 lOB Schapira M, Ramus M-A, Jallat S, Carvallo D, Courtney M. Recombinant aI-antitrypsin Pittsburgh (MefR-+Arg) is a p0tent inhibitor of plasma kallikrein and activated factor XII fragment. J Clin Invest 1985; 76:635-37 109 Travis J, Matheson NR, George PM, Carrell RW Kinetic studies on the interaction of aI-proteinase inhibitor (Pittsburgh) with trypsin-like serine proteinases. Bioi Chern HoppeSeyler 1986; 367:853-59 110 Nukiwa T, Brantly ML, Ogushi F, Fells GA, Crystal RG. Characterization of the gene and protein of the common a 1antitrypsin normal M2 allele. Am J Hum Genet 1988; 44:32230 III Cox D~ Woo SLC, Mansfield T. DNA restriction fragments associated with aI-antitrypsin indicate a single origin for deficiency allele Pi Z. Nature (London) 1985; 316:79-81 112 Cox DW DNA polymorphisms associated with alpha-I-antitrypsin and their clinical applications. In: Mittman C, ed. Pulmonary emphysema and proteolysis: 1986. New York: Academic Press, 1987; 119-32 113 Kidd VJ, Golbus MS, Wallace RB, Itakura K, Woo SLC.

208

114

115

116

117

118 119

120

121

122

123

124

125

126

127

128 129

130

Prenatal diagnosis of aI-antitrypsin deficiency by direct analysis of the mutation site in the gene. N Engl J Med 1984; 310:639-42 Petersen B, Kolvraa S, Bolund L, Petersen GB, Koch J, Gregersen N. Detection of alpha.-antitryspin genotypes by analysis of amplified DNA sequences. Nucl Acids Res 1988; 16:352 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et aI. Prime~directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239:48791 Okayama H, Curiel DT, Brantly ML, Holmes MD, Crystal RG. Rapid, non-radioactive detection of mutations in the human genome by allele specific amplification. (Submitted for publication) Brantly ML, Paul LD, Miller BH, Falk RT, Wu M, Crystal RG. Clinical features and natural history of the destructive lung disease with alpha-I-antitrypsin deficiency of adults with pulmonary symptoms. Am Rev Respir Dis 1988; 138:327-36 Sveger T. a.-antitrypsin defiCiency in early childhood. Pediatrics 1978; 62:23-25 Sveger T. Liver disease in alpha.-antitrypsin defiCiency detected by screening of 200,000 infants. N Engl J Med 1976; 294:1316-21 Eriksson S, Carlson J, Velez R. Risk of cirrhosis and primary liver cancer in aI-antitrypsin deficiency. N Engl J Med 1986; 314:736-39 Psacharopoulos HT, Mowat A~ Cook PJL, Carlile PA, Portmann B, Rodeck CH. Outcome of liver disease associated with a l antitrypsin deficiency (PiZ). Arch Dis Childhood 1983; 58:882-87 Chan CH, Steer CJ, Vergalla J, Jones EA. Alpha.-antitrypsin deficiency with cirrhosis associated with the protease inhibitor phenotype SZ. Am J Med 1978; 65: 978-84 Cox D~ Billingsley GD, Smyth S. Rare types ofa.-antitrypsin associated with deficiency. In: Allen RC, Arnaud ~ eds. Electrophoresis '81. Berlin: Walter de Grayter and Compan~ 1981;505-10 Esquivel CO, Vicente E, Van Thiel D, Gordon R, March ~ Makowka L, et ale Orthotopic liver transplantation for alphaI-antitrypsin defiCiency: an experience in 29 children and ten adults. Transplantation Proc 1987; 19:3798-3802 Hood JM, Koep LJ, Peters RL, Schroter G~ Weil R III, Redeker AG, et ale Liver transplantation for advanced liver disease with alpha-I-antitrypsin deficiency. N Engl J Med 1980; 302:271-75 Putnan C~ Porter KA, Peters RL, Ashcavai M, Redeker AG, Starzl TE. Liver replacement for alpha-I-antitrypsin deficiency. Surgery 1977; 81:258-61 Gadek JE, Fulmer JD, Gelfand JA, Frank MM, Petty TL, Crystal RG. Danazol-induced augmentation of serum alantitrypsin levels in individuals with marked deficiency of this antiprotease. J Clin Invest 1980; 66:82-7 Cohen AB. The clinical usefulness of different forms of alphaI-protease inhibitor. Am Rev Respir Dis 1986; 133:349-50 Hubbard R, Sellers S, Czerski D, Stephens L, Crystal RG. Efficacy and safety of augmentation therapy of aI-antitrypsin deficiency with monthly infusions of aI-antitrypsin. JAMA 1988; 260:1259-64 Hubbard RC, Stephens L, Crystal RG. Delivery of a1antitrypsin by aerosol: direct augmentation oflung anti-elastase defenses in aI-antitrypsin deficiency. Clin Res 1988; 36(3):625A

The AJpha,-antitrypsin Gene and its Mutations (Crystal et al)