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Defective splicing of mRNA from one COL1A1 allele of type I collagen in nondeforming (type I) osteogenesis imperfecta

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Defective splicing of mRNA from one COL1A1

allele of type I collagen in nondeforming (type I)

osteogenesis imperfecta.

M L Stover, … , M B McKinstry, D W Rowe

J Clin Invest.

1993;

92(4)

:1994-2002.

https://doi.org/10.1172/JCI116794

.

Osteogenesis imperfecta (OI) type I is the mildest form of heritable bone fragility resulting

from mutations within the COL1A1 gene. We studied fibroblasts established from a child

with OI type I and demonstrated underproduction of alpha 1 (I) collagen chains and alpha 1

(I) mRNA. Indirect RNase protection suggested two species of alpha 1 (I) mRNA, one of

which was not collinear with fully spliced alpha 1 (I) mRNA. The noncollinear population

was confined to the nuclear compartment of the cell, and contained the entire sequence of

intron 26 and a G-->A transition in the first position of the intron donor site. The G-->A

transition was also identified in the genomic DNA. The retained intron contained an in-frame

stop codon and introduced an out-of-frame insertion within the collagen mRNA producing

stop codons downstream of the insertion. These changes probably account for the failure of

the mutant RNA to appear in the cytoplasm. Unlike other splice site mutations within

collagen mRNA that resulted in exon skipping and a truncated but inframe RNA transcript,

this mutation did not result in production of a defective collagen pro alpha 1 (I) chain.

Instead, the mild nature of the disease in this case reflects failure to process the defective

mRNA and thus the absence of a protein product from the mutant allele.

Research Article

Find the latest version:

http://jci.me/116794/pdf

(2)

Defective Splicing

of mRNA from One COLlAl Allele of

Type

I

Collagen in

Nondeforming (Type I) Osteogenesis Imperfecta

M. L.Stover,D.Primorac, S. C.Liu,* M. B.McKinstry,andD. W. Rowe

Fromthe Departments ofPediatrics and *BiostructureandFunction, University ofConnecticut HealthCenter, Farmington, Connecticut 06030

Abstract

Osteogenesis imperfecta (OI) type I is the mildest form of heritable bone fragility resulting from mutations within the COLlAlgene.We studied fibroblasts established fromachild

with01typeI and demonstrated underproduction ofal(I)

col-lagenchains andal(I) mRNA. Indirect RNase protection

sug-gestedtwospecies ofal(I) mRNA,oneof whichwasnot col-linear with fully spliced al (I) mRNA. The noncolcol-linear

popula-tionwas confinedtothe nuclearcompartmentof the cell, and

contained the entiresequenceof intron 26 andaG-* A

transi-tion in the first positransi-tion of the intron donor site. The G-- A

transition was also identified in the genomic DNA. The

re-tained intron conre-tainedanin-frame stopcodon andintroduced anout-of-frame insertion within the collagen mRNA producing

stopcodons downstream of the insertion. These changes proba-blyaccountfor the failure of themutantRNAtoappearin the

cytoplasm. Unlike other splice site mutations within collagen mRNA that resulted inexonskipping andatruncated but

in-frame RNA transcript, this mutation didnotresultin produc-tion ofadefective collagen proal (I) chain. Instead, the mild

natureof the disease in thiscasereflects failuretoprocessthe defective mRNA and thus the absence ofaprotein product from

themutantallele.(J. Clin. Invest. 1993. 92:1994-2002.) Key words:osteogenesis imperfecta*RNA splicing-nuclearRNA

transport-RNase protection*premature stopcodon

Introduction

Osteogenesis imperfecta (01)1 isaheritabledisorder that

re-sults in bone fragility. Identification of mutations within the COLlA1 andCOL1A2 genesthat encode the chains oftypeI

collagen has beguntoprovideadetailed understanding ofthe

structural requirements for thetypeIcollagen molecule( 1,2).

Mutations that disrupt thetriple-helicalconfiguration of

colla-This workwasaposterpresentationatthe Americal Society ofBone and MineralResearch, Minneapolis, MN, September 30-October4, 1992.

Address correspondencetoDr. DavidW. Rowe, Department of

Pediatrics, School of Medicine, University ofConnecticut Health

Center,263Farmington Avenue, Farminton, CT 06030-1515. D. Primorac's permanent address is the University of Zagreb,

SchoolofMedicineatSplit, 58000 Split, Croatia.

Receivedfor publication2February1993andinrevisedform 30

April1993.

1.Abbreviations usedin thispaper: nt, nucleotide; 01, osteogenesis

imperfecta.

gen, including substitutions for glycine in the gly-x-y triplet

(3), partial

gene deletions (4), and exon skipping

(5),

pro-duce, dependingonthe locationof the mutation, arange of disease severity that extends from lethal

(01

type II) toseverely deforming(01typeIII)tomildly deforming

(01

typeIV) (6). In01 type I fractures are usually limited to the prepubertal years and are not associated with bone deformity. At times the fracture frequencyis not recognized as a distinctabnormality and thedisorder only comes to medical attention when early-onset osteoporosis develops in adult family members. Thus, mild forms of 01 and "familialosteoporosis"often coexist, in part because theunderlyingpathogenesis may be similar. For mutations in theaI(I) chain (COLlA 1), there appears to be a positional effect in which those at the COOH terminus are

morelikelytoresult in severe disease than those at theNH2 terminus (7). In fact, substitutions for glycine have been iden-tified near theNH2-terminalend ofthetriplehelix that result in nondeforming bone disease (type I01)and heritable osteoporo-sis(8). Mutations within the a2 (I) chain (COLIA2 gene) that produce mild 01 orosteoporosis (9)do notalwaysoccur atthe NH2-terminal end of the chain (10).

