Phenotypic Comparison of an Osteogenesis Imperfecta Type IV Proband with ade Novoα2(I) Gly922 → Ser Substitution in Type I Collagen and an Unrelated Patient with an Identical Mutation

BIOCHEMICAL AND MOLECULAR MEDICINE 62, 26–35 (1997)ARTICLE NO. MM972620

Phenotypic Comparison of an Osteogenesis Imperfecta Type IVProband with a de Novo a2(I) Gly922 r Ser Substitution

in Type I Collagen and an Unrelated Patientwith an Identical Mutation

Antonella Forlino,*,† Elena D’Amato,‡ Maurizia Valli,* Gianni Camera,§ Elizabeth Hopkins,†Joan C. Marini,† Giuseppe Cetta,* and Domenico A. Coviello‡,1

*Department of Biochemistry ‘‘A. Castellani,’’ University of Pavia, Pavia, Italy; †Section on Connective Tissue Disorders,Heritable Disorders Branch, NICHD, NIH, Bethesda, Maryland; ‡Institute of Biology and Genetics,

University of Genoa, Genoa, Italy; and §Human Genetic Center, Galliera Hospital, Genoa, Italy

Received June 6, 1997

fragility with several femur fractures, dentinogenesisWe examined the type I collagen synthesized by cul- imperfecta, wormian bone, and reduced height and

tured dermal fibroblasts from a patient affected with weight. We conclude that this phenotype is relatedosteogenesis imperfecta (OI) type IV. Both normal both to the location of this mutation and to the similarand abnormal trimers were produced. The mutant extent of matrix incorporation by the mutant chains.collagen molecules were excessively modified intra- Molecular and biochemical studies of unrelated indi-cellularly, had a melting temperature 47C lower than viduals with identical amino acid substitutions inthe control, were secreted at a reduced rate, and un- type I collagen resulting in either similar or dissimilarderwent delayed processing to mature a chains. clinical outcomes will make a significant contribution

Molecular investigations identified a G r A transi- to identifying the factors involved in the modulationtion in one COL1A2 allele, resulting in a Gly922 r Ser of the OI phenotype. q 1997 Academic Presssubstitution in the a2(I) chain. The proband’s muta-tion was demonstrated to arise ‘‘de novo’’ by the ab-

Osteogenesis imperfecta (OI) is a heritable connec-sence of the mutant allele restriction enzyme patterntive tissue disorder characterized by bone fragilityfrom parental genomic DNA.and deformity. The clinical severity is very broad,We analyzed the insoluble extracellular matrix de-

posited by long-term cultured fibroblasts from our pa- ranging from mild to lethal. Over 150 mutations intient and from a previously described unrelated indi- type I collagen coding sequences have been identifiedvidual who carries an identical substitution. In both in patients with the disease (1). The majority of thesecases, the mutant chain constituted 10–15% of the to- are point mutations, which cause the substitutiontal a chains deposited. by another amino acid for one of the glycine residues

We also present here the first detailed comparison which are present at the first position of each Gly-of phenotype between unrelated OI patients with anXaa-Yaa triplet composing the collagen helix (2,3). Theidentical collagen mutation. These two patients areglycine residues are important for helix folding. Sub-both Caucasian females, ages 8 and 9 years, each diag-stitutions for glycine cause a delay in helix formationnosed as type IV OI by the Sillence classification. Theyand excess glycosylation of the constituent chainshave a similar phenotype including moderate skeletal(4). A minority of OI collagen mutations cause single-exon skipping; small deletions and insertions as well1 To whom correspondence should be addressed at Istituto dias large deletions have also been reported.Biologie e Genetica, Viale Benedetto XV, 6, 16132 Genoa, Italy.

