available is not an upper limit. But it may beincreased significantly through the introduc-tion of seismic moment tensor informationand estimates of stress from geological andgeodetical investigations as well as from pastearthquakes.Our results indicate that only about one-

third of the strong earthquakes are precededby foreshocks that are separated in timefrom an independent shock by more thantM. For other modem catalogs of earth-quakes that are similar in quality to thecentral California CALNET catalog, thenumber of failures-to-predict is about thesame, that is, of the order of two-thirds (1,3,4).A modification of the above strategy for

prediction is called for in the case of theoccurrence of strong earthquakes. Strongearthquakes have the potential for serving asforeshocks of even stronger earthquakes, orthey may be the main shock in the sequence,just as weaker earthquakes can serve bothfunctions. However, the coda time tM isabout 15 minutes for an earthquake withmL = 5, and this time increases by a factorof about \/1T0 for a unit increase in earth-quake magnitude. If an earthquake withmL = 6 were to occur, with or withoutprior warning according to the scenarioabove, no alarm for a possibly even strongerearthquake would be sounded for abouttM = 50 minutes, which might be an unac-ceptably long delay for issuing a warning.This difficulty is circumvented if we reducethe dead time for large earthquakes to avalue less than the coda time.

This modification for strong earthquakesindicates that response strategies can also bedeveloped with time delays of the order ofseconds. As suggested by Heaton (20), itmay be possible to predict some large earth-quakes through the analysis ofsmall startingphases of complex events that later blossominto large earthquakes. These small startingphases are genuine earthquakes whose sig-nals overlap with those of their successorsand raise the probability level for a shorttime, thereby triggering an alarm for thelarger event. The number of these preshocksshould increase as tb-3/2, where tb is the timebefore the start ofthe main phase ofa strongearthquake (2-4). In the present method,we are not restricted to dead times of theorder of rupture times, but instead we areable to use longer delays of the order of afew minutes. With this procedure thereshould be far fewer failures-to-predict forvery strong earthquakes. Automated re-sponse strategies could take advantage ofthese predictions in a well-developed tech-nology.The differences between our proposed

forecasting technique and methods that use

19 JUNE i987..

empirically derived probabilities of fore-shock-main shock occurrence may be sum-marized as follows. (i) Since our model isbased on a formulation derived from a mul-tidimensional stochastic process, it is notnecessary to use arbitrary windows to ana-lyze seismicity, nor is it necessary to deleteaftershocks from a catalog to make the cata-log amenable to statistical analysis. There-fore, our forecasts are not dependent on apost-factum classification of earthquakesinto fore-, main, and aftershocks, a subdivi-sion that may not be possible in real time.(ii) Since the parameters of our seismicitymodel are obtained through a maximumlikelihood procedure, the model is optimalin a quantitative way. The choices of theparameters can be justified on the basis of awell-defined theoretical model of earth-quake occurrence (2, 10, 19). Furthermore,the model itself is consistent with all of theother aspects of statistical seismicity thathave been well documented, and it has notbeen derived for the sole purpose of devel-oping the foreshock-main shock relations.The model has only three parameters thatare adjusted to the properties of the localseismicity: the rate of occurrence of inde-pendent earthquakes and the coefficientsthat specify the occurrence of dependentearthquakes, ,u and a. In one sense theexponent 2/3 in Eq. 4 is also an adjustableparameter, but since this can be derived onformal grounds (3), we have considered it asfixed. (iii) The likelihood function we havederived allows us to estimate the effective-ness of the proposed prediction scheme interms of information content of an earth-

IN HIS CLASSIC STUDY OF INBORN ER-

rors of metabolism, Garrod (1) notedthat an unusually high proportion of

patients with alkaptonuria were progeny ofconsanguineous marriages. Almost immedi-

quake catalog. The procedures outlined herecan be adapted to predicting schemes otherthan the one we have used, as soon as thequality and quantity of the data describingthese precursors reach the stage where theycan be processed by similar multidimension-al statistical techniques.

REFERENCES AND NOTES

1. L. M. Jones, Bull. Seismol. Soc. Am. 75, 1669(1985).

2. Y. Y. Kagan and L. Knopoff, J. Geophys. Res. 86,2853 (1981).

3. mi preparation.4. , Geophys. J. R. Astron. Soc. 55, 67 (1978).5. W. Rinehart, H. Meyers, C. A. von Hake, Summaty

ofEarthquakeJData Base (National GeophysicalDataCenter, Boulder, CO, 1985).