The most common mechanism for 01 type I appears to be decreased synthesis of normal type I collagen molecules. The synthesis of proa 1 ( 1 ) collagen chains ( 11 ) and the steady-state level ofa1(I) mRNA appears to be reduced by - 50% (12,

13), suggesting that one of the two COLlA 1 alleles is "null." Wepreviouslyshowed that dermalfibroblasts fromsome indi-viduals withtype I 01containnormal or elevated amounts of

aI(I)mRNA inthenucleus( 14). Furthermore,anovel spe-cies ofa1(I)collagenmRNA present in the nuclear compart-ment of cells from one such child was not collinear with a cDNA probe (15). In this paper we show that a mutation withinasplicedonorsiteresultsininclusion oftheentire suc-ceedingintron in the mature mRNA that accumulates in the nuclear compartment. Apparently because no abnormal proaI(I) chains are synthesized from the mutant allele,the clinicalphenotypeof thischild is mild.

Methods

Description ofpatient. CF was a 13-yr-old female at the time of the dermalbiopsy (cell line 053). She hadbeenfollowed at the Newington Childrens Hospital (NCH) between 10 and 15 yr of age. She had

sus-tainedsevenfracturesbefore her first NCHvisit,allof which healed withoutdeformity. Subsequently, the fracture rate diminished and her primary problems were ligament strains and knee discomfort second-arytopatellar subluxation. Herheightwas atthe 50-75percentile. She hadblue sclera,ligamentous laxity,amildthoracicscoliosis (<10%), and normaldentition. Hercurrentage is28 yr, and she and her family, includinganaffected brother, have been lost to follow-up.

Cell culture. A fibroblast strain wasderived from askin explant

takenfromtheinner aspect of the upperarm.Fibroblastswere main-tainedaspreviouslydescribed ( 12). Confluent cultureswereharvested forprotein or RNAafter 48 hinfresh culture media supplemented

dailywith 25,ug/mlascorbic acid. 1994 M. L.Stover, D.Primorac, S.C.Liu,M.B.McKinstry, and D. W.Rowe

J.Clin.Invest.

© The American Society for Clinical Investigation, Inc.

0021-9738/93/10/1994/09 $2.00

(3)

Collagen analysis. Forexperiments analyzing collagen synthesis, the cultured cells wereradiolabeled with [2,3-3H]-L-proline (DuPont

Co., NEN Research Products, Boston, MA) as previously described ( 12 ). Collagen synthesis was determined from collagenase susceptible

countsin analiquot of the combined medium and cell extract (16). Therelative proportion of collagen al (I), a2(I), and al(III) chains

wasdetermined by interrupted SDS-PAGE electrophoresis of pepsin-treatedprotein extracts( 17).

Cellfractionationandnucleicacid extraction. Total RNA was ex-tractedfrom theconfluent fibroblasts cell layer using the SDS

protein-ase Kmethod(18).The nuclear and cytoplasmic compartments of the cellswereisolatedaspreviouslydescribed ( 14). Briefly, confluent fibro-blast cultures ( 12-100-mm petri plates) were scraped from the plate in 10mlof coldPBS, centrifuged,and resuspended in 15 ml of lysis buffer

(10mMTris,pH 7.5, 10 mM KCI, and 1.5mM MgCl2)containing 0.25%TritonX-100. The pelletwasdisrupted by dounce homogeniza-tion withatight-fitting pestle. Thesuspension was centrifuged for 10 minat2,200g and the supernatant was transferred to a 30-ml corex tube(cytoplasmicextract) containing 2.0mlof a lOXextraction buffer

(10% SDS,0.10 M Tris,pH 7.5, 0.05 M EDTA, and 500Mg/ml of

proteinaseK). The nuclear pellet was reextractedasecond time in5ml oflysisbuffer with Douncehomogenization. Aftercentrifugation, the supernatantwascombined with the first extract, producing a final con-centration of the extraction buffer of lx. The nuclear pellet was ex-tracted in 15 ml of lx extraction buffer. Subsequent steps of RNA isolationfollowed the SDSproteinaseKprotocol ( 18).

DNA was harvested from cultured fibroblasts in 1X extraction buffercontaining 100

Mg/ml

of proteinase K. The digest was extracted

sequentiallyin phenol/chloroform and chloroform and adjusted to 0.3

MNaOAc. Twotothree volumes of ethanol was layered over the

ex-tract,and the DNA was spooled from the interface, washed in 70%

ethanol, dried,and resuspended in water.

RNAanalysis.Therelative content ofal (I)and a2(I)mRNA in thenuclear and cytoplasmic compartments was assessed by dot hybrid-izationaspreviouslydescribed ( 14). The filters were hybridized with

al(I)-anda22(I)-specificcDNA probes (HF404[19]and HF32 [20]) afterlabeling bythe randomprimer method (21 ). The relative

inten-sity ofhybridization was quantitated in a Betascope G03 (Betagen, Waltham, MA) from the slope of the dilution curve of patient and control RNA.

