Alpha 1 antitrypsin deficiency caused by the alpha 1 antitrypsin Nullmattawa gene An insertion mutation rendering the alpha 1 antitrypsin gene incapable of producing alpha 1 antitrypsin

Amendment history:

Correction

(September 1989)

Alpha 1-antitrypsin deficiency caused by the

alpha 1-antitrypsin Nullmattawa gene. An

insertion mutation rendering the alpha

1-antitrypsin gene incapable of producing alpha

1-antitrypsin.

D Curiel, … , L Stier, R G Crystal

J Clin Invest.

1989;

83(4)

:1144-1152.

https://doi.org/10.1172/JCI113994

.

alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder associated with reduced

serum alpha 1AT levels and the development of pulmonary emphysema. An alpha 1AT

gene is defined as "Null" when no alpha 1AT in serum is attributed to that alpha 1AT gene.

Although all alpha 1AT Null genes have identical phenotypic consequences (i.e. no

detectable alpha 1AT in the serum), different genotypic mechanisms can cause the Null

state. This study defines the molecular basis for the alpha 1AT gene Nullmattawa, identified

and cloned from genomic DNA of an individual with the Null-Null phenotype and

emphysema resulting from the heterozygous inheritance of the Nullmattawa and

Nullbellingham genes. Sequencing of exons Ic-V and all exon-intron junctions of the

Nullmattawa gene demonstrated it was identical to the common normal M1(Val213) alpha

1AT gene except for the insertion of a single nucleotide within the coding region of exon V,

causing a 3' frameshift with generation of a premature stop signal. Family analysis using

oligonucleotide probes specific for the Nullmattawa sequence demonstrated the gene was

inherited in an autosomal fashion. Examination of blood monocytes demonstrated that a

normal-sized, 1.8-kb alpha 1AT mRNA transcript is […]

Research Article

Find the latest version:

al

-Antitrypsin

Deficiency Caused by the

al

-Antitrypsin

NUIlMattaWa

Gene

An

Insertion Mutation

Rendering

the

al

-Antitrypsin

Gene

Incapable

of

Producing

al

-Antitrypsin

D.Curiel,M. Brantly,E.Curiel,L.Stier,and R.G.Crystal

Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of

Health,

Bethesda, Maryland

20892

Abstract

al-Antitrypsin

(alAT) deficiency is a hereditary disorder

as-sociated

with reduced serum alAT levels and the development

of

pulmonary

emphysema.

An

alAT gene is

defined

as

"Null"

when no alAT in serum

is attributed to that alAT gene.

Al-though all alAT Null genes have identical phenotypic

conse-quences

(i.e. no detectable alAT in the serum), different

geno-typic mechanisms

can cause

the Null state.

This study defines

the

molecular basis for the alAT gene

Nullf,,,,,

identified

and

cloned from

genomic DNA of an individual with the

Null-Null

phenotype and emphysema resulting from the

heterozy-gous

inheritance

of the

Nullm.,w,

and

_Nullb.Iuigm

genes.

Se-quencing

of exons Ic-V and all exon-intron junctions of the

Null",,Vt.

gene demonstrated it was identical to the common

normal

Ml(Va1213)

alAT gene except for the insertion of a

single nucleotide within

the

coding

region of exon V, causing a

3'

frameshift

with

generation of a premature stop signal.

Fam-ily analysis

using oligonucleotide

probes

specific

for the

Nullmt,.

sequence demonstrated the gene

was

inherited in

an

autosomal

fashion.

Examination of blood monocytes

demon-strated that

a

normal-sized, 1.8-kb

alAT mRNA

transcript

is

associated with the

_Nul1tf,1,.

gene and in vitro translation of

mRNA

with

the

Null.,,,,

mutation showed it translated at a

normal rate

but

produced

a

truncated alAT

protein.

Addition-ally,

retroviral transfer

of the alAT Nulls wa cDNA

to

mu-rine

fibroblasts

demonstrated

no

detectable

intracellular

or

se-creted

alAT,

despite

the presence of alAT

Null,,twa

mRNA

transcripts.

These

findings

are

consistent with the concept that

the molecular

pathophysiology

of

_Nullf,,wa

is

likely

mani-fested

at a

posttranslational

level.

The

identification

of the

Null~t.,,,

gene

supports the concept

that

Null

a1AT

alleles

represent a

heterogenous group in which very different

mecha-nisms cause the

identical

phenotypic

state.

Introduction

al-antitrypsin

(alAT)'

deficiency

is

an

autosomal

hereditary

disorder, characterized

in adults

by

reduced

serum a

1

AT

levels and the

development ofpulmonary emphysema by

ages

30-45

(1,

2).

With

the

knowledge

that the

major physiologic

Addressreprint requests to Pulmonary Branch, National Heart, Lung, andBloodInstitute, Building 10,Room

6D03,

National Institutes of Health, Bethesda,MD20892.

Receivedfor publication29December 1987 and inrevisedform18 October 1988.

1. Abbreviations usedin thispaper: alAT, al-antitrypsin; DLCO, diffusing capacity;

FEV,,

forced

expiratory

volume in one

second;

FVC,forced vitalcapacity; TLC,totallungcapacity.

TheJournalofClinicalInvestigation,Inc. Volume83, April 1989, 1144-1152

role of

a

lAT is

to

inhibit neutrophil elastase,

we understand

that the

pathogenesis

of the

emphysema

associated with a

IAT

deficiency

results from insufficient

alAT,

which

provides the

lower

respiratory

tract

protection against the chronic burden

of elastase in the local milieu (3, 4). As

a

result,

over

several

decades, there is progressive

destruction of the alveolar

walls,

culminating

in the clinical

disorder, emphysema.

alAT

is coded for by

a

single

copy gene

of 12.2 kb

on chromosome 14 at

q31-32.3

(5-7). The coding exons of the

gene are

highly pleomorphic, with > 75 alleles described (1, 8,

9). The

alAT

phenotype is determined by codominant

ex-pression of the

parental alleles (1, 8, 10). The a IAT deficiency

state

associated with emphysema occurs only when there are

abnormalities in both ofthe parental genes (8, 10,

1

1). In most

cases there is some a1AT

detectable

in

serum, albeit in

re-duced

amounts

(1, 2, 8, 11). For example, the commonest

form

of alAT

deficiency associated with emphysema results

from the homozygous inheritance of the Z allele, a

circum-stance

that results in

aIAT

serum

levels 10-15% of normal (1,

8,

1

1).

There are

also

alAT

genes causing an absolute

defi-ciency

state

(1O, 12-23). This category of

a

IAT

genes,

referred

to as

the Null

alleles, represents

a

lAT genes

in which the gene

is present but does not code for an a

1

AT

molecule that is

detectable

in

the

serum

(1O, 19). Compared with the deficiency

alleles, the Null genes

are

rare,

with an estimated allelic

fre-quency of

<

0.001 in

Caucasians

of

Northern European

de-scent

(13).

Although

all

Null alleles were

originally

thought to

be

similar,

it is

now

recognized

that the

a

lAT gene can be

rendered Null

by

different

mutations (21-23).

The purpose

of

this

study

is to demonstrate

a

newly

recognized

type

of

muta-tional

event

that

can cause

the

a1 AT

Null state,

Nullmaw,,w.

a

nucleotide insertion

in a

coding

exon,

causing a

3' frameshift

with generation

of a stop signal many codons distal to the

mutation.

Interestingly, unlike

Nullbe,,innm,

in which a stop

codon

is associated with

a

lack of detectable alAT

mRNA

in

aI

AT-synthesizing cells,

the

Nullma,,,wa

gene codes for

detect-able

aIAT

mRNA,

but

no

detectable

a

lAT

protein

produced

by

these cells.