E-mail: [email protected]. We do not yet understand the exact mechanisms

261077-3150/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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27OSTEOGENESIS IMPERFECTA TYPE IV GLY922 r SER SUBSTITUTION

by which the primary defects in collagen result in to healthy unrelated parents and diagnosed with themoderately severe type IV form of OI, according tothe wide range of OI phenotypes. Several models

have been proposed in an attempt to clarify the rela- the Sillence classification (6). Birth weight was 2.77kg. Fractures of the right humerus and fourth andtionship between genotype and phenotype. A gradi-

ent model correlated increased severity of OI with a fifth left ribs were noted at birth. Other radiologicalfindings included femoral bowing, wormian bones,more C-terminal position of the mutation along the

chains (5). This model describes some sets of amino and generalized osteoporosis. Physical examinationrevealed blue sclerae, dentinogenesis imperfecta,acid substitutions well, especially along the a1(I)

chain, although several exceptions have been re- pectus carinatum, and scoliosis, which developed atage 3. The patient has incurred approximately 12ported. A regional model appears to describe the ge-

notype/phenotype relationship better for mutations femur fractures. Bailey rods were placed at age 2years. Radiographically, her growth plates havein the a2(I) chain (6). This model stresses the pres-

ence of local domains crucial for folding, stability, sharp margins.There is mild central compression of all thoracicand/or extracellular interactions. Seven alternating

regions of nonlethal and lethal phenotypes are delin- and lumbar vertebrae. The patient stood at 9 monthsand walked briefly at age 2 years. She is currentlyeated by the 68 mutations which have been identi-

fied in a2(I). unable to walk independently. At age 7.5 years, shehad the height of an average 4-year-old girl.As the collagen mutation map has become more

saturated, unrelated patients carrying the same mu- American proband. This Anglo–Saxon child is 9tation have been identified. Some of these shared years old and was diagnosed at birth with OI typemutations are associated with similar phenotypes, IV. Birth weight was 2.95 kg. She has true macro-while others have discordant clinical outcomes. This cephaly, with a head circumference average for ansuggests that phenotypic similarity or divergence adult female, blue sclerae, dentinogenesis imper-could result from discrete effects of other connective fecta, a mildly flared thoracic cage, and long bonetissue components, such as proteins which interact bowing. She was diagnosed with basilar invagi-with type I collagen in the extracellular matrix. nation at age 7 years, but has a normal neurological

Here we describe the third case of an OI patient examination.with a sporadic mutation causing a Gly922 r Ser This girl has incurred six femur fractures and re-substitution in the a2(I) chain. This Italian girl has quired three intramedullary rodding procedures. Ra-the moderately severe type IV form of OI (6). We diographically, her bones are osteoporotic with aclinically compared this patient with an American wide metaphyseal flare of her femurs. The growthgirl who carries an identical mutation. The biochem- plates of her long bones are open with well-definedical and molecular studies of the American girl have margins. A mild scoliosis is present with mild com-been previously reported (7; Proband A in the origi- pression of lumbar vertebrae and moderate compres-nal report). There is also a Canadian child with this sion of thoracic vertebrae. The patient has experi-mutation (8) on whom detailed clinical data are un- enced some learning difficulties. Independent ambu-available for comparison. The Italian and American lation was attained at age 3 years. She is able tochildren with Gly922 r Ser have similar phenotypes. walk 100 feet without gait aids and uses a wheel-

Associated with this similarity of phenotype are chair for long-distance mobility. Her growth rate hasbiochemical similarities of collagen helix overmodi- always been slow and her bone age has been consis-fication, extent of secretion of the mutant protein, tently 2 years less than her chronological age. At 8.5and deposition in extracellular matrix. Biochemical years of age, she had the height of an average 3-year-and clinical correlations of individuals who share old girl. Her maximal response to growth hormoneidentical mutations are important for understanding treatment was a 50% increase in her growth rate,the factors which modulate clinical outcome in OI. but she was unable to maintain this response beyond

1 year of treatment.MATERIALS AND METHODS

Cell CultureSubjects

Italian proband. The proband who is biochemi- Dermal fibroblast cultures were established froma skin punch biopsy of the Italian proband when shecally characterized here is an 8-year-old female, born


28 FORLINO ET AL.

was 40 days old, from an age-matched control and The cDNA synthesis was performed at 427C for 30min, followed by 35 cycles consisting of denaturationfrom her parents. Biopsies were performed after in-

formed consent. The cells were grown in Dulbecco’s at 957C for 1 min, annealing at 587C for 90 s, andextension at 727C for 90 s. The cDNA fragments weremodified Eagle’s medium supplemented with 10%

newborn calf serum, 200 U/ml penicillin, 0.2 mg/ml reamplified with two different internal oligonucleo-tides to obtain shorter fragments (16). Primer pairsstreptomycin sulfate and used in passages 2–15.AL26 (listed above) and AL29 5*-GGGAGACCG-