The methodforindirectRNaseprotectionof nuclear and cytoplas-micRNAusing single-stranded (SS)antisense cDNA toa I( 1 ) mRNA has been previously described (15). Briefly, hybridization of the sscDNA toRNA wascarried out at 500Cin20 Mlofhybridization

buffer(0.75MNaCl,1 mMEDTA,50mMHepes)(N-

[2-hydroethyl]-piperazine-AV-[2-ethanesulfonic

acid])for 3 h. The sample was diluted

to300Mlin RNasebuffer (0.2MNaCl,20 mMHepes,pH 7.5, and 2 mMEDTA)andincubated with 1

Mg

RNaseAand10Mg RNaseTlfor 30 minat37°C. TheRNase-resistanthybridswereextracted in SDS

proteinase K,ethanol precipitated, and reappliedto 1% agarose gel made up in 2.2Mformaldehyde.Theelectrophoresiswascarriedout at

roomtemperature ina25 mMimidazole,25 mM 3-N-morpholino]-propanesulfonicacid(MOPS),0.5 mM Na-EDTAbuffer,pH 7.0, for 3hat100V.TheRNAfragmentsresistanttotheRNasedigestionwere transferredto anitrocellulose filter withaPosiblot apparatus

(Strata-gene,LaJolla, CA)andidentifiedby hybridizationwith 2.5 x 106 cpm of[

32P04]UTP-labeled

cRNAprobeof HF404 transcribed fromasp64

vector(22).

The direct RNase protection wasperformed with a cRNA ofa

PCR-derived DNAfragmentthathad been clonedintopBSII-SK+

vec-tor(Stratagene). Nuclear and cytoplasmicRNA(7

Mg)

were hybrid-ized with 104cpmof thecRNAovernightat50°C in 50Mlofbuffer

containing0.4MNaCl, 10mMHepes, pH 7.3,and 1 mM EDTA. The

next day the solution was diluted to 300

Ml

of T2 buffer (50 mM NaOAc, pH 4.6,2 mM EDTA, and0.1 MNaCl) containing 5

M1

of crude T2 RNaseaspreviouslydescribed(23). Thefragmentsof the

protected probewereidentified byelectrophoresisina6.0%denaturing polyacrylamide gel. An RNA ladder of 0.16-1.77 kb (no. 56235A;

GIBCO BRL, Gaithersburg, MD) was end labeled with'-[32PO4]ATP

accordingtothe supplier's directions.

PCRofRNAand genomicDNA. 5.0

,g

of nuclear or cytoplasmic RNAwasreverse transcribed to cDNA using an al(1)-specific primer directedtosequences within the COOH-terminal propeptide as previ-ously described (8). The reaction was carried out in 50 mM Tris, pH

8.3,7mMKCl,3mM

MgCl2,

10mM dithiothreitol,and2.5,gof the primer. The mixture was heated at650Cfor 3min,cooled, adjusted to contain

500MgM

dNTPs, and 500Uof Moloney murine leukemia virus (M-MLV) reverse transcriptase. The incubation lasted for 1 h at 370C and was followed by phenol/chloroform extraction and ethanol precip-itation. The sample was dissolved in 20,lwater and 1 Ml was used for PCRamplification. In the case of genomic DNA, 1 Mg of DNA was used in the PCR reaction. The oligonucleotide pairs for each set of amplificationsaregiven in Table I.

ThePCR mixture contained 50 mMKCl, 10mM Tris, pH 8.3, 1.5 mMMgCl2,0.0 1%gelatin,and 2.5 UAmpliTaq (PerkinElmerCorp., Norwalk, CT) inafinal reaction volume of 84 Ml. The mixture was heated to 940Cfor5min,after which the 16 Ml containing 200 gM

dNTPs(hotstart[24])wasadded and thesamplewascycled25 times withdenaturationat940Cfor 1 min,hybridizationat620C for 1 min,

andpolymerizationat720C for 1 min inaDNAthermalcycler(Perkin

Elmer-Corp.). Theproductswereextracted inphenol/chloroformand

1:10of the original reaction volumewasappliedto a5%

polyacryl-amide TBEmini-gel.

For directDNAsequencingof PCRproducts,weused the dsDNA CycleSequencing System (GIBCOBRL). PCR products were cloned either withG-tailinginto C-tailedpUC9vector(Pharmacia Fine Chem-icals, Piscataway, NJ)orbyblunt endcloning into Sma site pBSII-SK+. Forsequencingof the clonedDNA,weused theSequenase Re-agent Kit(no.70750;United States Biochem.Corp.,Cleveland, OH).

Results

Total collagen synthesis by the fibroblasts derived from the patient (Table II)was4.0%oftotal protein synthesis. For com-parison, three cell strains derived from patients with severe forms ofthediseaseare shown. We have observed significant variation in the rate and percentage of collagen synthesis in partdependentonthe ageofthe donor. The range of synthesis innon-OI controlcells is 5-7% (5). In most of the cell strains obtained frompatientswith 01 type I, a reduced rate of total collagen synthesis isobserved.