Methods

Identification

ofthe Null-Nullphenotypein twoindex cases. The Null-Null alATphenotypewasidentified intwosisters(indexcase1 and indexcase2)usingacombination ofisoelectricfocusingofserum at

pH 4-5toidentifyalAT alleles,serumalATlevels(radial

immuno-diffusion),andfamily analysis (Fig. 1) (10).Toconfirm that the index

cases weretrulyNull-Null,theserum wasalsoevaluated for the

pres-enceof aIAT usingan enzyme-linked immunoassay sensitiveto 2

nM2

(24). Thetwoindexcasesboth had clinical evidence of emphy-sema.

2.a1ATlevelsexpressedinmilligramsper deciliterarebasedonthe

commonly used commercial

standard,

whereasthose inmicromolar

are based on a true laboratory

standard;

the commercial standard overestimates aIAT levelsby35%(seereference24for

details).

Figure

1.

Family

tree

demonstrating

M2 Null Ml Null the inheritance of Null alleles.

147 ₁₂₉ a AT_phenotypesweredetermined

by evaluating

thealAT patternsin isoelectric

focusing

ofserum,alAT

levels inserum,andfamily analysis.

r

_-l

l

1

1

l

,

j

%

Family

membersnotavailable for

r

0[2

'_-o

2O*___,* @ 0 '",,8 ,_,_J@ s t) 2 linedevaluation are indicatedcirclesorsquares.Thebybroken-two M2Null Null Null M2Null

129 0 138

Indexcase 1

Null Null

o

Index case 2

MI Null 166

Null Null M2Null

0 138

indexcases are indicatedbyarrows.

Thephenotype is listed below each family membertogetherwith the serumaIATlevel(milligramsper

deciliter).

Index case 1, a 39-yr-old female who had had dyspnea for 10 yr, had no smoking history. Severe pulmonary emphysema was docu-mentedby physical examination showing a hyperresonant chest and

distantbreath sounds; chest x ray demonstrating hyperinflation, flat-tenedhemidiaphragms, and a marked loss of vascularity at both bases; xenon-127ventilation scan showing abnormal retention of gas in the lower lobes and a technetium-99m perfusion scan depicting loss of vascularity in the same regions; pulmonary function testing (25) re-vealing that vital capacity(VC)was 42%predicted, total lung capacity (body plethysmography) was 122% predicted, forced expiratory vol-ume in 1 s(FEVI)was25%predicted,FEV1/forced(F) VC was 50% observed(69% predicted value), and diffusing capacity (DLCO; cor-rected for volume and hemoglobin) was 41% predicted.

Index case 2, a 31-yr-old female without pulmonary symptoms, had nosmoking history. The physical examination and chest x ray werenormal. The presence of mild emphysema was documented by a xenon-127ventilationscandemonstratingabnormalretention of gas in the lower lobes, atechnetium-99m perfusion scan showing comple-mentary lossofvascularity, and lung function testing revealing VC was 93% predicted,TLC was 114% predicted, FEVI was 78% predicted,

FEV1/FVC

was65% observed (82% predicted), and DLCO was 87% predicted.

EvaluationofgenomicDNAusingoligonucleotide gene

probes.

To determine if the Null-Nullphenotypesof the indexcases weredueto

the inheritance of the alAT Null alleles Nullbellingham (21) or

NUllgrnit,

fll. (22), genomicDNAof the indexcasesand relevant con-trolswereevaluatedusinggelhybridizationwitholigonucleotidegene probes by the methodofKiddetal.(26)asmodified by Satohetal. (21). Genomic DNAofindex case 1, a normal

M1(Val2I3)

homozygote

(27),a

_Nul1eii,,jam

homozygote,andanMl(Val213)

_Nullg,,ite

fajls

het-erozygoteweredigestedwith the restriction endonuclease PstIandthe DNA waselectrophoresed on 0.7% agarose (8

_;ig/lane).

Thegels were

hybridizedat55°Cwitholigonucleotidegeneprobescenteredonthe sequence of interest (seeFig. 2,top for the sequences used).The

oligo-nucleotide probes were

5'-labeled

with [32P]ATPand separatedfrom

unincorporatedlabelon an8%polyacrylamide-8M ureadenaturing gel. After hybridization, thegels were washed at 60°C in 25 mM

sodiumphosphate buffer,pH7.0,0.45MNaCl,2.5mMEDTA,0.1%

SDSfor 15 min andautoradiographedat-70°C for2 wk.

Cloning

and sequencing

of

the

Nullmnaawa

gene. After

oligonucleo-tideanalysisofgenomic DNAof indexcase 1 demonstrated that the Null-Null state in this family was caused by inheritance of the

Nullbjiji&m

alleletogether with a previously unidentifiedNullallele

(referredtosubsequently as

NulImaI..tt

basedonthebirthplaceof index

case1). The

NullmatItw.

genewascloned fromgenomicDNAof index

case I by conventional methods.UsingDNAfrom skinfibroblasts,a

cosmid library was prepared by inserting size-fractionated partial

Mbo I-digestedDNA into the vector C2RB (28) with subsequent

packaging andtransfection into Escherichia coli 1046 as previously

described byvanOmmenet al. (29). The primary library was plated

out on 20 90-mm dishes at adensity of 40,000 colonies per dish.

Duplicatefilterswerepreparedand the filtershybridizedunder stan-dard conditionsusinga32P-labeled humana1ATcDNAprobe.Clones

containingtheregion oftheaIAT genewereconfirmedbyrestriction enzymeanalysis usingEco RI. To select for theNullmanawa allele, cos-mid clonescontainingtheaIAT genewereevaluated

using

oligonucle-otidegeneprobesasdescribedabove. Because the genotype of index

case1 had beendefinedasheterozygous

_{Nullbiiingham}

Nullmattawa,

those clones which didnotcontainthe

NUllbelfinam

sequenceatresidue 217 by definition containedthe

Nullmatta,

allele. Asingle40-kb cosmid clone that contained the entire alAT

Nullmatt,,.

geneplus flanking regions was selected and subcloned into

pUC19

by standard tech-niques (30). The recombinant subclones obtained contained 0.5 kb

encompassingexonIc, 1.6 kb

encompassing

exonII,2.4kb

encom-passingexonsIIIandIV,and 1.1 kb

encompassing

exonV(see

Fig. 2,

top, for the overall normala1ATgenestructure).Thesesubcloneswere usedastemplates forsequencing

using

thedideoxynucleotide chain

terminationmethod(31)withbidirectional

primers

of15-mer

oligonu-cleotides(30). Sequencingincluded 150bp5'toexon

Ic,

exonsIc-V

togetherwith the intron-exonjunctions,and 40bp3'to exonV.

Demonstration

of

inheritanceof the

Nu11,na,,awa

allele. To

demon-strate the inheritance ofthe

Nullmatwa,8

mutation, oligonucleotide

probeswere prepared that were complementary to both the region

centeredat residue 353 in exon Vwith the insertional mutation in

Nullmawa

and tothe normal sequence in this region. Genomic DNA from index case 1, index case 2, thefatherof the index cases, and an

Ml(Val213)

homozyogotecontrol were cut with Pst I and evaluated with labeled oligonucleotide probes using the methods described above.

Evaluationof

aJAT

mRNA transcripts

from

cellsexpressing the

aJAT

gene. Togain insightinto the consequences of the

NuIlman,,a

mutation, blood monocytes, cells known to normally express the

alATgene(32, 33)wereevaluatedforthe presence of alATmRNA

transcripts. Monocyteswereevaluated fromindex case 2 and com-paredwiththosefromanormal

MI(Val2'13)

homozygote control.

Al-though index case 2 (like indexcase 1) is a

_{Nullbninom-Nullmanawa}

heterozygote, the consequences of the

Nullmata,

allele could be di-rectly evaluated independent of the

_{Nullbeffinham}

allele because (a) the parental

a1AT

genes are codominantly expressed, i.e.,

a1AT

gene

expressionof one allele is independent of the other parental allele (1, 8, 10); and(b)the

_{Nullbifingham}

gene is known to be associated with no

aIATmRNA transcripts and no production of aIATby blood mono-cytes(20).