Biochemical Studies TTGAGTCCA-3 * (nt 3578–3595) and AL28 5*-TAA-AGGGTCACCGTGGCTTC-3 * (nt 3439–3458) andFibroblast proteins were labeled for the indicatedAL27 (listed above) were used for COL1A1, andtime with [2,3-3H]proline (Amersham). Type I colla-primer pairs AM1 (listed above) and AL30 5*-CCT-gen obtained after pepsin digestion of ethanol-pre-TATCGCCACGAATGCC-3 * (nt 3146–3164) andcipitated medium and cell layer proteins was ana-DC12 5*-CAAGAGGTCCTAGTGGCCCAC-3 * (ntlyzed on 6% SDS–urea–PAGE. CNBr peptide analy-3119–3139) and DC13 (listed above) were used forsis was performed by treating gel slices containingCOL1A2. Amplification cycles were performed as de-the separated a1 and a2 chains with 25 mg/mlscribed in the previous PCR. SSCP analysis of theCNBr. The samples were electrophoresed on 10%amplified products was performed. The samplespolyacrylamide–SDS–urea gels.were denatured at 957C for 3 min and electropho-Collagen secretion and thermal stability were as-resed on a 5% acrylamide gel without glycerol at 47C.sayed as described (9,10). Procollagen maturationFragments were visualized by silver stain.was evaluated as described (11) in the presence of

COL1A2-amplified cDNA corresponding to nt0.05% dextran sulfate (12). To study insoluble ma-3123–3732 was subcloned into the PCR 2.1 vectortrix formation, cells of both probands were plated(TA Cloning Kit, Invitrogen). SSCP analysis wasand grown for 48 h. They were then labeled for an-performed on the PCR-amplified subclones for alleleother 48 h in serum-free medium containing 100 mg/identification. The sequences of both alleles were de-ml ascorbic acid and 0.05% dextran sulfate (13).termined using the dideoxy chain terminationAfter the medium was removed and the cells weremethod (Sequenase, USB). Direct sequencing waslysed, the insoluble matrix was recovered in electro-performed on a product from a separate RT-PCR,phoresis sample buffer and analyzed on SDS–urea–purified by Magic Prep (Promega), using the Seque-PAGE as described (11).nase kit (USB). Genomic DNA was extracted fromproband’s and parents’ peripheral blood as describedMolecular Studies(17). Exon 46 of COL1A2 was amplified using two30-mer primers identical to the 5*-end and comple-Fibroblast total RNA was extracted using the gua-

nidium thiocyanate method (14). Reverse transcrip- mentary to the 3 *-end of this exon, as described (7).PCR products were digested by NciI and analyzedtion–polymerase chain reaction (RT-PCR) was used

to amplify collagen cDNA. First-strand cDNA from on an 8% acrylamide gel. Allele determination inparents and proband was also performed by PCRboth type I collagen transcripts was synthesized us-

ing 500 ng of total RNA with AMV-reverse tran- amplification of the hypervariable regions of Apo Band Ig-JH genes, as described (18).scriptase (Promega) in a total volume of 20 ml. The

numbering system for the nucleotides of COL1A1and COL1A2 has been defined by Dalgleish (15). Two RESULTSreverse primers were used: AL27 5*-CGAACCACA-TTGGCATCATC-3 * (nt 3774–3793) for COL1A1 Biochemical Analysis of Type I Collagenand DC13 5*GAGAAATCACAGTATACTTTG-3 * (nt3712–3732) for COL1A2, respectively. SDS–urea–PAGE of pepsin digested type I colla-

gen from cultured skin fibroblasts of the Italian pro-PCR amplifications were performed with TaqDNA polymerase (Promega) using two sense prim- band showed both normally and slowly migrating

a1(I) and a2(I) chains (Fig. 1A). Densitometric anal-ers: AL26 5*-GGTGAACCTGGCAAACAAGG-3 * (nt3057–3076) for COL1A1 transcript amplification ysis revealed that the overmodified, slowly migrat-

ing chains were partially retained in the cell layerand AM1 5*-TCTTCTTGGTGCTCCTGGTA-3 * (nt2734–2753) for COL1A2, respectively. (Fig. 1B). The extent of overmodification, confirmed