Therelative proportion oftype I and type III collagen was determinedonpepsin-treated radiolabeled proteinextracts of themedium and celllayer(Fig. 1). The ratio was elevated to 0.38and 0.45intwoexperiments(controlpatientsrangefrom 0.15 to 0.25). Because an equal amountoftotal collagen is loadedintoeachelectrophoresislane, the elevation in the colla-gen type III-to-Iratioreflects underproduction of type I colla-gen. The electrophoretic mobilities ofthe a I(I) and a2(I) chains from thepatient samplewere identical to those of the controlsample.

Therelativecontentofa 1(I) and a2 (I) mRNA was deter-mined bydotblothybridizationandcomparedwith the ratio of control cells(Fig.2). The slopeofthelineofdotdensitywhen thefilterwashybridizedto an a 1(I)or a2(I)cDNAprobewas

compared withtheslopeofacontrol RNAappliedtothe same

filter.Inthisassay, the

alI

(I)/a2(I)mRNAratio ofthecontrol is normalized to 1. Whenthe slope ofthe al(I) and a2(I) mRNA derived from cell strain 053 was compared with the standard (std),the al(I)/stdwas0.30 and the a2(I)/stdwas

1.19, makingthealI (I)/a2(I)ratioofthepatient0.25.Thus,

the amountofa 1(I)mRNA presentinthetotal cellularRNA

(4)

Table I. Oligonucleotide Pairs Used to Amplify al(I)mRNAEncoded by HF404

NT* 3-Oligonucleotide 5'

Oligonucleotide pair/exonsamplified 5'Oligonucleotide-3' NT*

1:35-39 2541 AGCAGGACCATCAGCACCAGGGGAT CCCGGCCCTGCTGGCTTTGCTGGCC 2948

2:30-36 2113 TTTCGGCCACGACTAACCAGG AGACCTTGGCGCCCCTGGCCCCTCT 2621

3:25-31 1800 TTGCACACCACGCTCGCCAGGGAAA TCAAGATGGTCGCCCCGGA 2159

4:24-28 1746 AGCAGGGCCTTGTTCACCTCTCTCG AGCCCTGGCAGCCCTGGTCCTGATG 2005

5:19-23 1330 GCTTCACCGGGACGACCAGTC GTATTGCTGGTGCTCCTGG 1708

*Thenucleotide number is determined from the a

1(I) collagen

mRNAstartsite.

cells have a 2:1 ratio of al(I)/a2(I), the amount of alI (I) of thepatient sample is reduced to< 1:1.

Analysis of this patient and other cell lines with 01 type1

indicatedthat theaI(I)/ a2 (I) collagen mRNA ratio extracted from isolated nucleiapproximated or exceeded 2:1 in contrast to the low ratio from cytoplasmic compartment ( 14). This observationsuggestedadefect in a 1(I) mRNA transport simi-lar to that observedinsplicing defects associated with

f3-thalas-semia (25, 26).AnindirectRNaseprotectionwasdevelopedto detect insertions withincollagenmRNA( 15).Itrelieson pro-tection of the mature mRNA with a single-stranded antisense cDNA.TheprotectedorcleavedRNAis detected byNorthern hybridization ofthe RNase-digested products. Nuclear and cy-toplasmicRNA wasprotected with a 1.8-kb cDNA fragment derived from HF404 that encodes exons 19-43 (Fig. 3). In

RNA from cell strain 053, the expected 1.8-nucleotide (nt) band andtwo smaller bands(1.4and 0.4 nt)were visible in

nuclear RNA.Onlythefully protectedband was present in the cytoplasmic RNAand wassimilar in sizeto RNA extracted fromnormal cells.

TocharacterizetheabnormalRNAspecies, nuclear RNA was reversetranscribed and then PCRamplified insegments to spantheregion encoded by HF404 (Fig. 4). Of the five PCR primersets used(Table I),two(sets 3 and4) produceda sec-ondhighermolecularweight ( - 150bp larger) band from the

patientbut notthe control RNA. Athirdband well above the 1.3-kb marker was also unique to these oligonucleotide sets and probably represents a heterodimer produced when two complementary strands of differentlengths hybridized during

TableII. Production of Total Collagen by Dermal Fibroblasts

DerivedfromPatients with Various Forms of OI

Collagen Total protein Percent Cellline synthesis synthesis collagen

dpm/mgDNA

053(This case) 372 1,729 3.98 118 (Lethal01) 1,385 4,042 6.34

144(TypeIII01,age 5) 818 2,264 6.69

147(TypeIII01,age 46) 693 2,292 5.59

the laterounds ofamplification. Both primersets amplifya

cDNAsegment that hasintron 26 in common.

The PCRproducts ofthe normalandmutant RNAwere

cloned. DNAsequencingrevealed thepresenceofthe intron 26

(Fig.5A)in thelarger species. Furthermore,theexpectedfirst

positiondonor site Gwasreplaced byanA. This mutation was

confirmed when the corresponding region of the genomic DNA wasisolated by PCRand foundtobe presentin - 50% of

the clonessequenced (Fig.5 B).

