Nonrandomassociation II - PNAS · Vol. 82, pp. 5465-5469, August 1985 Genetics Nonrandomassociation...
Transcript of Nonrandomassociation II - PNAS · Vol. 82, pp. 5465-5469, August 1985 Genetics Nonrandomassociation...
Proc. Nati. Acad. Sci. USAVol. 82, pp. 5465-5469, August 1985Genetics
Nonrandom association of a type II procollagen genotypewith achondroplasia
(restriction fragment length polymorphism/Southern blot/dwarfism/human type II collagen)
CHARIS E. L. ENG*, RICHARD M. PAULIt, AND CHARLES M. STROM*:
*Department of Pediatrics, Joseph P. Kennedy, Jr., Mental Retardation Research Center, Committee on Developmental Biology and Committee on Genetics,The University of Chicago, Chicago, IL 60637; and tDepartments of Pediatrics and Medical Genetics, The University of Wisconsin, Madison, WI 53706
Communicated by A. A. Moscona, May 6, 1985
ABSTRACT Achondroplasia is an autosomal dominantdisorder that involves defective endochondral bone formation.Type H collagen is the predominant collagen of cartilage. Wefound a HindIH polymorphic site in the normal Caucasianpopulation by using the type II procollagen gene probepgHCol(II)A. The presence of this site yields a 7.0-kilobase (kb)band; its absence yields a 14.0-kb band. We found a significantdeviation in genotype distribution and allele frequencies in apopulation of unrelated individuals with sporadic achondro-plasia, compared with the normal control population. TheHindIll genotype frequencies in 32 individuals with achondro-plasia are 0.41 for the 7/7 genotype (controls, 0.08), 0.34 forthe 7/14 genotype (controls, 0.54), and 0.25 for the 14/14genotype (controls, 0.37). The apparent equilibrium excess ofthe "7" allele in individuals with achondroplasia may reflecteither a predisposition for the mutation that causesachondroplasia or it could be the result of the achondroplasia-causing mutation. In either case, these findings suggest anassociation of the type II procollagen gene with achondroplasia.
Type II collagen is the major collagen of cartilage. The typeII collagen molecule is composed of three al(II) (Col2AJ)chains arranged in a triple helix (1). Type II collagen may playa role in endochondral bone formation by stabilizing devel-oping structures (2). During normal limb development, thelimb buds contain type I collagen (2). Subsequently, type IIcollagen becomes the predominant form during chondrogen-esis. As the cartilage is replaced by bone, type I collagenagain predominates (2). The human type II collagen moleculehas yet to be purified in quantities sufficient for amino acidsequencing.Strom and Upholt (3) have isolated genomic clones corre-
sponding to the human al(II) procollagen gene. Previouswork utilizing the human type II procollagen gene probepgHCol(II)A has revealed the presence of a polymorphicHindIII site in the normal Caucasian population at a genefrequency of 0.35 (4). The subclone pgHCol(II)A containscoding sequences corresponding to triple helical amino acids892-1014 and continuing at least through the carboxyl-terminal telopeptide (3). The presence ofthe polymorphic siteyields a 7.0-kilobase (kb) band, in contrast to the usual14.0-kb band (4). Previous work using single and sequentialrestriction enzyme digestions as well as DNA sequenceanalyses, demonstrated that the polymorphic HindIII site islocated within the type II procollagen gene (ref. 4; unpub-lished results). Analysis of an extended family demonstratedthat this allele segregated in a mendelian fashion (4). Thedistribution of the HindIII genotypes in the overall normalpopulation, as well as the normal Caucasian population wasfound to be in a Hardy-Weinberg equilibrium (4).
Achondroplasia is a short-limbed dwarfing condition (5). Itoccurs with a frequency of 1 in 26,000 births and it is the mostcommon nonlethal dwarfing condition (5). It is inherited as anautosomal dominant disorder with 100% penetrance (6).Approximately 90% of all cases occur to parents of averagestature (sporadic) and therefore represent new mutations (5).The estimated mutation rate has been calculated to be 1.4 x10-5 (7). Individuals who are heterozygous for achondro-plasia can be identified by their characteristic clinical andradiographic features (5-9). Microscopic examination ofachondroplastic cartilage has revealed an abnormality in thegrowth plate of endochondral bones (10, 11). However, theprecise mechanism of disruption of this bone morphogenesishas yet to be elucidated. Gene dosage analyses using thehuman type II procollagen genomic probe pgHCol(II)A wereperformed on DNA samples from 36 achondroplastic pa-tients. Gene deletions were demonstrated in 4 of thesepatients with achondroplasia (ref. 12; unpublished results).The present study examines the relationship betweenachondroplasia and the human type II procollagen gene in 32Caucasian achondroplasts without type II collagen genedeletions by restriction fragment length polymorphism anal-ysis using pgHCol(II)A as a probe.