FIG. 1. Analysis of type I collagen synthesized by cultured dermal fibroblasts. (A) Type I collagen obtained after pepsin digestion ofmedium and cell-layer protein was analyzed on 6% SDS–urea–PAGE. A double width band (a1(I)*, a2(I)*) of both a chains is presentin medium and cell layer of the proband (P). C is an age-matched control. (B) Short-term pulse of dermal fibroblasts from a normalsubject and from the OI proband. Dermal fibroblasts from a control (N) and the proband (P, normal protein; P*, mutated protein) werelabeled for the time indicated and collagen was prepared from the medium and cell layer. For each time, the plot indicates the percentageof a chains present in the medium with respect to the total a chains present both in the medium and in the cell layer. The data are theaverage of two independent experiments. (C) Pulse–chase experiment. Dermal fibroblasts from control (C) and proband (P) were labeledwith tritiated proline for 4 h; the medium was then replaced with fresh medium containing cold proline and incubation continued for thetimes indicated. For each time point procollagen was prepared and run under reducing conditions on 5% SDS–urea–PAGE. Densitometricscanning showed a lower relative amount of pC-a1(I) with respect to pro-a1(I) for the proband than for control.

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30 FORLINO ET AL.

by CNBr peptide mapping, suggested a C-terminallocation of the molecular defect. Thermal unfoldingof helices was assayed as loss of trypsin resistance.About 50% of the proband’s type I collagen was un-folded at 387C, which is 47C below the thermal stabil-ity of normal type I collagen (427C) (data not shown).On the basis of the protein data, the search for themutation was confined to the carboxyl-terminal re-gion of the triple helix domain.

Molecular Analysis

The cDNA synthesis and PCR amplifications ofboth COL1A1 and COL1A2 mRNAs yielded the ex-pected fragments: 737 bp for AL26/AL27 and 999 bpfor AM1/DC13. Shorter fragments of 539 and 355 bp,respectively, were obtained using AL26/AL29 andAL28/AL27 for COL1A1. Fragments of 431 and 610bp were obtained using AM1/AL30 and DC12/DC13for COL1A2. SSCP analysis of the amplified prod-ucts was performed on a 5% acrylamide gel withoutglycerol at 47C. A series of extra bands was detectedfrom the 610-bp fragment of COL1A2 which spannedfrom 3123 to 3732 nt. This fragment was subclonedand the two alleles were differentiated by SSCP, un-der the conditions described above. Sequencing iden-tified a G r A transition at nt 3173 in one allele,which converted the Gly922 to a Ser (Fig. 2A). The

FIG. 2. cDNA sequences and genomic DNA analysis. (A) DNAmutation was confirmed by direct sequencing of ansequences of mutant and normal COL1A2 cDNA of the proband.

RT-PCR product obtained from an independent RNA The arrow indicates the G r A transition at nt 3173. (B) Analysissample (data not shown). The mutation abolished an of the COL1A2 mutation in the patient and her family. Exon 46

of proband (2), father (3), and mother (4) was PCR-amplified fromNciI restriction site. The complete digestion of theleukocyte DNA. The PCR products were digested by NciI and ana-parents’ genomic DNA by NciI indicated that thelyzed on an 8% acrylamide gel; they were compared to fX174 RF/proband bears a de novo mutation (Fig. 2B).HaeIII marker fragments (1). Only the proband had the 107-ntfragment resulting from lack of NciI digestion of the mutant allele.

Consequences of the Mutation for Type I CollagenFunction

Analysis of type I collagen (Fig. 1A) showed that, of procollagen processing to pC-collagen, which isthe product remaining after removal of the procolla-after long-term labeling, the overmodified chains

were partially accumulating in the cell layer. Short- gen N-propeptide by N-proteinase. The SDS–urea–PAGE analysis of the medium procollagen of theterm pulse and pulse–chase experiments were per-

formed to verify the cellular retention. The mutant proband showed only traces of pCa1-collagen com-pared with a control incubated under the same con-chains were barely detected in the medium during

a 6-h pulse, and the retention of these mutant chains ditions (Fig. 1C). Densitometric scanning of the flu-orogram showed that, after a 6-h chase, partiallyalso impaired secretion of normal trimers (Fig. 1B).