6. Y. Kagan and L. Knopoff, Phys. Earth Planet. Inter.14, 97 (1977).

7. D. Vere-Jones,J. Phys. Earth 26, 129 (1978).8. K. Aki, in Earthquake Predition, An International

Review, D. W. Sunpson and P. G. Richards, Eds.(Maurice Ewing Series, no. 4, American Geophysi-cal Union, Was7hington, DC, 1981), p. 566.

9. F. Papangelou, Z. Wahrsceinlichkeststheorie er.Geb. 44, 191 (1978).

10. Y. Y. Kagan and L. Knopoff, Geopiys. J. R. Astrmn.Soc. 88, 723 (1987).

11. I. Rubin, IEEE Tran. Inf: Theory IT-18, 547(1972).

12. T. Ozaki, Ann. Inst. Stat. Math. B31, 145 (1979).13. D. Vere-Jones and T. Ozaki, ibid. B34, 189 (1982).14. Y. Y. Kagan and L. Knopoff, Proc. 8th Int. Con{f

Earth Ensg. 1, 295 (1984).15. S. M. Marks and F. W. Lester, U.S. Geol. Surp. Open-

File Rep. 80-1264 (1980), and references therein.16. See (4), p. 69.17. W. H. K. Lee et al., U.S. Geol. Surp. Open Fie Rep.

(1972).18. W. L. Ellsworth, BuU. Seismol. Soc. Am. 65, 483

(1975).19. Y. Y. Kagan, Geopys. J. R. Astron. Soc. 71, 659

(1982).20. T. H. Heaton, Scienc 228, 987 (1985).21. Supported in part by grant CEE-84-07553 from the

National Science Foundation. We acknowledgc use-fiu discussion with G. M. Molchan. Publication2962, Institute ofGeophysics and Planetary Physics,University of Caliormia, Los Angeles, CA 90024.4 August 1986; accepted 1 April 1987

E. S. Lander, Whitehead Institute for Biomedical Retsearch, Nine Cambridge Center, Cambridge, MA

02142; Department of Biology, Massachusettnsimtof Technology, Cambridge, MA 02139; and HaaUniversity, Cambridge, MA 02138.D. Botstein, Departnent of Biology, Massachusetts Inwstitute of Technology, Cambridge,MA 02139.

REPORTS.

Homozygosity Mapping: A Way to Map HumanRecessive Traits with the DNA of Inbred ChildrenEiuc S. LANDER AND DAVID BOTSTEIN

An efficient strategy for mapping human genes that cause recessive traits has beendevised that uses mapped restriction fragent length polymorphisms (RFLPs) and theDNA of affected children from consanguineous marriages. The method involvesdetection of the disease locus by virtue of the fact that the adjacent region willpreferentially be homozygous by descent in such inbred children. A single affectedchild of a first-cousin marriage is shown to contain the same total information aboutlinkage as a nuclear family with three affected children. Calculations show that itshould be practical to map a recessive disease gene by studyingDNA from fewer than adozen unrelated, affected inbred children, given a complete RFLP linkage map. Themethod should make it possible to map many recessive diseases for which it is;impractical or impossible to collect adequate numbers of-families with multipklaffected offspring.

on

Sep

tem

ber

21, 2

008

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

ately, Bateson (2) supplied the Mendelianexplanation, providing an early success for thethen newly rediscovered theory of heredity.Theory subsequently predicted and observa-tion has confirmed that the rarer the disease,the more pronounced is the effect (3).Our strategy for gene mapping relies on

the related fact that in a child of a consan-guineous marriage affected with a recessivegenetic disease, a region of many centimor-gans (cM) spanning the disease locus isalmost always homozygous by descent. Afew other regions may also be homozygousby descent in any given child, but theseregions will vary from one child to another.Searching for regions that are consistentlyhomozygous by descent would be expectedto provide a powerful strategy for mappinga recessive gene (4).A complete restriction fragment length

polymorphism (RFLP) linkage map of thehuman genome (5) may soon be available.We suspected that such a map might make itfeasible to detect homozygosity by descent(by looking for regions in which a numberof adjacent RFLPs were all homozygous)and thereby to map recessive traits in hu-mans without the need for family studies.Below, we provide a rigorous justificationfor this intuition.