MATERIALS AND METHODS
Populations Sampled. Genotypic frequencies of polymor-phic sites associated with the human type II procollagen geneprobe pgHCol(II)A were previously determined throughsampling of 48 unrelated Caucasians (4). Similar analyseswere performed on DNA obtained from peripheral bloodsamples from 32 nonselected, independently ascertained,unrelated, Caucasian volunteers with achondroplasia. Elev-en of these individuals fulfilled all criteria for the diagnosis ofachondroplasia through clinical and radiographic examina-tion by the authors. The remainder were examined andphotographed and had clinical features consistent with thediagnosis of achondroplasia; of this latter group, the majority(14/21) had had previous diagnostic confirmation at variousmajor skeletal dysplasia centers.DNA Probes. The genomic subclone pgHCol(II)A contains
a 2.0-kb EcoRI/BamHI insert in pBR322 (3). DNA sequenceanalyses revealed that the insert contains coding sequencesspecifying amino acids 29-79 at the carboxyl-terminalpropeptide [the numbering scheme corresponds to that usedin the chickan a1(I) procollagen gene in which amino acidsare numbered sequentially, beginning with the first residue ofthe carboxyl-terminal telopeptide as described by Fietzekand Kuhn (13) and as discussed by Sandell et al. (14)]. Thissubclone also contains sequences coding for the entire
Abbreviation: kb, kilobase(s).tTo whom reprint requests should be addressed.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
kb
4.8- -as@Saaattas*a_- 5 -gam-a _lemaen na _
FIG. 1. Autoradiograms of BamHI-digested genomic DNA isolated from 32 patients with achondroplasia hybridized with pgHCol(II)A.
carboxyl-terminal telopeptide and triple helical amino acids892-1014 (of a total of 1014) (3, 4).The human genomic subclone pG44 was generously sup-
plied by L. Kunkel and contains a 2.6-kb EcoRI/HindIIIinsert in pBR322 and has been localized to Xq24-Xqter (15).The human genomic subclone HgP115 was generously
supplied by Y. W. Kan and J. C. Chang and contains a1.15-kb EcoRI insert in pBR322 (16).Southern Hybridization. DNA was prepared from 10-20 ml
of heparinized or EDTA-anticoagulated peripheral blood asdescribed (4). Restriction endonuclease (Bethesda ResearchLaboratories) digestions and appropriate control experi-ments to eliminate the possibility of incomplete digestionswere performed as described (4). After overnight incubationat 370C, the DNA samples were electrophoresed at 6 V/cmin 0.7-1.0% agarose gels for 6-8 hr, depurinated, denatured,and transferred to Zetabind filters (AMF, Cuno, Meriden,CT) by the method of Southern (17). Prehybridization,hybridization, and washing procedures have been described(4).
Inserts of pgHCol(II)A, pG44, and Hg,8115 were preparedand made radioactive by nick-translation in the presence of[a-32P]dNTPs (Amersham) to a specific activity of 107 cpmper gg of DNA as described (3). All hybridizations werecarried out in 50% formamide at 46°C under the ionicconditions described (3).
Densitometry. Densitometric analysis was performed onautoradiograms shown in Figs. 4 and 5, using an LKB Model2202 densitometer and an Apple HIe Interface with Geiscan(LKB) software.
Statistical Test. x2 tests of significance using RxC contin-gency tables were performed.
RESULTSDNA samples isolated from individuals with achondroplasiawere digested with eitherBamHI or EcoRI and the fragmentswere resolved by agarose gel electrophoresis. The DNA wassubsequently denatured, transferred to Zetabind filters, andhybridized to radiolabeled pgHCol(II)A, a probe correspond-ing to the carboxyl-terminal telopeptide of type II
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15kb
pgH9ol(II)A.