These findings were confirmed after a 4-h pulse-label processed a1 chains accounted for 25–27% of totalproa1(I) in control but only 7–10% in the proband’sfollowed by a chase up to 24 h. In the proband, the

pro-alpha chains synthesized during the pulse re- cells. The rate of processing was also measured asthe percentage of fully processed a1(I) chains, withquired 6 h to clear from the cells, whereas control

chains cleared after 2 h. respect to the sum of unprocessed, partially pro-cessed, and fully processed proa1(I) chains in fibro-The chase experiments showed a decreased rate



Clinical Comparison of the Patients

Both the Italian and the American children withthe Gly922 r Ser substitution presented with clini-cal features characteristic of Sillence type IV OI.Within the range of clinical severity compatible withtype IV OI, these girls have quite similar clinicalfindings. In both patients the disorder was recog-nized at birth, with similar long bone and rib find-ings (Table 1). The patients also had similar birthweight, length, and head circumference.

Both children have moderate susceptibility to frac-tures, incurring several femur fractures and requir-ing intramedullary rodding. The radiographic ap-pearance of their femurs is similar, with generalizedosteoporosis and metaphyseal flare (Fig. 4). Both pa-FIG. 3. Matrix deposition in the presence of dextran sulfate.tients also have wormian bones of the skull and ver-Fibroblasts were plated and labeled as stated under Materials and

Methods. The insoluble matrix left after Triton treatment of cell tebral compression (Fig. 5), although the scoliosis islayer was recovered and run on a 6% SDS–urea–PAGE under more severe in the Italian proband. Both have thenonreducing conditions. The formation of insoluble matrix was con- growth deficiencies characteristic of type IV OI.firmed by the presence of type I collagen dimers (b) and fibronectin

When the Italian proband had a chronological age(FN). The arrow indicates the broadening of a1(I) in proband (P).of 7.5 years, her weight was that of an average 3-year-old girl and her 108-cm height was that of anaverage 4-year-old. When the American child had ablasts cultured in the presence of 0.05% dextran sul-chronological age of 8.5 years, she had the weight offate. The presence of the dextran sulfate polymeran average 4-year-old girl and the 93.3-cm height ofconcentrates the newly synthesized procollagen inan average 3-year-old.the pericellular region accelerating its natural pro-

The phenotypes of the two children are not identi-cessing in vitro. These experiments showed that 75–cal. The American girl has true macrocephaly and90% of collagen chains in controls, but only 50% inbasilar invagination while her Italian counterpartthe proband, were present as a1(I) (data not shown).has a proportionate cranium. Moreover, in spite ofIn addition, the bands corresponding to both aher large cranium and more extreme short stature,chains were clearly overmodified.the American patient is a functional ambulatorHowever, when the patient’s cells were labeled inwhile the Italian girl requires a wheelchair. Ofthe presence of 0.05% dextran sulfate for a longer

period of time, that is 48 h, type I collagen was fullyprocessed to mature alpha chains. After 48 h label- TABLE 1ing in the presence of 0.05% dextran sulfate, electro- Clinical Comparison of the Two Patients:phoretic analysis of the insoluble matrix remaining the Italian and the American Probandsafter cell lysis showed complete processing of procol-

Physical characteristics Italian Americanlagen and formation of covalent crosslinks (Fig. 3,b band), confirming that a matrix was formed. In

Blue sclerae / /addition, overmodified collagen chains were evident Dentinogenesis Imperfecta / /in the proband’s matrix and amounted to about 10– Low mineralization skull / /

Wormian bone / /15% of total collagenous chains. The extracellularGeneral Osteoporosis / /matrix deposited by long-term cultured fibroblastsMultiple fractures / /from the American proband yielded a similar rate of Macrocephaly / /

incorporation of the mutant protein. Furthermore, Basilar invagination 0 /the densitometric scanning revealed that there was Vertebral compression / /

Short stature / /a lower amount of insoluble matrix proteins, suchReduced weight / /as fibronectin and type I collagen, deposited in theIndependent ambulation 0 /patient cell line compared to that of the control.