Blood monocytes wereisolated from index case 2 and a normal

M1

(Val2"3)

homozygoteusing adherence purification of mononuclear cellsobtained by monocytapheresis. Blood mononuclear leukocytes were harvested by cytapheresis on a cell separator (model 2297; IBM Instruments, Inc., Danbury, CT) during continuous centrifugation by standardtechniques (34). The mononuclear cells were separated from

150-mm tissue culture plates for 1 h at 370C. The nonadherent cells were removed and the plates were washed three times with PBS, pH 7.4. Theresulting adherent cells were harvested by scraping, washed threetimes,withPBS, and replatedasabove.The resulting cell popula-tions were >90% monocytes (morphology and nonspecific esterase

staining) and had >95% viability (trypan blue exclusion). alAT mRNAtranscripts in the monocytes were evaluated by Northern

anal-ysis(33). Total cellular RNA (10 ,Ag/lane) prepared by guanidine HCl extraction followed by CsCl centrifugation was electrophoresed in

aga-rose gels under denaturing conditions, transferred to nitrocellulose filters, hybridized with a32P-labeledalAT cDNA probe, and

autora-diographed.

Analysis of translation of NUilmattawa aJATmRNA transcripts. In vitro translation analysis was used to examine the effect of the

Nullmat.awa

mutationonthe translation of the al AT mRNA transcript intoprotein.Toaccomplishthis, ana1 AT

_Nullmat.awa

cDNA was con-structed fromanormal alAT

Ml(Val2"3)

cDNA(pPBO1)by standard

techniques. BoththismutantalAT cDNA andanormalMl(Val213)

aIAT cDNA were usedtoconstructplasmidsfor use in the Riboprobe (Promega-Biotec) SP6 polymerase in vitro transcription system. The M

l(Val213)

a1AT cDNAcontaining plasmidand the

Nullmant,.

alAT

cDNA containing plasmid servedas templates forgeneration of capped,poly-adenylated syntheticalATmRNAtranscriptsof normal

MI(Val213)

type or mutant

Nullmantwa

type, respectively (35). After

purification, 1 ,ug of each alAT mRNA specieswasused todirect translation (1 hat37°C) inarabbitreticulocyte lysate system

(Pro-mega-Biotec)with [35S]methionine used asa label. Aliquots ofthe reaction mixtureswereanalyzed bySDS-acrylamidegel

electrophore-sisandfluorographyandquantified bylaserdensitometry.

Retroviralgenetransfer

of

human

MJ(Val213)-type

and

Null,,xtawav

type alATcDNAs. To examineposttranslationaleventsinal AT

bio-synthesis,

Ml(Val2

3)and Nullmatawatype alAT cDNAs were

trans-ferredtomurine fibroblastsusing retroviralgene transfer. The

retro-viral vectors used to insert the full length M

l(Val213)-type

and

Nullma.,,a-type

alATcDNAs into the genome ofmousefibroblasts were constructed from the N2 vectoraspreviouslydescribed (36). The final retroviral vectors (pN2-alAT/Ml and pN2-a lAT/Mattawa)

contained(5'to3')theSV40earlypromoter and fulllengthhuman alAT cDNAsofM

l(Val213)

typeor

NUllmatawa

type, respectively, in-serted into the Xho Isite of N2. Sequence analysisconfirmed that

pN2-alAT/Mattawa differedfrompN2-alAT/Ml by onlythesingle

nucleotide insertioncorresponding to the

Nullmatwa

mutation. The helpervirus-freepackagingcell line

_0t2

(37)wasusedtopackage

tran-scriptsfrompN2-alAT/Ml and

pN2-a1AT/Mattawa

intoecotropic

infectious viralparticlesthatwerethenusedtoinfect NIH-3T3 cells.

Polyclonal populations ofinfected NIH-3T3 cellswerescoredat> 100

colonies/10-cm plateand were

equivalent

for the human alAT

Ml(Val2'3)-type

and

Nullmat5,wa-type

cDNA

containing

cell popula-tions,referred to asNIH-3T3/alAT-Ml and

NIH-3T3/alAT-Mat-tawa,respectively.

alAT mRNA levels in mousefibroblasts. HumanalAT mRNA

transcriptswereidentified in

NIH-3T3/a

lAT-M1 and NIH-3T3/

alAT-Mattawa cells by

cytoplasmic

dot

hybridization (38).

Unin-fected NIH-3T3 cells were used ascontrol. Extracted cytoplasmic

RNAwasdenatured andappliedtonitrocellulose filters witha mini-fold apparatus(Schleicher&Schuell, Keene, NH)inserialdilutions. Thefilterswerehybridized usinga32P-labeled human alATcDNA probe. Exposure of theautoradiogramswasfor 48 hat-70°C.

Synthesisand secretionofhuman alA T by mouse

1ibroblasts.

The

synthesisandsecretionof human alAT byNIH-3T3/alAT-Ml and

NIH-3T3/alAT-Mattawa cells was evaluated by immunoprecipita-tion of

[35S]methionine-labeled

alAT as previously described (36).

NIH-3T3/alAT-Ml andNIH-3T3/alAT-Mattawacells were plated (5X105cells per 60-mm plate); the next day, each plate was incubated for 10 min in methionine-free Iscove's minimal essential medium

containing 10% dialyzed calf serum, pulsed with 250 ,uCi of

[35S]-methionine (600 Ci/mmol) for 30 min, then chased with nonselective media supplemented with 1 mMunlabeledmethionine for 120min.

Tonormalize thetwostudy cellpopulations, aliquots of supernatants

containingI05dpmandlysates containing 106 dpm of TCA-precipita-bleprotein were compared. Immunoprecipitation, gelelectrophoresis,

andfluorographywere aspreviouslydescribed(33).

Results

Evaluation of index case

I

for the presence

of the

Nullgraniie

falls

and

the NUllbellingham alleles.

Using

oligonucleotide

probes

spe-cific forthe sequence defining the

_Nulluanite

falls

mutation, the

Nullbellingham mutation,

and the

corresponding

normal se-quences of these regions, it was apparent thatthea1AT

geno-type of

index

case 1 was heterozygous Null-Null containing the

Nullbeillingham

mutation

at one allele and a differentalAT Null

mutation

in

the

other allele (Fig. 2). In this regard, genomic DNA

of

index case 1 had a sequence that was normal inthe

regions

defining the

_NU11granite

fallsmutation (compare lane 6

withlane 5, along with the controls in lanes 1-4). Incontrast,

the

genomic

DNA

of index

case 1 clearly contained the

Nullbellingham

mutation in one gene (lane 12), and a normal sequence in

the

Nullbeingham

mutation region in the other (lane 11, along with controls lanes 7-10). Together, these findings

indicate that index

case 1 must be a heterozygous Null-Null,

with

one

_Nullbeingham

alleleand another, differentalAT Null

allele, which

possesses the normal sequence at positions

214-220 (the region of

the

Nullbellingham

mutation).

Identification

ofthe Nuilmattawa mutation by sequence

analy-sis.

Sequencing of

exons Ic-V, all exon-intron junctions, and the 3'

flanking region of the

cloned a1 AT

_Nullmatawa

gene of

index

case 1

demonstrated

that it was identical to that of the

normal

Ml

(Val2'3)

a1 AT gene except for a

single

nucleotide

insertional mutation in

the exon V coding region at residue 353 of the

primary

amino acid sequence (Fig. 3). The 217

region

of the

Nullmattawa

allele, the region of the

_{Nullbellingham}

mutation

site,

was

similar

to the normal

a1AT

Ml(Val213) gene. This novel mutation causes a 3' frameshift, resulting in

an

altered

reading frame commencing

at the codon for amino acid 353. The new

nucleotide

sequence codes for a

signifi-cantly

altered

amino acid

sequence distal to the insertion site

and

terminates in

apremature stop signal at new codon 376.