Thecloned reverse transcriptase PCR fragments

encom-passingexons25-28 derivedfrom oligonucleotideset 3 were

used to generatearadiolabeledantisense cRNAprobes comple-mentary to the retained intron andthenormally spliced

tran-script. Theseprobeswerehybridizedtonuclear and cytoplas-micRNAderived from thepatientand control cell strain(Fig.

Delay Unreduced Reduced

1 2 3 4

-Y1(I111)unreduced

_pm% -(x1(II1)reduced

mm

_mI

U..

~--c1(1)

oif wow':2*_1 x_-a2(I)

Figure1.SDS-PAGEof radiolabeled collagen from patient and con-trolfibroblastcultures.Thecells werelabeled for 24hand the cell layer and media combined for analysis. The sample was treated with

pepsintodestroy the noncollagen proteins before the gel electropho-resis. The typeIIIcollagen is separatedfrom type I based on the

unique intrahelicalcysteinedisulfide bonds within typeIIIcollagen. Therelativeratio of theaI

(111)/al

(I)wasobtained by quantitative

densitometryof the pattern inlanes3 and4. Lanes1 and 3, patient; lanes 2 and4, control.

(5)

al(I)mRNA

6-=

5-o 4.-I

3-

2-0I

I I I I I I I II-I

0 1 2

glTotalRNA Ratio PatientlStd=0.3

1

2

a2(1) mRNA

I I I I I I I II I

0 1 2

pgTotal RNA

RatioPatientlStd=1.19 Patienta((1) / ca2(1) mRNA=0.25

Figure 2. Plotof dot hybridization densityandtotal RNAobtained

from patient- and control-cultured fibroblasts.TheslopefortheaI (I)

mRNAofthepatient islessthanthecontrolwhile thatofa2(I)is similartocontrol.Thisfinding is consistent withareduction ofthe

a1(I)mRNAthat accumulateswithinthefibroblasts ofthepatient.

6). The probe containing the included intron protected the

predicted 442-nt fragmentinthe nuclear (Fig. 6 A, lanes 4 and 6)butnotthecytoplasmiccompartment(Fig.6A, lanes 3and

5) ofthepatient RNA. Noevidence of the 442-ntbandwas presentinthe controlRNA(Fig. 6 A, lanes 7 and 8). The RNA encoded by the normal COLlA1 allele from the patientaswell asallthe RNA from thecontrol cell linedidnot protectthe

intron 26-containing probe. Instead the probe was cleaved

producingtwofragments (209 and 90 nt) collinear with the flankingexons. The cRNAprobe derivedfrom the normally spliced allele was protected equally well by total fibroblast

RNAfrom the patient (Fig. 6 B, lane 6) and control (Fig. 6 B, lane 5), indicating thatexonskippingwasnotoccurringtoa significant degreeas aresult of the donorsitemutation(27). The intensity of the normally spliced transcriptinthe patient

total RNAextractis significantly lessthan the control, con-firming the lowamountof normala1(I) mRNA that ispresent

inthiscellstrain. Discussion

The fibroblasts derived from this patient hadaG-- A

transi-tionatposition + 1 of intron 26 in oneof thetwoCOLlA

1

2

3

4

5

A B

A

B C

A B

C

A

B

A

B

C

Figure 3. Indirect RNase protection of nuclear and cytoplasmical (I)

mRNAextractedfrom patient's cul-turedfibroblasts. The fully protected ( 1.8-nt) bandpresentin both

com-partmentsisidenticaltothe band foundin normal fibroblasts. However, thelower bands ( 1.4 and 0.4 nt) that

werepresentinthe nuclear RNA (lane

1) but absent from the cytoplasmic RNA(lane 2)wereuniquetothis

pa-tient.

allelessothatthe donorsplicesitewasinactivated. Asa

conse-quenceofthis mutation, all of intron 26wasretained and the frame-shifted mRNA transcript contained severalstopcodons within and downstream of the intron insertion. Analysis of RNAtranscriptsfrom other multiexongenessuchas

dihydro-folate reductase(28)and triosephosphateisomerase(29)have shown thattransportof the abnormaltranscriptinto the

cyto-plasmwasreduced when thestopcodonwaslocatedin allbut

the terminalexon.The behavior of themutantal(I) mRNA

in the 053cells isconsistent with the model thatastopcodon

before thepenultimateexonprohibitstransportfrom the nu-cleus,since therewas noevidence of abnormal transcript

accu-mulation inthecytoplasm.

M

1.30 1.00

0.80

0.60

Figure4. PCR of reverse-transcribed cDNAusing

nuclearRNA extracted from control- and

patient-cultured fibroblasts.(Lanes A)PatientRNA; (lanes B) control RNA; (lanes C)clonedal(I)cDNA. The

0.31 newband ispresentin lanes 3 A and 4 A, and is

0.27 - 150bp

larger

that theexpectedband. The

hetero-dimerband is also present in these lanes. Theprimer 0.1 9 pairsaregiven: (lanes 1)exons35-39; (lanes2)exons

-0' 12 30-36; (lanes 3)exons25-31; (lanes 4)exons24-28; (lanes 5)exons19-23.(Lane M)PhiX HaeIII DNA

markers.

DefectiveSplicing ofal(I)mRNA in OsteogenesisImperfectaType 1997

6-c 5-*

4-'r

3-._ d

2-01

1

-8..

(6)

-Patient Control

GAT C GAT C

ftm.