14.03M $ t Y
FIG. 2. Autoradiograms of DNA (from a sample of 15 of the 32achondroplastic dwarfs) digested with HindIII and hybridized withpgHCol(II)A.
procollagen. All 32 achondroplastic samples yielded a single4.8-kb BamHI band (Fig. 1) and a single 3.7-kb EcoRI band(data not shown). There were no exceptions. The BamHI andEcoRI bands are the sizes usually observed in the normalpopulation (3, 4). When these achondroplastic DNA sampleswere digested with HindIII, transferred to filters by theSouthern technique, and hybridized with pgHCol(II)A, thethree band patterns observed in normal populations (4) wereseen: a single 7.0-kb band (Fig. 2, lanes 1-6; Fig. 3, lanes1-7), a single 14.0 kb band (Fig. 2, lanes 13-15; Fig. 3, lanes13-17), or a 7.0-kb band and a 14.0-kb band (Fig. 2, lanes7-12; Fig. 3, lanes 8-12). In the filter shown in Fig. 2, theintensity of the 14.0-kb band appears to be less than that ofthe 7.0-kb band for individuals with the 7.0/14.0 genotype.This was not a consistent observation, however. To ensurethat a single 7.0-kb band is not due to a 14.0-kb fragment beingretained in the gel after DNA samples were transferred tofilters, the following experiments were performed. Aftertransfer was complete, the gel was restained with ethidiumbromide and visualized under ultraviolet light. No visiblestaining was observed in each gel. Most experiments involv-ing the DNA samples from individuals with the 7.0/7.0genotype were repeated several times with consistent results,even in gels with equal intensity of 7.0 and 14.0 hybridization.In addition, results were consistent with and withoutdepurination.The frequencies of the three HindIII genotypes in this
population of achondroplastic patients is compared withthose found previously in normal Caucasians (Table 1). Thedistribution in both populations is consistent with aHardy-Weinberg equilibrium (P 0.2 in the general popu-lation and P 0.1 in the achondroplastic population thatthese distributions would be observed by chance whencompared with expected Hardy-Weinberg distributions cal-culated from the observed allele frequencies in each popu-lation). However, comparison of the distribution of geno-types between these two groups shows a significant deviationfrom expectation in the achondroplastic group (P 0.002).Similarly, analyses of allele frequencies show a significantexcess of the "7" allele in the population of individuals with
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
kb
9.~~~~~~~~~~~~~~~A1bt
14.0
7.00y
?]r:W4 ti;:#-FIG. 3. Autoradiograms of DNA (from 17 of the 32 achondro-
plastic dwarfs) digested with HindIII and hybridized withpgHCol(II)A.
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Table 1. Distribution of HindIII genotypes in a normalCaucasian population and in individualswith achondroplasia
AchondroplasticControl population* population
(N= 48) (N= 32)N Frequency N Frequency
Genotypet14/14 18 0.37 8 0.257/14 26 0.54 11 0.347/7 4 0.08 13 0.41
Allelet7 34 0.35 37 0.5814 62 0.65 27 0.42
*Data from ref. 4.tContingency table-derived x2 for genotype data = 11.97, df = 2, P
0.002.4Contingency table-derived x2 for allele frequencies = 7.81, df = 1,P -0.005.
achondroplasia in comparison with the control Caucasianpopulation (P 0.005).
Since a portion ofthe apparent excess ofthe "7" allele (andthe 7/7 genotype) might be secondary to deletion ofone ofthetwo type II procollagen genes, gene dosage analysis wasperformed on the DNA samples from all 21 achondroplastswith either the 7.0/7.0 genotype or the 14.0/14.0 genotype, aswell as 4 individuals with the 7.0/14.0 genotype. Patientsheterozygous at this locus (7.0/14.0 genotype) cannot pos-sess a deletion of the gene in this region. DNA samples weresubjected to BamHI and/or EcoRI restriction digestions,transferred to filters by the method of Southern, and hybrid-ized with a combination of pgHCol(II)A, pG44 (the X-linkedgenomic probe) and/or Hgf3115 (the A-globin genomic probe)(Figs. 4 and 5). The probe pG44 is X-linked, while the al(II)procollagen gene has been localized to chromosome 12 byhybridization of pgHCol(II)A to various mouse-human cellhybrids (18). Comparisons of the collagen:pG44 and colla-gen:Hgt3115 hybridization ratios allow the collagen genedosage to be measured. The 25 achondroplasts analyzeddemonstrated normal collagen gene dosage (Figs. 4 and 5;Table 2). Since females have twice the gene dosage for theX-linked probe compared to males, the collagen:pG44 ratios
A1 2 3 4 5 6 7
I.4!T qlXkb
6.6
4.8I.