32 FORLINO ET AL.

FIG. 4. X-rays of the lower long bones of the probands. (A) Previously described American proband; (B) Italian proband describedin this communication. Both patients show moderate osteoporosis, tibial bowing, and metaphyseal flare. Bailey–Dubow rods have beenplaced in each proband’s femurs bilaterally and diaphyseal atropy is present.

course, these differences in motor function are also and C-terminal ends. The secretion of the mutanttrimers, as evaluated by pulse experiments, was alsoinfluenced by the aggressiveness of rehabilitation ef-impaired. Finally, there was a 47C decrease in theforts. The variability in phenotype of the patients isTm of the helix, as assayed by trypsin resistance.well within the OI type IV range and the two pro-

The present case is the third report of a Gly922 rbands may be considered to have similar outcomesSer substitution in OI patients. Recently, identicalto date.mutations have been detected in unrelated patientsat a number of sites along the type I collagen helix.DISCUSSIONBiochemical and clinical comparisons can now be

In this report, we describe a sporadic, moderately performed. These comparisons provide the opportu-severe case of type IV OI, caused by a G r A transi- nity to better understand the complexity of the ele-tion at nt 3173 of one COL1A2 allele. This base ments involved in the wide clinical manifestation ofchange results in a Gly922 r Ser substitution in the the disease. The two previous cases with a Gly922 r

a2(I) chain synthesized by the mutant allele. The Ser substitution, according to available data, dis-entire collagen triple helix of the Italian proband played the same biochemical abnormalities as thewas overmodified due to slow folding of the collagen present case. In addition, this case and the pre-molecules. This was associated with a reduced rate viously identified American proband have the sameof procollagen processing which suggested an exten- rate of incorporation of the abnormal protein into the

extracellular matrix (10–15% of the total a chains)sion of the conformational alteration at both the N-



sonal communication) with type II OI. Three of thefive sets, Gly238 r Ser (21; Byers, Nuytinck andDe Paepe, personal communication), Gly586 r Val(13,22), and Gly859 r Ser (23; Nuytinck and DePaepe, personal communication), have nonlethaloutcomes but vary across a broader clinical range.For example, Gly238 r Ser cases range from mildtype I OI to severe type III. The only instance ofidentical mutations with an outcome which does notfit the regional model is Gly811 r Ser (Byers, per-sonal communication), with outcomes of both lethaltype II and moderate type IV OI.

The situation is more complicated for the muta-tions in the a1(I) chain, where the regional modelwould require a minimum of 10 regions along thechain and must tolerate 17 exceptions. Thirteen setsof identical mutations have been reported in a1(I).In two of those sets, Gly352 r Ser (7,24,25; Byers,personal communication) and Gly862 r Ser (26,27,28; Byers, personal communication), the out-comes include both lethal and nonlethal forms. Inboth lethal cases, however, the probands may havehad the severe II/III form of OI. In seven sets, allpatients have nonlethal forms, but with variabilityof expression ranging from mild type I to type III orIV. Finally, there are six sets in which the outcomes,FIG. 5. Lateral views of spine of probands. (A) Previouslyeither lethal or nonlethal, have been identified asdescribed American proband; (B) Italian proband described in

this communication (proband A). Both probands have moderate having the same Sillence type.central compression of all thoracic vertebrae; additionally, the These sets of identical mutations which result inItalian proband has central compression of lumbar vertebrae. phenotypic differences represent an opportunity to

investigate the role of other factors in the clinicalexpression of OI. Transcriptional and translational

during long-term fibroblast culture. Deposition of factors in the cells as well as molecular and biochem-the mutant chains into the extracellular environ- ical interactions with modifying genes or proteinsment appears to be crucial for the production of se- may play a role. A possible explanation for the muta-vere tissue pathology (19). tion pairs in which there is type I OI versus a clini-

The regional model which has been proposed to cally severe form is the occurrence of mosaicism incorrelate phenotype and genotype in OI predicts that the mildly affected patient. Second, even within themutations in the same location will result in the regions defined by the regional model, the effect ofsame phenotype. This model associates lethal and the same mutation can be modulated by quantitativenonlethal outcomes with the presence of discrete, and structural differences in collagen fibril forma-alternating domains along the helical region of the tion and interaction of fibrils with the noncollage-collagen chains (Fig. 6). For the a2(I) chain, the 68 nous proteins in the extracellular matrix. Since col-identified glycine substitutions and single-exon lagen is packed into higher order structures, disrup-splicing defects describe four lethal and three nonle- tions of interactions with bone matrix moleculesthal regions. Of the six instances of identical muta- such as osteonectin, osteopontin, or bone sialopro-tions in a2(I) in unrelated individuals, five would fit tein may be involved. Variable expression could alsothe regional model with outcomes broadly described be the result of abnormalities in other bone proteinsas lethal or nonlethal. Two of the five sets have the themselves. For example, a possible decorin defectsame Sillence type: Gly922 r Ser with type IV OI was reported in a patient with a Gly415 r Ser substi-

tution in the a1(I) chain of type I collagen, whichand Gly625 r Asp (20; Nuytinck and De Paepe, per-