Demonstration ofinheritance ofthe

NUilmattawa

gene.

Evalu-ation of genomic

DNA

of

the

index

casesand the father of the

index

cases

using synthetic oligonucleotide

probes centered at

the

353

region

and

specific for

the

_NUllmatawa

mutation se-quence or

the

corresponding

normal sequence,

demonstrated

that the

NUllmatuawa

nucleotide insertion

mutation had been

inherited

in

an

autosomal

fashion (Fig.

4). In this context,

specific hybridization

of the

Nullmatawa

probe to the genomic DNA

of index

case 1 (lane 2), index case 2 (lane 4), and the

father (lane 6), yielded

a 1.

1-kb

band corresponding to the Pst I

fragment

containing

the mutated sequence in exon V.

Geno-mic

DNA

of both index

cases and the father also specifically

hybridized

with the

normal 350-356 probe. In the case of the

father,

the

alternate allele

is

M2

(Fig.

1) and thus is identical to the normal

probe in

the 350-356 region. Likewise, even

though

both

index

cases

have

the

_{Nullbellingham}

allele as the alternate

allele, they

are normal in the 350-356 region (the

Nullbiingham

substitution mutation

is centered at residue 217). Based on

this analysis, it

may be deduced that the genotype of the mother

is

M

lNullbeiingam

despite the lack of direct analysis

of

her DNA. Together, these observations confirm both the

1

kb

m

1 6 24 -kb

IA

I_I

IB IC

_{e S}

II

n~~II

III

IV

I

V

V

157 158 159 160 161 162 163 164 214 215 216 217 218 219:

lie

Asn Asp Tyr Val Glu Lys Gly Thr Thr Val Lys Val Pro I

57-164probe TC AAC GAT TAC GTG GAG AAG G

Normal214220probe

ACC ACC GTG AAG GTG CCTA

*eFalls probe

TC

AAC GAT TAG TG GAG AAG G

NUlBellingham

probe ACC ACC GTG TAG GTG CCTA

lie Asn Asp stop Thr Thr Val stop

Ml(VaI213) Index

NUIlGranite

Falls CaSe 1

Normal Normal

157-164 Nullf 157-164

Nullgf

Ml(VaI213) M1(VaI213)

Normal

214-220

NUlIlb

NUIlBellingham

NUIIBellingham

Normal 214-220

NUlIlb

Ml(Val213)

Ml(VaI213) Normal

157-164

Nullgf

2.4

kbo-220 Met kTG

kTG

Index

case 1

Normal 214-220

Nullb

1.6kb_- w

1 2 3 4 5 6 7 8 9 10 11 12

Figure2. Evaluation of indexcase1 for thepresenceoftwopreviouslydescribedNullalleles,Null.gnit5 fails(22)andNullb,1ingh..m(21).The

oligo-nucleotidegeneprobesused included:probescenteredonthe

_NU11gnit

failsdefect(Nullgf)orthecorrespondingnormal sequence forresidues

157-164(Normal,57-164)andprobesthat centeredonthe_Nullmiin,,mdefect(Nullb)orthecorrespondingnormal sequencefor residues 214-220

(Normal2l4

220). Shownatthe top is thestructureof theaIATgene.ExonsIA-kcontain untranslatedregions (forhepatocytealATexpression, the 3'part ofexonIcandashort 5'segmentofexon_II;for mononuclearphagocyte expression,IA, B1,Icandashort 5'segmentofexonII;see

references 6, 7,and9),whereas thecoding regionsfor theproteinareinexonsII-V (6).Both thesequencesof theoligonucleotide probesand thecorrespondingcoded amino acids in theprimary proteinstructurearedepicted;themutationalchangesareunderlined. Theprobeswere

la-beled and usedtoevaluate PstI-(P) digested genomicDNA from various sources; Pst Iwasused because itseparatestheexonswiththe

Null-granitefalls andNullbellinghammutations(seediagramandtext).Lane 1,genomicDNA of an

MIl(Val213)

homozygotecontrol evaluated with

Nor-Mal,57164

probe. Lane2,identicaltolaneIexcept evaluated withNullgranitefallsprobe. Lane3, genomicDNAofMl(Val213)Nullgrt falls

hetero-zygote evaluated withNormal,57-164probe.Lane4,identicaltolane3exceptevaluated withNullg,.,,iefallsprobe.Lane5,genomicDNAofindex

case I (see Fig. 1)evaluated withNormal157164probe.Lane6,identicalto lane5 exceptevaluated withNull.je,,fallsprobe.Lane7, genomic DNA ofanMIl(Val2'3)homozygotecontrol evaluated withNormal214220probe. Lane8,identicaltolane7exceptevaluated with

Nulhb,jinglam

probe. Lane9, genomicDNA of

_Nulljignm

homozygoteevaluatedwithNormal214220probe. Lane10,identicaltolane9except

evaluated withNullbeiinghamprobe.Lane11, genomicDNAof indexcaseI evaluatedwithNormal214220probe.Lane12,identical to lane 11 ex-cept evaluated withNullbeinghamprobe.

autosomal inheritance of the

NUllmaa

allele in this family

and theheterozygous

Nullbelingham-Nullmatawa

phenotype of the

indexcases.

Evaluation

of

alA

T-producing

cells

for

mRNA

transcripts

associated with the

Nuilmatawa

gene. Because the

_Nullbeeffio.

allele is associated withthecompleteabsence ofa_1ATmRNA

in alAT producing cells (20), any alAT mRNA transcript

exhibitedbyblood monocytes of the indexcasemustrepresent the exclusiveexpressionofthe

Nullma.,,,

allele.Inthisregard,

Northernanalysisof RNA ofindexcase2showed thata1AT

mRNAtranscriptwasexpressed(Fig. 5). Furthermore, itwas

1.8 kb long (lane 2),similartothe alAT mRNAtranscriptsof

a normal Ml(Val213) homozygote (lane 1).It appears,

there-fore, that thepresenceofa stop codonnearthe 3' end of the

a1ATmRNAmoleculegeneratedby the

Nullmatt,,,

mutation

is not associated with the absence of alAT mRNAasis the

case for the

_Nullinghanm

mutation,in which the stopsignalis

generatednearthecenterof thecoded aIAT mRNA.

1 kb

m

P P P P

IA

IB IC II III IV V

5'

H}O - I _{- 3}

3'

348

349

350

351

352

353

354

355

356

Ala

Gly

Ala

Met

Phe

Leu

Glu

Ala

lle

G--G

C

-T -G G

EG

-G c -c _A

T

-G

T

-T

T T -A

G

A -G

.G c .-c

A

_/

T -A

Ml

(Val213)

G ATC

NUIlMattawa

G ATC

II.

40

...t

c-

]

Ala

._ T

_VW

G-GJ

Gly

G

_NNOW

c

_Ala

_ c

IP A

-

T

_Met

_m*

T-=

T

Phe

T

_-Tl

Phe

A] G

_Arg

HGi

c_

Gly

XAT

His

C

352 354 356 358 360 362 364 366 368 370 372 374 376

Ml(Val213) Phe Leu Glu Ala lie Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met lie Glu

...TTTTTAGAGGCCATACCCATGTCTATCCCCCCCGAGGTCAAGTTCAACAAACCCTTTGTCTTCTTAATGAATGAA...

3' Frameshift

TTTTTTAGAGGCCATACCCATGTCTATCCCCCCCGAGGTCAAGTTCAACAAACCCTTTGTCTTCTTAATGAT ..A..