B1 2 3 4
kb i>'t5 6
6.0 * * i
3.7 & ;
2.4 a 4at
FIG. 4. Autoradiograms of BamHI-digested (A) and EcoRI-digested (B) DNA hybridized with a mixture of pgHCol(II)A, pG44,and Hg,8115. (A) The 4.8-kb band represents the pgHCol(II)Ahybridization, the 6.6-kb band represents the pG44 hybridization,and the 2.4-kb band represents the Hgf115 hybridization. Lanes: 1,female control; 2, male control; 3, 4, 6, and 7, female achondroplasts;5, male achondroplast. (B) The 3.7-kb band represents thepgHCol(II)A hybridization, the 3.4-kb band represents the pG44hybridization, and the 6.0-kb band represents the globin hybridiza-tion. Lanes: 1, female control; 2, male control; 3-6, femaleachondroplasts.
for females should be half that of males. All theachondroplasts have the same pgHCol(II)A:pG44 hybridiza-tion ratios as controls of the same sex. The collagen:pG44ratios of all females are half those of the males, thusdemonstrating the ability of this technique to differentiatebetween one and two gene copies per diploid genome. Thecollagen:Hgf3115 ratios of all the achondroplasts are similarto those of the controls. There were no exceptions. Thus, thehigh frequency of the 7.0/7.0 HindIll genotype is not due togene deletion.
DISCUSSIONThese data demonstrate a discrepancy in the distribution ofa type II procollagen HindIII polymorphic site in individualswith achondroplasia compared with a control population.Since the vast majority of achondroplasia results from newmutations (31 of 32 in the group studied here had parents with
1 2 3 4 5 6 7 8 9kb
6.6 *A*i in
4.8 .e
1 2 3 4 5kb
6.6SU WW
4.8 _ m m _ _m
1 2 3 4 5 6 7 8
kb
3.7 ** 4&,0O3.4 S Sm a SO
4 0.a0
a
A B C
FIG. 5. Autoradiograms of Southern filters ofBamHI (A and B) and EcoRI (C) digestions ofDNA hybridized with a mixture of pgHCol(II)Aand pG44. (A) The 4.8-kb band represents the pgHCol(II)A hybridization and the 6.6-kb band represents the pG44 hybridization. Lanes: 1, femalecontrol; 2, male control; 3, 4, 7, and 9, male achondroplasts; 5, 6, and 8, female achondroplasts. (B) Lanes: 1, female control; 2, male control;3, female achondroplast; 4 and 5, male achondroplasts. (C) The 3.7-kb band represents the pgHCol(II)A hybridization and the 3.4-kb bandrepresents the pG44 hybridization. Lanes: 1, female control; 2, 3, and 5-7, female achondroplasts; 4, male control; 8, male achondroplast.
Genetics: Eng et al.
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Table 2. Summary of densitometric analyses of Southern hybridization
Fig. 4A
DNA source
Female controlFemale achondroplast 1Female achondroplast 2Female achondroplast 3Female achondroplast 4Male controlMale achondroplast
pgHCol(II)A/ pgHCol(II)A/Lane pG44 ratio Hg,3115 ratio
1 0.323 0.274 0.316 0.377 0.352 0.595 0.50
DNA source
2.6 Female control2.1 Female achondroplast 52.3 Female achondroplast 62.2 Female achondroplast 72.4 Female achondroplast 82.3 Male control2.1
Fig. 4B
pgHCol(II)A/ pgHCol(II)A/Lane pG44 ratio Hg,8115 ratio
1 0.263 0.274 0.305 0.286 0.282 0.56
0.811.10.981.21.00.95
Fig. SA Fig. 5B Fig. 5CpgHCol(II)A/ pgHCol(II)A/ pgHCol(II)A/
DNA source Lane pG44 ratio DNA source Lane pG44 ratio DNA source Lane pG44 ratioFemale control 1 0.72 Female control 1 0.27 Female control 1 0.81Female achondroplast 9 5 0.69 Female achondroplast 12 3 0.24 Female achondroplast 13 2 0.88Female achondroplast 10 6 0.77 Male control 2 0.46 Female achondroplast 14 3 0.81Female achondroplast 11 8 0.81 Male achondroplast 6 4 0.44 Female achondroplast 15 5 0.80Male control 2 1.3 Male achondroplast 7 5 0.51 Female achondroplast 16 6 0.79Male achondroplast 2 3 1.3 Female achondroplast 17 7 0.85Male achondroplast 3 4 1.4 Male control 4 1.5Male achondroplast 4 7 1.6 Male achondroplast 8 8 1.6Male achondroplast 5 9 1.7
normal stature), this discrepancy cannot be secondary tolinkage disequilibrium. Since the achondroplastic populationallele frequencies appear to be consistent with Hardy-Wein-berg equilibrium, the difference must arise from an excess ofthe "7" allele.The "7" allele, while present in excess in the achondro-
plastic population, is neither absolutely necessary nor in itselfsufficient to result in achondroplasia, because 25% of studiedachondroplasts have the 14/14 genotype and 8.5% of thenormal population are 7/7.