Consider a child with coefficient of in-breeding F, defined (6) as the chance that agiven locus will be found homozygous bydescent or, equivalently, the fraction of thechild's genome expected to be homozygousby descent; for sibling, first-cousin, andsecond-cousin marriages, F = 1/4, 1/16,and 1/64, respectively. Imagine a diseasecaused by a recessive allele at a single Men-delian locus, where disease alleles have fre-quency q in a population in Hardy-Wein-berg equilibrium. If the child is affected,then homozygosity by descent at the diseaselocus is almost surely at fault; the probabilityis a = Fql[Fq + (1 - F)q2]. To see this,note that Fq of all inbreds will be homozy-gous by descent at the disease locus andaffected, while (1 - F)q2 will not be homo-zygous by descent at the disease locus butwill be affected due to random meeting ofdisease alleles. Provided that q is small com-pared to F, a is very close to 1.While the region actually around the dis-

ease locus is homozygous by descent withprobability a 1, unlinked regions are ho-mozygous by descent with a probability ofonly F (<< 1). Consider an RFLP so poly-morphic that homozygosity for any alleleimplies homozygosity by descent. Each timethat such an RELP were observed to behomozygous in an inbred affected child, onewould obtain an odds ratio of a:F in favorof the hypothesis that the disease gene istightly linked to the marker, as compared to

ix68

the hypothesis that it is unlinked. For exam-ple, finding homozygosity for such anRFLP in the affected progeny of a first-cousin marriage (F = 1/16) yields odds of16:1 in favor of linkage. If the RFLP is infact tightly linked, then homozygosity willbe consistently observed and odds of 163 :1in favor of linkage-well over the acceptedthreshold (7) of 1000:1 required for prov-ing linkage-will be obtained after studyingonly three such affected children.While one cannot count on having a

single highly polymorphic RFLP exactly atthe disease locus, a similar effect can beaccomplished by the use of a genetic map ofadequately polymorphic RFLPs spacedthroughout the human genome.

Specifically, homozygosity mappingwould be performed as follows. To test thehypothesis that the disease gene maps to agiven interval, we would examine the DNAofeach affected inbred child for homozygos-ity at (say) six consecutive RFLPs-three tothe left and three to the right of the interval.There are 64 possible outcomes at the sixloci denoted HHH.HHH, HHH.HH-,HHH.H- -, and so on, where "H" denoteshomozygosity, " " denotes heterozygosity,and "." indicates the interval being tested forlinkage. For each outcome, we would com-pute the probability PI that the outcomewould occur if the disease actually maps tothe interval and P2 that the outcome wouldoccur if the disease is actually unlinked. Theodds ratio in favor of linkage is then P1 :P2.Odds ratios from independent cases canthen be multiplied until the threshold of1000:1; for the customary "LOD score"(loglo of the odds ratio) the threshold is 3.

d - e ~ ~ ~ -~~ ++ od-----O.00.O + 0000

_ed_d

+XO ~~~~<>z ~~+0000-

-o.o.,d4-o. =I -HH.H--

Fig. 1. Sample pedigree showing a first-cousinmarriage producing a child affected with a reces-sive disease, due to homozygosity by descent for adisease-causing allele (d). For the purpose ofhomozygosity mapping, one is only assumed toobserve the pattern of homozygosity and hetero-zygosity in the child; in the case shown, examin-ing six RFLPs flanking the region would yield thepattern -HH.H- -.

Computing PI and P2 constitutes multi-locus linkage analysis ofseven loci (six mark-ers and the disease gene) in a pedigree (Fig.1) with a great deal of missing information.These calculations can be performed by stan-dard programs that compute multilocuslikelihoods (8), although they might bequite time-consuuming. Recently, however,Lander and Green (9) introduced an effi-cient algorithmn that makes it convenient toperform the analysis in seconds. A computerprogram, called HOMMAP, has been writ-ten implementing this algorithm for proge-ny of sibling or cousin marriages.

Consider, for example, a child of a first-cousin mating affected with a rare recessivedisease. By rare, we mean that the frequencyq of the disease allele is small enough so thata 1. Suppose that a genetic map wereavailable consisting of RFLPs spaced every10 cM throughout the human genome, eachwith a probability h = 0.5 of being foundhomozygous at random in the populationfrom which the child is drawn. For eachoutcome, we used HOMMAP to computethe probabilities of observing an outcomegiven that the disease gene lies in the centerof the region (PI) and given that the diseasegene is unlinked (P2). A partial list (given inorder ofLOD score) is shown in Table 1.