W. _Wh v"

A.

-0

x

wU

a 0

x

Iw

... GOT OTT CCC GOR CCC CCT GOC OCT OTC

... Gly Ual Hyp Gly Pro Hyp

Gly

Ala Ual

Intron 26 a

gtaagtatct cctttccatc cctacctcct tccgattgct

gccccggcac tttcgtctcc ctgcaggagg ggtgcta...

OAT GAR ORO OCT ...

Asp Gly Glu Ala ...

Muta nt All

IelIe

N o

rmalI

A

IelIe

G A T C G A T C

t~~~~~~~~~~~r ~ ~ ~ ~. L

0

_' . o~~~~~~~~~~~~C

dGGTTaagac

GGCCTTCstagttN

Figure 5. DNA sequencingofthemutation within intron 26.(A)

DNAsequencingof cloned PCRfragmentsfromthepatientand controlnuclearRNA. Theabnormalpatientalleledemonstrated the

presenceofacomplete intron 26 withaG->Atransition in the+ 1

donorposition. The patientalso hadsequencedemonstratingnormal

splicing of intron26. Thepartialsequenceofexons26 and 27 and intron26 isgiven below the figure. (B)DNAsequencingof cloned

alleles ofpatient053. The G-. Atransition thatwasobserved in the

nuclearRNAwasconfirmedinthegenomicDNA. Thepartial se-quenceofexonandintron26 isgivenbelow thefigure.

Aframeshiftorpremature stopcodon that results froma

mutation within an exon would be expected to produce the sameeffect as aretainedintron, althoughanexample of this

type of mutation has not been

reported.

All ofthe defined mutations ofCOL I A 1 orCOLI A2that inducedaframeshift did notresult inapremature stop codon before the terminal exon(30-33).Lowlevelsofal(I)mRNA(basedonreversed ratio ofa1 / a2 mRNAratio of1:2)wereobserved withaframe shift inexon 51that inactivatedthe authentic translation termi-nation codonandextended the translated producttothe first polyadenylation signal (34). A

potential

mechanism for the lowaI(I)mRNA seen in this case isanindirect effecton

splic-ing secondary to the altered

transcriptional

termination

sig-nal(35).

Infibrillarcollagen genesall theexonsthat encodethe tri-ple-helical portion of theachainarein-framecassettes of the Gly X-Y

triplet starting

with the first G of the

glycine

codon and ending withthelast nucleotide of theY

position

amino acid.Thecollagen mutationsthat causeabnormalities of

splic-ingwereinitially discovered because of

synthesis

ofshorteneda

chains.Theshortened chain resulted fromexon

skipping

due to amutation in eitheranintron donororacceptor site

(Table

III). These mutations canresult in more severe forms of01 becausetheshortenedRNA

transcripts

are in

frame,

are trans-portedto thecytoplasm, andaretranslated into afunctional although truncatedachain. The shorteneda

chain

is

incorpo-rated into a trimeric moleculethat interferes with

secretion,

proteolytic processing, and

fibrillogenesis,

thus

accounting

for the strongdominantnatureofmutations of structural macro-molecules(36).

Anexplanation for whyasimilar mutation inadonorsite results in exon skipping in the casescited above and intron retention inour case comesfrom themodelofexondefinition developed by Robbersonetal. (37) and Talerico and Berget (38).Inthis model,thefirststepisthebinding of U2RNA to a potential intronacceptor site followed by thesecond step, in which U I RNA andspecific proteins identifyand bind to the strongest

adjacent

downstream donor site(39). Thisprocess definesthe exonsand initiatesthe

splicing

machinerytoexcise theintervening intronsequence.IfU1RNA identifiesan ade-quate donorwithin 300 bases (theupperlimit in size for inter-nal exons [40]), then an exon isdefined (Fig. 7 a) andthe intervening intronsarespliced. Withamutantdonorsite, ifthe intron islarge and U 1 RNA does not detect an adequate down-streamdonorsite within 300bases, no acceptable exonis de-fined, so thatit and itsflankingintrons are spliced out of the maturemessage (exonskipping; Fig. 7b).However, if the in-tron issmall, then U 1RNA willidentifytheauthentic donor site ofthesubsequent downstream intron. This definesthetwo exonsand theincluded intronas anacceptable"exon."Thus, theentire intron is retained in themature RNA(intron

reten-tion; Fig.

7 c). Thismodel accountsforwhy a mutant donor sitecanresult ineitherexonskippingorintron retentionand also

predicts

that allintronacceptorsite mutations willresult inexonskipping,because thedefiningprocess to identify an exonisinitiated fromthe acceptorsite.

Approximately halfof the introns withintheaI(I) geneare

< 150bp, makingtherisk fora donorsite mutation leading to a retained intronacommonpossibility. There are 25

exon/in-tron/exon

combinations

thatare <300bp, thesizelimit for an acceptableexon if the donorsite oftheincluded intron was inactivated. 17ofthecandidate siteswouldinduce a frame shift

mutation,

while5 of the in-frame introns containanin-frame stopcodon. The

phenotype of

a

patient

harboringamutation that introduces aframe shift-producing or stop

codon-con-1998 M. L. Stover, D. Primorac, S. C. Liu, M. B. McKinstry, and D. W. Rowe

A

(D Cs c 0

Cs

Exon 26

Exon27

GOT CCT OCT GOC ARA

Gly Pro Ala Gly Lys

(7)

A

M 1 2 3 4 5 6 7 8 9 M

-1.38

-0.78

-0.40

I I

o

--*

0- x.. * i..