NUlIMa"taw Phe Phe Arg Gly His Thr His Val Tyr Pro Pro Arg Gly Gin Val GIn Gin Thr Leu Cys Leu Leu Met Aspstop,

352 354 356 358 360 362 364 366 368 370 372 374 L3Z6J

Figure3.Identificationof

Nullmatt,,,

mutation inexonVbysequenceanalysis. (A)SchematicrepresentationofthealATgene, seelegendto Fig.2for details. The PstI (P)subclonesused forsequencingareindicated above thegene;the actualareassequencedareshownassolidlines

below thegene.Thesequence wasidentical to that of theaIATMl(Val2'3)geneexcept foraregioninexonV.(B) Evaluation of the348-356 regionofexonVfor theMl(Val213)and

Nullma.,,w

alleles.Athymidineinsertion(arrow)inthe codon foramino acid353 is the singlechange

in theNullmat,. allele.(C)The nucleotide insertionin codon 353causes aframeshift in the3'direction,which alters thereading frame and generatesastopsignalatthenewcodon 376.

TranslationoftheNuilmatawa alATmRNAtranscript. Eval-uation of the translation oftheNullmatwa aIATmRNA tran-scriptdemonstrated that itwas_capableofdirectingthe

synthe-sis ofanalATprotein productinafashioncomparableto the

normal Ml(Val213) alAT mRNA transcript, except that, as

expectedforamutationcausingastop codon,the translation

productwastruncated(Fig. 6).Inthiscontext, invitro

trans-lation ofasyntheticalAT Ml(Val213) mRNAtranscript

di-rected synthesis ofa 47-kD aIAT protein species (lane 1), correspondingtothenonglycosylatedalATproteinincluding

the 24-amino acid signal peptide. In contrast, the a AT

Nullma.tawa

mRNAtranscriptdirectedthesynthesisofa45-kD

aIAT protein species (lane 2), corresponding to theprimary

translation productofaIATminus the 19 amino acids 3' to

the premature termination codon of the NUllmattawagenethat

arenottranslated intoprotein.Quantificationof the amounts

of alAT directedby the Nullmaawa mRNAdemonstrated it

was similarto that directedbythe normal mRNA(P> 0.2;

two-tailedttest).It thusappearsthat the insertional mutation of the Nullmattawa gene does not alter the capacity of the

Nullm.,twa

mRNAtranscripttobetranslated intoproteinand

that,therefore,themolecularpathophysiologyaccountingfor the aMAT Null state associated with thisgene is likely not manifestatthe translationallevel.

HumanalA Tgeneexpressioninmurinejibroblasts

modi-fied to contain the human aJAT of MJ(Val213)-type and

Nullmtawa-typecDNAs. Retroviralgenetransfer of the human

alAT cDNAs of the normal M

l(Val2'3)-type

and the mutant

1148 D.Curiel,M.Brantly,E. Curiel,L.Stier,and R. G. Crystal

A

B

348

349

350

351

352

353

354

355

356

p p p p

2.4 ₁

p p

1.1 kb

Normal350-356

probe

NUIlMaftawaprobe

Index case 1

Normal 350-356 Nullm

1 .1

kb--1 2

ExonV

350 351 352 353 354 355 356 Ala Met Phe Leu Glu Ala lie GCC ATG mTr TTA GAG GCC ATA GCC ATG mT UT AGA GGC CAT Ala Met Phe Phe Arg Gly His

Index case 2

Normal 350-356 Nullm

Father

Normal 350-356 Nullm

.*

3 4 5 6

Figure 4.Determination ofthe in-heritanceoftheNullma,,awaallele usinganoligonucleotide probe spe-cific fortheNUilmattawa mutation. Synthetic21 -meroligonucleotide

probescenteredin exonVaboutthe

NUilmattawamutation (Nullm)or the

corresponding normalsequencein theregion codingforresidues 350-356

(Normal350356)

were used toevaluate genomic DNA. Shown

arethesequencesof the probesand

thecorrespondingcodedamino acid

sequenceoftheprimary protein

structure.Thenucleotide insertion in

Nullmattaw

isunderlined. Geno-micDNA wasdigested withPst I

(P), an enzymethat cuts on either

side ofexonV, renderinga1.1-kb fragment. Lane1, genomicDNAof indexcase1 evaluated withthe

Ml

(VaI213)

Normal350356probe.Lane 2,

identi-Ml

(Val213)

cal to laneI except evaluated with the Nullmattw.probe.Lane3,

geno-Normal mic DNA of index case 2 evaluated 350-356 Nullm with the

_Normal350356

probe. Lane

4, identicaltolane 3except evalu-atedwith theNullmattawaprobe.

Lane5,genomic DNAof the father of the indexcasesevaluated with the Normal 350-356probe.Lane6, identicaltolane 5exceptevaluated with the Nullmattawaprobe.Lane 7,

genomicDNAofMI

(Val213)

homo-zygotecontrol evaluated with the Normal350-356 probe.Lane8, identicalto lane 7except evaluated 7 8 with theNullmatawaprobe.

Nullma.n..

type to

murine

fibroblasts

demonstrated that the

aIAT

Null

state

associated with

the

_Nullmant.,

gene

could

be

reproduced in

cellsthat do not normally produce human

pro-teins,

and donot

normally produce

any

form

of

aIAT

(Fig.

7).

Cells

modified

to

contain

the normal alAT M

l(Val213)-type

cDNA

exhibited

humanaIAT gene

expression

atthe mRNA and

protein

level.

In contrast,

whereas the cells

modified

to

contain the

alAT

Nuilmaa,,a-type

cDNA expressedequivalent

Ml

(Vat213)

Index

Ml

(Vat213)

case 2

1.8

kb.-1

Figure5.Northernanalysis of alATtranscriptsin blood

monocytesofindexcase2and

Ml(Val213)

homozygote

con-trol.Total cellularRNA

(10

*

_zsg/lane)

wasevaluated

using

a

32P-labeled

human alAT cDNAprobe.Lane1,

MIl(Val2

3)homozygote.Lane

2,indexcase2. Theposition

andaveragelengthof normal

Ml(Val213)

alATmRNA tran-2 script is indicated.

amounts

of

alAT mRNA

transcripts,

no aIAT

protein

prod-uct

could

be

detected.

In

this

regard, cytoplasmic

dot

analysis

of

RNA

isolated

from the cell

populations

using

a

32P-labeled

aIATcDNA

probe demonstrated

comparable levels of

a

lAT

mRNA

transcripts in the NIH-3T3/alAT-Ml

and

NIH-3T3/

alAT-Mattawa

cells

(Fig. 8 A). This

finding

is

consistent with

the

concept

that

the

Nullmatawa

alAT mRNA

transcript

has

comparable

stability

tothe normal

Ml(Val213)

alAT mRNA

M1(Val213)

Nulllan.,a

Figure 6.Invitro translation of

Nullma.t,.

and

Ml(Val213)

alAT mRNAtranscripts. Synthetic alATmRNAtranscriptsderived by in vitro

transcription

of

Nullmatt,.

and

Ml(Val213)

alAT-kD type cDNAs, respectively, were

47-o- _usedtodirect

alAT

_protein

syn-45-s- _{thesis in}

arabbit

reticulocyte

ly-satesystemwith[35S]methionine

as alabel. Shownarefluorograms

ofSDS-acrylamide gel analysisof

1 2

[35S]methionine-labeled alAT.

Lane1,

Ml(Val2

3)aIAT. Lane2,

Nullmatt.w,

alAT.Thepositionsof the47-and 45-kD

primary

trans-lation products ofMI

(Val213)

and

Nullmat,.,

mRNAs,respectively,

areindicated.

pBR 322 S'LTR

300bp

Figure 7. DNAplasmidmapof thepN2-alAT/Ml andpN2-alAT/ Mattawaretroviralvectorscontainingfull-lengthhumanal

-antitryp-sin cDNAs ofMl-typeand

Nullma.,awa-type,

respectively.The

pN2-alAT/Ml andpN2-alAT/Mattawavectors wereformedfrom

pN2bycombining (5'-3') the SV40early promoter and the forward

orientation of thea1ATcDNAsof

Ml(Val213)

type and

NUllmalwa

type,respectively, into the XhoIsite.The

Ml(Val2

3)-typeand

Nullmatawa-type

a

lAT

cDNAsdifferby asingle-nucleotide insertion

causingthe replacement of leucine by phenylalanineataminoacid

position353ofthe coded protein anda3'frameshift withthe

genera-tionofapremature stopcodonatposition 376.

transcript.