34 FORLINO ET AL.

FIG. 6. Diagram of collagen mutations identified in more than one OI patient.

In From Genotype to Phenotype (Humphries SE and Malcomresulted in a more severe OI phenotype than in anSE, Eds.). Oxford: Bios Scientific, 1994, pp. 49–65.unrelated patient described in the literature (24,

4. Engel J, Prockop DJ. The zipper-like folding of collagen tri-29,30). Analysis of the extracellular matrix organi-ple helices and the effects of mutations that disrupt thezation of bone in OI patients and binding studies ofzipper. Annu Rev Biophys Chem 20:137–152, 1991.

the mutant collagens will help clarify the pathophys-5. Byers PH, Wallis GA, Willing MC. Osteogenesis Imperfecta:

iology of the disorder. Translation of mutation to phenotype. J Med Genet 28:433–442, 1991.

6. Sillence DO. Osteogenesis Imperfecta nosology and genetics.ACKNOWLEDGMENTSAnn N Y Acad Sci USA 543:1–15, 1988.

7. Marini JC, Lewis MB, Wang Q, Chen KJ, Orrison BM. Ser-We extend our gratitude to the patients described in this paperine for glycine substitutions in type I collagen in two casesand their families for their cooperation. This work was supportedof type IV Osteogenesis Imperfecta (OI): Additional evidenceby MURST 40% and 60% and by CNR, Rome, Progetto Bilateralefor a regional model of OI pathophysiology. J Biol ChemItaly/USA.268:2667–2673, 1993.

8. Sztrolovics R, Glorieux FH, van der Rest M, Roughley PJ.REFERENCESIdentification of type I collagen gene (COL1A2) mutationsin non-lethal Osteogenesis Imperfecta. Hum Mol Genet

1. Byers PH. Osteogenesis Imperfecta. In Connective Tissue 2:1319–1321, 1993.and Heritable Disorders (Royce PM and Steinmann B, Eds.).

9. Valli M, Mottes M, Tenni R, Sangalli A, Gomez Lira M, RossiNew York: Wiley-Liss, 1993, pp. 317–350.A, Antoniazzi F, Cetta G, Pignatti PF. A de novo G to T

2. Kuivaniemi H., Tromp G, Prockop DJ. Mutations in collagen transversion in a pro-alpha 1(I) collagen gene for a moderategenes: Causes of rare and some common diseases in humans. case of Osteogenesis Imperfecta. Substitution of cysteine forFASEB J 5:2052–2060, 1991. glycine 178 in the triple helical domain. J Biol Chem

266:1872–1878, 1991.3. Dalgleish R. Mutations in type I and type III collagen genes.



10. Bruckner P, Prockop DJ. Proteolytic enzymes as probes for 22. Bateman JF, Hannagan M, Chan D, Cole WG. Characteriza-tion of a type I collagen alpha 2(I) glycine-586 to valinethe triple-helical conformation of procollagen. Anal Biochem

110:360–368, 1981. substitution in Osteogenesis Imperfecta type IV. Detectionof the mutation and prenatal diagnosis by a chemical cleav-11. Valli M, Rossi A, Forlino A, Tenni R, Cetta G. Extracellularage method. Biochem J 276:765–770, 1991.matrix deposition in cultured dermal fibroblasts from four

probands affected by Osteogenesis Imperfecta. Matrix 23. Rose NJ, Mackay K, Byers PH, Dalgleish R. A Gly 859 r

13:275–280, 1993. Ser substitution in the triple helical domain of the alpha 2chain of type I collagen resulting in Osteogenesis Imperfecta12. Bateman J, Golub SB. Assessment of procollagen processingtype III in two unrelated individuals. Hum Mutat 3:391–defects by fibroblasts cultured in the presence of dextran394, 1994.sulphate. Biochem J 267:573–577, 1990.