209-1 ...- -3 9. 0

-- 209 1~l43 ~90

H--

3-_I

3

M 1 2 3 4 5 6 M

I I I

1

I,- 7JJ~-O

-. 40

-1.38

-0.78

-0.53

-0.404 I I 1 1

g .l [rI -I. I I IJ

I I l I _ o

go -- -0.16

1241 25 1 Z6 27 I28

-*-- 379

I

taining retained intron would beexpectedtobe similar regard-less of the site of the intron. However, variability might be observed if theexon/intron/exon size approachesorexceeds 300bases. At thatpointexon definitionwould not be

deter-mined invariably and the entire domain would be either

re-tained orskipped.Diseaseseveritywould reflect theproportion of mRNA that contained theskippedexon.

Review of published data ofcollagen splicing mutations is consistent with the exon definition model, with one possible exception (Table III). All acceptor mutations resulted inexon

skipping (49, 50),asdid donor mutations where the

exon/

in-tron/exon size(scanningdomain)was >300 nt(5, 45-48). In a number ofthesecases(49)the mRNAproductofthe mutant

allelewas lower than predicted bythe model. It seemslikely thatcryptic splice sites mayhave been used andproducedsome

transcripts containing frame shift mutations that were not

transported to the cytoplasm. Since nuclear RNA was not

ex-aminedinthese cases, thispossibilityneeds further

examina-tion.

The exon definition model may require modification to accommodate an exon 14 donor site mutation in COLlAl (50). Here intron retention is predicted that would insert three in-frame stop codons and produce an OI type I phenotype. Instead, exon 14 was skipped and resulted in a lethal phenotype (Table III). Apotential explanation for thisexception isthe natureofthe mutation. This donor site mutationwaslocatedat position + 5, which is a lesshighly conserved consensus base andproducedeither normalsplicingor exonskipping.Normal splicingwasdecreased at elevated temperatures. This suggests thattheexon-definingmechanism canbedisruptedifthere is weakbinding ofU1 RNA atthe mutantdonorsite. Inthose transcriptswhere U 1 binds the mutant site but does not result in cleavage,there isinterference withsplicing of theupstream intron such that theupstream intronforms alariat with the downstream branchpointresultingin exonskipping. Coopera-tionbetweenbranchpointsand donorsitesindeterminingthe extentofexonskippinghas been observedinadifferentcontext (39, 51). Other factors suchasstrength ofcompeting donor and acceptor splice sitesand exon size also influence which splicing alternative will predominate,and may accountfor the heterogeneity of diseaseseverity observed in mildto moder-ately deforming01 that result from mutations that affect splic-ingofcollagen mRNA. Theexception observed in this case suggests that anincluded intronwillonlybe observedwhen the donorsite iscompletelyinactivatedand thescanning mecha-nismprogresses to the nextdownstream strong donorsite.

Analysis of patients with a null collagen allele has been difficultbecauseprotein datapointingtothesite ofapotential

*1i

Figure6.DirectRNase protection ofRNAfrompatient and control fibroblast strains.(A)RNAwashybridized with the intron

26-con-tainingcRNAprobederived fromcloning and sequencingexperiment discussedinFig.5 b. Twoseparatepreparations ofnuclear and

cyto-plasmicRNAfromthepatient's fibroblast (lanes 3-6)wereanalyzed.

Theprobeis 522nt(lanesIand 9) and is incompletely digestedby the RNasetreatment(lane 2). Itprotectsa442-nt fragment ofRNA from thenuclearcompartmentfromthepatient fibroblasts(lanes4

and6),whichisnotfoundinthe cytoplasmiccompartmentfromthe

samecellpreparations (lanes 3and5).RNAfromacontrolcellstrain

didnotshowthis band(lane 7, cytoplasmic; lane 8,nuclear).The

smallersize results from RNasecleavageof thepolylinker portionof thecloningvector.Theprobeisnotfully protected by normally splicedRNAand is cleaved intotwoexon-containing fragments

con-tainingexons24-26(209 nt)andexons27-28(90 nt).Thetwo

fragmentswerepresentinall lanescontaininga1(I) collagenmRNA.

Kinased RNAmarkersareinthe lanes M. Theprobeand the

ex-pectedband sizesarediagrammedbelow thefigure. (B)RNase pro-tection with the introncontaining(lanes 1-3)andfully spliced (lanes 4-6)cRNAprobes.Lane 1,introncontaining probealone;lane2, patient cytoplasmic RNA;lane3, patientnuclearRNA;lane4, spliced probe alone;lane5,totalRNAfromcontrolcells;lane 6 total

RNAfrompatientcells. Kinased RNA markersareinlanesM.A

diagramof thefully splicedcRNAisgivenbeneath thefigure.