However,

despite

the

presence

of

a

1 AT mRNA

transcripts which differ from

M

l(Val2"3)

alAT

mRNA

tran-scripts

by

only

a

single

nucleotide

insertion, murine fibroblasts

containing

the

Nullmttaw5-type

cDNA

exhibited

no

detectable

human a IAT protein

(Fig.

8B). In this context,

labeling

with

[35S]methionine

for 30

min

followed

by

a

120-min

chase

showed

that the

murine

fibroblasts containing

the

Ml(Val213)-type alAT cDNA secreteda 52-kD mature form of human aIAT

specifically immunoprecipitated by

anti-a

lAT

anti-body

(lane

1).

In

marked

contrast, no

secreted human

aIAT

could be detected

from the

Nullmallwa

cDNA-containing

cells

(lane

2).

Analysis

of the cellular

lysates

after

a

30-min pulse

labeling

with

[35S]methionine

for the

NIH-3T3/alAT-Ml

cells

showeda

50-kD

precursor form of humana1 AT

specifi-cally

immunoprecipitated by

anti-a lAT

antibody (lane 3).

In contrast,

examination of

lysates

of

NIH-3T3/alAT-Mattawa

cells

demonstrated

no

detectable

intracellular

alAT

(lane 4).

Thus,

although

it is conceivable that the truncated

a

1AT

pro-tein associated with the

Nullmallawa

gene is

stable but

not

de-tected

by

the

polyclonal

anti-alAT

antibody used,

it ismore

likely

that if

a

truncated

protein

is

produced, it is degraded

rapidly

and thus not

detected.

Discussion

Although

the consequences

of all

al AT

Null alleles

are

identi-cal,

i.e.,

no detectableserum

alAT,

it is

becoming

apparent

that

several different

mutational

eventsmay

render

an a

1

AT gene Null. To

date, all

result

from mutations in

exons

coding

for

the mature

form of

the a l AT

protein.

Nullbewnam

results from a

nucleotide

substitution mutation in exon III,

causing

the

substitution of

a stop

signal

for

a

lysine residue

(Lys2"7

AAG -.

stop2"7

TAG) (21).

In contrast,

_NUll1granie

_falls

results

from

a

single

nucleotide

deletion mutation

inexonIIatamino

acid

position

160

causing

a5'frameshift of the

_reading

frame

with

the

consequent

generation

of a

premature stop

signal

at

the codon

position

of the mutation

(Tyr'60TAC

Val'6'GTG,

deletion of C in codon TAC for residue

160,

and 5'

frameshift

forming

stop160TAG) (22).

The

Nullhong kong

allele is a TC dinu-cleotide deletionat

Leu318

causing

a5'

frameshift

with

genera-tionof a

premature

termination codon at

position

334

(23).

Thea

1

AT

Nullmaawa allele presents yet another mechanism;

a Nullallele

resulting

from a

single-nucleotide insertional

muta-tion within the

coding region

of exon

V, causing

a distal 3'

frameshift,

different amino acids

substituting

forresidues 353

-I

375,

and a

premature

stop

codon

at

residue 376. Thus, the

heterogeneity

in the Null

mutations

is

diverse.

Consistent with

this

concept,

wehave

recently observed

an

entirely different

cause of the Null

state,

an

inherited deletion of the entire

aIAT

gene

_{(NU1ldeleCim0}

procida;

Takahashi, H., and R. Crystal,

unpublished observation).

A

NIH-3T3

NIH-3T3/a1AT-Mi

9 @ *

NIH-3T3/a1AT-Mattawa *-**

B

Extracellular NIH-3T3 NIH-3T3 a1 AT-MI al AT-Mattawa

kDa 52. Elms

1

Intracellular NIH-3T3

NIH-3T3

a1AT-Mi

a1lAT-Mattawa

kDa 50, * *

3 4

2

Figure 8. Evaluation of humanalATgeneexpressionin murine fi-broblastsmodifiedtocontain thea1AT cDNAs of M

l(Val2'13)type

andNullanw5-type. (A)Identification of alATmRNAtranscriptsin mousefibroblastscontainingtheintegrated

Ml(Val2'13)-type

and

Nullm.,w,-type

human alAT cDNAs. Shownaredatafrom

NIH-3T3/alAT-Ml andNIH-3T3/alAT-Mattawacells andas

con-trol,unmodified NIH-3T3 cells.CytoplasmicRNAwasevaluated for humana1ATmRNAtranscripts usingcytoplasmic dot hybridiza-tionanalysiswitha32P-labeleda1ATcDNAprobe.(B)Synthesis

andsecretion of humana1 ATbymousefibroblasts modifiedto

con-tain the human alAT cDNAs ofM

l(Val2'13)-type

and

Nufllmattwa-type.ShownarefluorogramsofSDS-acrylamide gel analysisof

35S-labeledproteinsisolated from lysates andsupernatantsof NIH-3T3/ alAT-Ml andNIH-3T3/alAT-Mattawacells (30 minpulsewith

[35S]methionine

and 120 min chasewith label-freemedia)and

im-munoprecipitatedwithananti-a 1ATantibody.Lysateswere

ana-lyzeddirectlyafter the 30-min pulse period andsupernatantsafter

the120-min chaseperiod.Lane 1,NIH-3T3/alAT-Ml,supernatant. Lane2,NIH-3T3/alAT-Mattawa,supernatant.Lane3,NIH-3T3/

a1AT-M 1,lysate.Lane4,NIH-3T3/alAT-Mattawa,lysate. The

po-sition of the 52-kD secreted form and 50-kD intracellular form of

alATareindicated.

Insertional

mutations

generating

a Null state have been described in other

protein deficiency

disorders. Subsets of the 130

thalassemia

genes have been identifiedinethnicAsian

In-dians and Chinesethat result from differentsinglenucleotide

insertional

mutations within the

coding

region

of the

_fl-globin

gene (39, 40). Recently,Hidaka andcolleagues (41)have

iden-tified

an insertional mutation in a

splice junction

in the

human adenine

phosphoribosyltransferase

gene,

causing

aber-rant

splicing. Interestingly,

the inserted nucleotide

resulting

in theNullmataswaallele is athymidinethat occurs at the terminus

of a short

polymeric

sequenceof

repeating thymidines.

This is

consistent with theobservation in studies of

bacteriophage

T4

mutants

that

nucleotide insertion mutations

generating

fra-meshifts

generally

occurinthe

setting

of short

repeating

poly-meric

sequences

(42-45),

a

phenomenon

which has been

ex-plained

on

the

basis of

slippage mispairing

during

transcrip-tional

replication (42,

46).

Consistent with this

concept,

a

tetrameric or

pentameric polythymidine

sequence has been

shown to bea frameshift mutational hotspotboth in vivo

(44,

45) and ininvitro systemsevaluating mutagenesis

(47,

48).

The

insertional mutation

of the

_Nullmauawa

gene

generates

a

3' frameshift withan altered

reading

frame distal to and

in-cluding

codon353.This altered

reading

frame terminates ata

premature stop

signal

at new codon

376,

19 codons

upstream

from the normal stop signal. Interestingly, the presenceofa

premature stop

signal

in the

_Nullmatwa

allele is associated with anormal-sized a1AT mRNA

transcript.

In

contrast,

the pre-maturestop

signal

of

Nullbeilingham

is associated with the

com-plete absence

of

detectable

aIAT mRNA froma

lAT-express-ing cells (20). Perhaps

an

explanation

for this difference is that

the

premature stop

signal

of the

_Nullbiingham

alleleoccurs near

the centerof the aIAT mRNA

transcript;

the lack ofaIAT

mRNA

in

this

setting

may

be

the consequence of mRNA

mol-ecules

not

protected

at the 3' end

by polysomes,

as hasbeen

proposed

for

some

of the

130 thalassemia mutations in which no

_f3-globin

mRNA

is detected

(49, 50).