24. Bateman JF, Moeller I, Hannagan M, Chan D, Cole WG.13. Forlino A, Zolezzi F, Valli M, Pignatti PF, Cetta G, BrunelliCharacterization of three osteogenesis imperfecta collagenPC, Mottes M. Severe (type III) Osteogenesis Imperfecta duealpha 1(I) glycine to serine mutations demonstrating a posi-to glycine substitutions in the central domain of the collagention-dependent gradient of phenotypic severity. Biochem Jtriple helix. Hum Mol Genet 3:2201–2206, 1994.288:131–135, 1992.14. Chomczynski P, Sacchi N. Single-step method of RNA isola-

tion by acid guanidinium thiocyanate–phenol–chloroform 25. Mackay K, Byers PH, Dalgleish R. An RT-PCR-SSCP screen-extraction. Anal Biochem 162:156–159, 1987. ing strategy for detection of mutations in the gene encoding

the alpha I chain of type I collagen: Application to four pa-15. Dalgleish R. The human type I collagen mutation database.tients with Osteogenesis Imperfecta. Hum Mol GenetNucleic Acids Res 25:181–187, 1997.2:1155–1160, 1993.16. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiyat. De-

26. Virdi AS, Loughlin JA, Irven CM, Goodship J, Sykes BC.tection of polymorphisms of human DNA by gel electrophore-Mutation screening by a combination of biotin-SSCP andsis as single-strand conformation polymorphisms. Proc Natldirect sequencing. Hum Genet 93:287–290, 1994.Acad Sci USA 86:2766–2770, 1989.

27. Namikawa C, Suzumori K, Fukushima Y, Sasaki M, Hata A.17. Coviello DA, Bertolini S, Masturzo P, Ghisellini M, TiazzoRecurrence of Osteogenesis Imperfecta because of paternalK, Zambelli F, Stefanutti C, Torcia F, Pachi A, Ricci G, etmosaicism: Gly862 r Ser substitution in a type I collagenal. Chorionic DNA analysis for the prenatal diagnosis ofgene (COL1A1). Hum Genet 95:666–670, 1995.familial hypercholesterolemia. Hum Genet 92:424–426,

1993. 28. Zhuang J, Tromp G, Kuivaniemi H, Castells S, Bugge M,18. Decorte R, Cuppens H, Marynen P, Cassiaman JJ. Rapid Prockop DJ. Direct sequencing of PCR products derived from

detection of hypervariable regions by the polymerase chain cDNAs for the pro alpha 1 and pro alpha 2 chains of type Ireaction technique. DNA Cell Biol 9:461–469, 1990. procollagen as a screening method to detect mutations in

patients with osteogenesis imperfecta. Hum Mutat 7:89–99,19. Bateman JF, Golub SB. Deposition and selective degrada-1996.tion of structurally abnormal type I collagen in a collagen

matrix produced by osteogenesis imperfecta fibroblasts in 29. Mottes M, Gomez Lira MM, Valli M, Scarano G, Lonardo F,vitro. Matrix Biol 14:251–262, 1994. Forlino A, Cetta G, Pignatti PF. Paternal mosaicism for a

COL1A1 dominant mutation (alpha 1 Ser-415) causes recur-20. Byers PH, et al. Mosaicism in Osteogenesis Imperfecta. Vrent Osteogenesis Imperfecta. Hum Mutat 2:196–204, 1993.International Conference on OI, Oxford, 1993.

21. Rose NJ, Mackay K, Byers PH, Dalgleish R. A Gly 238 r Ser 30. Dyne KM, Valli M, Forlino A, Mottes M, Kresse H, Cetta G.Deficient expression of the small proteoglycan decorin in asubstitution in the alpha 2 chain of type I collagen results in

osteogenesis imperfecta type III. Hum Genet 95:215–218, case of severe/lethal Osteogenesis Imperfecta. Am J MedGenet 63:161–166, 1996.1995.


Documents

Phenotypic Comparison of an Osteogenesis Imperfecta Type IV Proband with ade Novoα2(I) Gly922 → Ser Substitution in Type I Collagen and an Unrelated Patient with an Identical Mutation