Defective Splicing ofal(I) mRNA inOsteogenesis ImperfectaType I 1999

B

(8)

14-Table III. OutcomeofReported Splicing Mutations

Outcome Scan

Intron No. Intronmutation domain Predicted Observed Clinicalphenotype

al(I)mRNA

6(45) Donor(-1) 345 Exon6skip Exon 6skip EDS VII

14(50) Donor(+5) 213 Intron 14inclusion (+) Exon14partial skip* Lethal 01 16 (41) Acceptor Exon 17skip Exon17skip Moderate 01 21(42) Acceptor Exon 22skip Exon 22skip Severe nonlethal OI 26(This case) Donor(+ 1) 251 Intron26inclusion (+) Intron 26inclusion (+) Mild(typeI)OI

47(46) Donor (+ 1) 482 Exon 47skip Exon47skip Lethal01

a2(I) mRNA

6(47) Donor(+ 1) 749 Exon6skip Exon 6skip EDS VII 6(48) Donor(-1) 749 Exon6skip Exon 6skip EDS VII 10 (43) Acceptor Exon11 skip Exon 11 skip EDSVII/Mild01 12 (5) Donor(+2) 1399 Exon 12skip Exon 12skip Moderate OI 27(44) Acceptor Exon28skip Exon28skip Lethal01

Theintron containing an acceptor or donor site mutation is designated in columns 1 and 2. Thescanningdomain(exon/intron/exonsize)isonly

given for donor sitemutations because it is this type of mutation in which thescanningdomain appearstobeimportant.Thepredictedand actual outcomesof the acceptorordonormutationareindicated. Whetheraretained intron would containastop codonorinduceonebyaframe shift within the mature RNA isgiveas + or -.Thediseaseseverity observedas aconsequence of the mutation isgiven.EDSVIIis Ehlers-Danlos syndrome, type VII. *Thisexception is discussed in thetext.

'acceptor 5' donor 3'acceptor 5 donor 3' acceptor 5' donor a

Normal

Splicing

3' acceptor 5' cnor 3'

acceptor

qdonor 3'

apetor

5'donor

%% I I e0

% I I

.

a

a

c IntronInclusion

Exon defined because scan domainc300bp

lI

3' acceptor 5'cdbpor 3'acceptor 3'acceptor 5'donor

b Exon skipping /

I

4 1

4% I

4 I

4.

Exon undefined because scan domain>300bp

3'acceptor5'dohr 3' acceptor 3'adceptor 5'donor

4 .

USE BLACK INK LANCASTER PRESS

Figure7.Outcome ofsplicing with a mutation that inactivates a donor site. U2 RNA (open circle) identifies the3'acceptor site and subsequently U 1 RNA(filled circle),andassociated proteins identify the downstream donor site. (a) Normal splicingoccurswhen exons areidentified and theinterveningintronsareremoved.(b)Exonskippingoccurswithadamageddonorsite. If thescanningmechanism doesnotidentifyan ade-quate donor within 300bpof the acceptorsite,theregionisnotconsideredan exonand theregionisspliced.Theoutcomeis that theexon

ad-jacenttothemutantdonorsite isskipped.(c)Introninclusionalso can occurwithadamaged donor site. If thescanningmechanism does

identifyastrongdonorsite within 300 bp of the acceptor site, then theexonis defined by thepositionof the donor site. If thenewdonorsite is thenextdownstream exon, then the entire intron is includedaspart of theenlargedexon.Ifanadequatedonor isidentified within theintron,

thencryptic splicingandinclusion ofapartialintronareobserved.

(9)

mutation are lacking. Initialcharacterizationofnuclear RNA not only suggestspotentialmechanisms to account for the un-derproduction ofmature mRNAfromthe mutant allele, it pro-videslocalizationof an unprocessed intron that might not be obtained from analysis oftotalRNA because ofthe enrichment oftheabnormal transcript in the nuclear compartment. The difficulty of detecting null-producing mutations in cDNA de-rived fromtotal RNA is illustrated by the presence of 2 abnor-malof 19 sequenced cDNAtranscriptsofthe 2.5-kb ferroche-lastase mRNA (52), < 2% of the expected cystic fibrosis trans-membrane conductance regulator transcript in bronchial epithelial cells detected by mutation specific oligonucleotide hybridization (53), and < 5% of a frame shift-producing exon skip mutation ofthe 5.3-kb lowdensity lipoprotein receptor mRNA (54). Itis likelythatframe shift mutationsthat result from insertionsordeletionswithin exons of COLlA 1 will also accountfortype I 01 but these will be hard to localize by the RNaseprotection techniques used in thiscase. PCRof frag-mentsofnuclear mRNAenriched forthepresenceofthe mu-tanttranscript followed byananalysis usingtechniques capa-bleof detectinga l-bp changewillberequiredtoidentify such cases.Discovery of mutations withintheregulatory domains of theCOLIA 1gene thatleadtoreducedtranscriptionalactivity will be an even greater challenge because the regions essential for theactivity of thisgene in collagen-producingtissues are not yetclearlydefined(55). Thesewillbeimportant abnormal-itiestoidentifybecause it is likely that subtle mutations that result in apartially null allele of theCOLlA1 gene may be a commoncauseoftype I 01 andheritable forms ofosteoporosis.

Acknowledgements

This workwassupported by National Institutes of Health grant

AR-30426. This workwasdone in partas fulfillment ofD.Primorac's

Ph.D.requirement at the University of Zagreb.

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References

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