In this

regard,

the

Nullmatuwa

allele

is

associated withapremature stop

signal

near

the

terminal

part

of the

aIAT mRNA molecule. In this

setting,

the occurrence of the stop

signal

atadistal

position

may allow

sufficient

protection by

polyribosomes

such that

astablea

lAT

mRNA

transcript

results.

As

the

a

lAT

Nullmattawa

allele

is associated with

alAT mRNA

transcripts, it is of

interest that

no

alAT

translation

product in

association

with this allele could be detected from

cells with the

_Nullmattwa

mutation.

In

vitro translation

analysis

ofthe

Nullmalawa

mRNA

transcript

demonstrated that

it

directs

synthesis

of

a

truncated

a1AT

molecule. The coded

transla-tion

product

contains alterations of

the

primary protein

struc-ture

predictive

of structural

instability (51).

Presumably, the

conformational changes affected

by these

alterations

generate

a

protein species incapable of secretion.

This could

occur on

the

basis of

intracellular aggregation

within

a

subcellular

com-partment or

alternatively,

secondary to

intracellular

degrada-tion of the

nascent

protein.

The alAT

Nullhong

kong gene has

recently

been

shown

to

result

in a

truncated

a1AT

protein

which

aggregates

within

the

cell

at

the

level

of

the rough

endo-plasmic reticulum (23).

Incontrast, not only can noa

lAT

be detected as a

secretion product from

a1

AT-expressing

cells

with

the

_Nullmatwa

gene, but

additionally,

no detectable

intra-cellularform ofalATcanbeidentifiedaggregatingwithin the

cell.

Thus,

it is

likely

that

translation of

the

NUllmattwa

mRNA results in

synthesis

of

a

structurally unstable,

truncateda1AT

molecule,

with the consequence that the altered

aIAT

protein

is

degraded

within

the cell and thus

not

present

intheserumin

detectable

amounts.

Acknowledaments

WearegratefultoSusanLeitman,M.D.and thenursesof the National Institutes of Health Clinical Center Blood Bank for

carrying

out

cyta-pheresis

for blood monocytes.

M.BrantlyisaParker B. Francis Fellow in

Pulmonary

Research.

References

1.Gadek,J.E.,and R. G.

Crystal.

1982.

al-antitrypsin deficiency.

In The Metabolic Basis ofInherited Disease. J. B.

Stanbury,

J. B.

Wyngaarden, D. S.Fredrickson, J. L.

Goldstein,

andM. S.

Brown,

editors. McGraw-HillBook

Co.,

NewYork. 1450-1467.

2. Morse, J. 0. 1978.

Alpha,-antitrypsin deficiency.

N.

Engl.

J.

Med.

299:1045-1048;

1099-1105.

3.

Gadek,

J.E.,G.A.

Fells,

R. L.

Zimmerman,

S. I.

Rennard,

and R. G.Crystal. 1981. Antielastases of the human alveolarstructures:

Implications

for the

protease-antiprotease theory

of

emphysema.

J.

Clin.Invest.68:889-898.

4.

Janoff,

A. 1985. Elastases and

emphysema:

currentassessment of the

protease-antiprotease

hypothesis.

Am. Rev.

Respir.

Dis.

132:417-433.

5.Rabin, M.,M.Watson,V.

Kidd,

S. L.C.Woo,W. R.Breg,and F. H. Ruddle. 1986.

Regional

locationof

al-antichymotrypsin

and a1-antitrypsingeneson human chromosome 14. Somatic Cell Mol. Genet. 12:209-214.

6.Long, G.L.,T. Chandra, S.L.C. Woo, E. W.

Davie,

andK.

Kurachi. 1984.Completesequence of the cDNA for human

al-anti-trypsinand the gene for the S variant.

Biochemistry.

23:4828-4837.

7. Perlino, E., R. Cortese, and G. Ciliberto. 1987. The human

a l-antitrypsin gene is transcribed from two different promoters in

macrophages andhepatocytes. EMBO (Eur. Mol. Biol.

Organ.)

J. 6:2767-2771.

8.

Fagerhol,

M.K.,and D. W. Cox. 1981. The Pi

polymorphism:

genetic, biochemical,

and clinical aspects of human

al-antitrypsin.

Adv.Hum.Genet. 11:1-62.

9. Brantly, M., T.

Nukiwa,

and R. G.

Crystal.

1988. Molecular basis ofa

l-antitrypsin deficiency.

Am.J.Med.84:13-31.

10.

Cox,

D.W.,A.M.

Johnson,

and M. K.

Fagerhol.

1980.Report

of nomenclature

meeting

for

al-antitrypsin. INSERM,

Rouen/Bois-Guillaume. 1978. Hum. Genet. 53:429-433.

1 1.Kueppers,F. 1978. Inherited differences inalpha-I

_{-antitrypsin.}

InGeneticDeterminants of

Pulmonary

Disease. S.D.

Litwin,

editor. MarcelDekker, Inc.,NewYork. 23-74.

12.Talamo,R.C.,C.E.Langley,C.E.Reed,andS. Makino. 1973. a

_{l-antitrypsin deficiency:}

avariant withnodetectableaI

-antitrypsin.

Science

(Wash.

DC).

181:70-71.

13.Laurell,C.-B.,T.Sveger,andC.-G.Ljunggren. 1974.

al-anti-trypsin deficiency:

Pi genotypeZO, SO, and MO.Acta Paediatr. Scand.63:855-857.

14.Schandevyl, W.,A.Hennebert,G.Leblanc,A.deCoster,J.C.

Yernault,G.Achten,M.Ledoux,andJ. J.Buneaux. 1975.

Alpha-l-antitrypsin deficiencyof PiOO typeandconnective tissuedefect.InL'

alpha-l-antitrypsine

etlesysteme Pi.J.-P.

Martin,

editor.

INSERM,

Paris. 97-107.

15.Martin,J.-P. 1975.Furtherexamplesconfirmingtheexistence of PiNull

(Pi-).

Path.Biol. 23:521-524.

16.Lieberman, J.,L.Gaidulis,and L.A.Schleissner. 1976.

Inter-mediate

alpha-l-antitrypsin deficiency

resultingfromanullgene (M-phenotype).Chest. 70:532-535.

17. Larsson,C. 1978.Intermediate

alpha-l-antitrypsin deficiency,

Pi M-. Acta.Med. Scand.204:353-356.

Familial casesof al-antitrypsin deficiency (PI NULL Type). Nippon NaikaGakkaiZasshi. 67:50-56.

19.Muensch, H., L.Gaidulis, F. Kueppers, S. Y. So, G. Escano, V. J. Kidd, and S. L. C. Woo. 1986. Complete absence of serum Alpha-l-antitrypsin in conjunction withanapparently normal gene structure.Am.J. Hum. Genet. 38:898-907.

20. Garver, R. I., Jr., J.-F. Mornex, T. Nukiwa, M. Brantly, M. Courtney, J.-P.LeCocq, and R. G. Crystal. 1986. Alpha-l-antitrypsin deficiency and emphysema caused by homozygous inheritance of non-expressingalpha-l-antitrypsingenes. N.Engl. J.Med. 314:762-766.

21. Satoh, K., T.Nukiwa,M.Brantly,R. I.Garver, Jr.,M. Court-ney, M.Hofker,and R. G.Crystal. 1988.Emphysemaassociated with complete absence ofal-antitrypsin in serum andthe homozygous

inheritance ofstop codon inan al-antitrypsin codingexon.Am. J. Hum.Genet. 42:77-83.