Trivial explanations for this observed association seemunlikely. Both populations were randomly accessed. Achon-droplasia occurs in all Caucasian ethnic groups (19), makingit unlikely that the distribution differences are secondary todifferences in ethnicity in the control and achondroplasiapopulations (which was not, however, controlled).Two general alteratives are suggested by these data. The
"7" allele may, in some as yet undetermined way, predisposeto the mutation that results in achondroplasia (20, 21). Thiswould mean that unaffected individuals with the "7" allelewould be at higher relative risk to undergo the germinalmutation that would result in achondroplasia in their off-spring. Alternatively, in patients with a "7" allele, the "7"may result from the change that causes achondroplasia. Thelatter hypothesis implies heterogeneity of the mutationalevents resulting in achondroplasia. This is consistent with theobservation that 4 of 36 patients with achondroplasia showeddemonstrable deletions involving pgHCol(II)A (12). Thismolecular heterogeneity of the syndrome is not surprising inlight of the fact that most cases of achondroplasia aresporadic. In addition, the clinical course observed inachondroplasia is quite variable.We are currently attempting to differentiate between the
two alternatives through analyses of a large number of familytriads of unaffected parents and affected offspring. The firstalternative predicts that these parents would show a signif-icant excess of the "7" allele; the second alternative predictsthat the genotype patterns of these families would sometimesbe inconsistent with mendelian predictions. We have studiedfour family triads. The parents of the four achondroplastshave a genotype distribution similar to that of the normalpopulation, while the offspring possess a distribution similar
to that of the achondroplast population. But these four triadsrepresent too small a sample for statistical significance.
It is possible that the polymorphic HindIII site lies withinor in the vicinity of sequences crucial for events leading to themutation resulting in achondroplasia. These sequences mayfacilitate recombinant events, which may somehow result inachondroplasia. Such sequences have been described in bothprokaryotes and eukaryotes. Examples include the X se-quences and X-like sequences of Escherichia coli (22) andbacteriophage X (23) and the switch regions of mammalianimmunoglobulin genes (24, 25). Furthermore, it is possiblethat the HindIII site forms part of a signal, be it a portion ofa triplet codon or a donor/acceptor splice sequence. In sucha case, only a fraction of the HindIII sites would be expectedto have the mutated genotype. Conversely, not all themutations affecting the function of the signal would beexpected to result in the formation of the HindIl consensussequence. As an illustration, a mutation creating a newtermination codon could result in achondroplasia. For ex-ample, the sequences TAAGCTT, TGAGCTT, and TAGC-TT would all result in achondroplasia but only the firstsequence would create a new HindIII site (AAGCTT).Whatever the explanation for the excess "7" allele may be,the observations cited in this paper imply a relationshipbetween the type II procollagen gene and achondroplasia.
We wish to thank the members of the Little People of America,especially District 6, for their support and cooperation. We areindebted to T. Christides and C. Belles for expert technical assist-ance. C.E.L.E. is a recipient of the American Heart Associa-tion-Borg Warner Medical Student Research Fellowship. C.M.S. isa Hartford Fellow. This work was supported by March of DimesGrant 6-35160, Sprague Foundation, Schweppe Foundation, AmocoFoundation, and National Institutes of Health Grants AM-33910 andHD-04583.
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