22.Nukiwa, T., H.Takahashi,M.Brantly,M.Courtney,and R.G. Crystal. 1987.al-antitrypsin

NUlgrnie

fal1s,

anonexpressing

al-anti-trypsingene associated withaframeshifttostop mutation inacoding

exon.J.Biol. Chem. 262:11999-12004.

23.Sifers, R.N., S.Brashears-Macatee, J. V. Kidd,H.Muensch, andS. L. C. Woo. 1988.Aframeshift mutation results inatruncated

al-antitrypsin that is retained within therough endoplasmic reticu-lum. J.Biol. Chem.263:7330-7335.

24. Wewers, M. D.,M. A. Casolaro, S. E. Sellers,S. C.Swayze,

K. M.McPhaul, J. T.Wittes, and R. G.Crystal. 1987.Replacement

therapy foralpha1-antitrypsin deficiencyassociated withemphysema.

N.Engl. J. Med.316:1055-1062.

25.Fulmer, J. D., W. C.Roberts,E. R.VonGal,and R. G.Crystal.

1977.Smallairwaysinidiopathic pulmonaryfibrosis.Comparisonof morphologic and physiologic observations. J. Clin. Invest. 60:595-610.

26. Kidd, V. J., M. S. Golbus, R. B. Wallace, K. Itakura, and S. L. C. Woo. 1984.Prenatal

diagnosis

ofaI

-antitrypsin deficiency by

direct analysis ofthe mutation site in the gene. N.

Engl.

J. Med.

310:639-642.

27.Nukiwa,T., M.Brantly,F.

Ogushi,

G.Fells,K.Satoh,L.

Stier,

M. Courtney, and R. G. Crystal. 1987. Characterization of the Ml

(Ala2l3)

type ofa1-antitrypsin, anewly

recognized

common "nor-mal"al-antitrypsin haplotype. Biochemistry. 26:5259-5267.

28. Bates, P. F., and R. A. Swift. 1983. Double cos site vectors:

simplifiedcosmidcloning.Gene(Amst.).26:137-146.

29.vanOmmen, G. J. B.,A.C.Arnberg, F. Baas, H. Brocas, A.

Sterk,W. H. H.Tegelaers, G. Vassart, and J. J. M.Vijlder. 1983. The human thyroglobulin gene contains two 15-17 kb introns near its 3'-end.Nucleic AcidsRes. 11:2273-2285.

30.Nukiwa,T., K.Satoh,M. L.Brantly,F.Ogushi,G.A.Fells, M.

Courtney,and R.G.Crystal.1986.Identificationofasecondmutation in theprotein-codingsequence oftheZ-Type alpha-I-antitrypsingene. J.Biol. Chem. 34:15989-15994.

31. Vieira, J., and J. Messing. 1982. The pUC plasmids, an

Ml

3mp7-derived system for insertion mutagenesis and sequencing

withsynthetic universal primer. Gene(Amst.). 19:259-268.

32. Perlmutter, D.H.,F. S.Cole,P.Kilbridge,T.H.Rossing,and H. R.Colten. 1985.Expressionof theal-proteinaseinhibitor gene in human monocytes and macrophages. Proc. Nati. Acad. Sci. USA. 82:795-799.

33. Mornex, J.-F.,A.Chytil-Weir,Y.Martinet,M.Courtney,J.-P.

LeCocq,and R.G.Crystal. 1986.Expressionofthealpha-l-antitrypsin

gene in mononuclearphagocytes of normal and alpha-l-antitrypsin

deficientindividuals. J. Clin. Invest. 77:1952-1961.

34. Hester, J. P., R. N. Kellogg, and E. J. Freireich. 1983. Mononu-clearcellcollection by continuous flow centrifugation. J. Clin. Apher-esis. 1:197-201.

35. Krieg, P. A., and D. A. Melton. 1984. Functional messenger RNAs areproduced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 12:7057-7071.

36. Garver, R. I., Jr., A. Chytil, S. Karlsson, G. A. Fells, M. L. Brantly, M. Courtney, P. W. Kantoff, A. W. Nienhuis, W. F. Ander-son,and R. G. Crystal. 1987. Production of glycosylated physiologi-cally "normal" human

a,-antitrypsin

by mousefibroblasts modified byinsertion of a human

a,-antititrypsin

cDNA using a retroviral vec-tor.Proc.Natl.Acad. Sci. USA. 84:1050-1054.

37. Mann, R., R. C. Mulligan, and D. Baltimore. 1983.

Construc-tionofaretroviruspackagingmutant andits use to produce helper-free

defectiveretrovirus.Cell.33:153-159.

38.White,B. A., and F.C.Bancroft. 1982. Cytoplasmic dot hybrid-ization: simple analysis of relative mRNA levels in multiple small cell ortissue samples. J.Biol. Chem.257:8569-8572.

39. Kazazian, H. H., Jr., S. H. Orkin, S. E. Antonarakis, J. P. Sexton, C. D. Boehm, S. C.Goff,and P.G. Waber. 1984. Molecular characterizationofseven,B-thalassemiamutations in Asian Indians. EMBO(Eur.Mol. Biol.Organ.) J. 3:593-596.

40.Cheng, T.-C., S. H.Orkin,S. E. Antonarakis, M. J. Potter, J. P. Sexton,A.F.Markham, P. J. V. Giardina, A. Li, and H. H. Kazazian, Jr. 1984.

fl-Thalassemia

inChinese: use of in vivo RNA analysis and

oligonucleotide hybridizationinsystemic characterization of molecu-lardefects. Proc.Natl.Acad. Sci. USA.81:2821-2825.

41.Hidaka, Y., T. D. Palella, T. E. O'Toole, S. A. Tarle, and W. N. Kelley. 1987. Humanadenine phosphoribosyltransferase.

Identifica-tionof allelic mutationsatthenucleotidelevel as a cause of complete

deficiencyof the enzyme. J.Clin.Invest. 80:1409-1415.

42.Streisinger,G., Y. Okada, J. Emrich, J. Newton, A. Tsugita, E. Terzaghi, and M. Inouye. 1966. Frameshift mutations and the genetic code. Cold Spring HarborSymp. Quant.Biol.31:77-84.

43. Okada, Y.,G. Streisinger,J.Emrich,J.Newton, A. Tsugita,

and M. Inouye. 1968. Frame shift mutationsnearthebeginning ofthe lysozyme geneof bacteriophage T4. Science(Wash. DC). 162:807-808.

44.Streisinger, G.,and J. E. Owen.1985.Mechanisms of

spontane-ousandinduced frameshift mutation inbacteriophageT4.Genetics. 109:633-659.

45.Okada, Y.,G. Streisinger,J. E.Owen,J.Newton,A.Tsugita,

and M.Inouye. 1972. Molecular basis ofamutationalhot spot in the

lysozymegene ofbacteriophageT4.Nature(Lond.).236:338-341. 46. Roth, J. R. 1974. Frameshift mutations. Annu. Rev. Genet. 8:319-346.

47. Kunkel, T.A. 1985. The mutationalspecificityof DNA poly-merase-,Bduringinvitro DNA synthesis.J. Biol. Chem. 260:5787-5796.

48. Kunkel, T. A. 1986. Frameshift mutagenesis by eucaryotic

DNApolymerasesinvitro. J. Biol.Chem.261:13581-13587. 49.Chang,J.C.,and Y. W. Kan. 1979. ,B thalassemia,anonsense

mutation inman.Proc.Natl. Acad. Sci. USA. 76:2886-2889. 50.Maquat, L.E.,A.J.Kinniburgh,E.A. Rachmilewitz,and J. Ross. 1981. Unstable,-globinmRNAinmRNA-deficient° thalasse-mia.Cell.27:543-553.

51.Loebermann,H., R.Tokuoka,J.Deisenhofer,andR.Huber. 1984.Human

a,-proteinase

inhibitor:crystalstructureanalysisoftwo crystalmodifications,molecularmodel andpreliminary

analysis

of the implications for function. J. Mol. Biol. 177:531-556.