In order to estimate the power of themethod, it is necessary to compute the ex-pected LOD score (ELOD). If the intervalactually contains the disease gene, theELOD will be Y PI1(w)z(w), which turns outto be 0.31. Thus, to achieve an LOD scoreof 3, about ten (= 3/0.31) such inbredaffected children will on average be re-quired.

If the interval under study is unlinked tothe disease gene, however, the ELOD willbe I P2(w)z(w), which turns out to be-0.35; about six children will be needed toachieve the customary threshold (6) of -2for exclusion.The results ofmany similar calculations are

summarized in Fig. 2, which displays theexpected number of inbred offspring [proge-ny of brother-sister (Fig. 2A), first-cousin(Fig. 2$), or second-cousin (Fig. 2C) mar-riages] required to map recessive diseases withRFLP linkage maps ofdifferent powers. Witha map of RFLPs that is only modestly poly-morphic (h = 0.5) and spaced every 15 cM,about 15 such inbreds should suffice forhnkage mapping. With more highly polymor-phic RFLPs, more densely spaced RFLPs, orboth, between five and ten such individualsshould suffice. Of course, other instances ofdose inbreeding (for example, uncle-niece,F = 1/8) are also useful and results are inter-mediate. The analysis above assumes no cross-over interference; the presence of positiveinterference would tend to increase the power

SCIENCE, VOL. 236

on

Sep

tem

ber

21, 2

008

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

Table 1. Probability of different patterns of ho-mozygosity given that the disease gene lies in thecenter ofthe region [PI(Q)] or is not linked to theregion [P2(m)]. Markers are spaced every 10 cMand are homozygous 50% of the time at random."H," homozygosity; "-," heterozygosity; ".," theinterval being tested for linkage. Probabilities areshown as percents.

Outcome, X PI(w) P2(w) LOD, (z)(w)

HHH.HHH 21.5 3.3 +0.81HHH.HH- 9.0 2.2 +0.62- HH.HHH 9.0 2.2 +0.62HHH.H- H 5.4 2.0 +0.43H-H.HHH 5.4 2.0 +0.43HHH.H-- 4.7 2.0 +0.37--H.HHH 4.7 2.0 +0.37-HH.HH- 3.8 1.7 +0.35HHH.--- 1.3 1.6 -0.09---.HHH 1.3 1.6 -0.09H--.HHH 1.4 1.7 -0.08HHH.--H 1.4 1.7 -0.08- - - -- - - 0.07 1.3 -1.24

of the method.It might seem that more distant inbreed-

ing (for example, a mating ofsecond cousinsrather than of siblings) would yield moreinformation about linkage, since unlinkedregions would be even less likely to behomozygous by descent. However, two seri-ous problems limit the usefulness of moredistant consanguinity: (i) Because of theincreased number of opportunities for re-combination, a denser RFLP map is re-quired to extract the full information (Fig.2). (ii) As the coefficient of inbreeding Ffalls relative to the allele frequency q, so toodoes the probability that the disease is actu-ally the fault of homozygosity by descent.Eventually, the disease locus will not behomozygous by descent frequently enoughto make detection possible. This effect isshown in Fig. 2D, which displays the rela-tive increase in the number of inbred affect-ed children required for homozygosity map-ping, as q increases, for different degrees ofinbreeding.The following prescription, for example,

will ensure at most a doubling of the re-quired numbers in Fig. 2, A to C: the allelefrequency q must be less than about 0.07 forprogeny of sibs to be useful; q < 0.02 forprogeny of first cousins; q < 0.008 forprogeny of second cousins. Since most re-cessive diseases have q < 0.02 [this beingroughly the frequency of the cystic fibrosisallele in the United States (10)], progeny offirst-cousin matings and closer should begenerally quite efficient for homozygositymapping. Progeny of second-cousin mat-ings should be collected for relatively rarerdiseases.We should emphasize that LOD scores

obtained via homozygosity mapping are au-thentic LOD scores, as used in human ge-netics: they may be directly added to LOD

19 JUNE I987

scores obtained from traditional family stud-ies, since each represent likelihood ratiosobtained by comparing the same twohypotheses. In this connection, it is strikingto note that the ELOD obtained from asingle affected child from a first-cousin mar-riage is equal to that of an outbred nuclearfamily with three affected, in the limit of adense RFLP map; in both cases, six meiosesare under study, with two being used toinfer phase of the recessive disease allele.The main advantage of homozygosity

mapping is thus that it provides a way tomap a recessive disease, even when-as isfrequently the case-families with multipleaffecteds are scarce. Since inbred children areoverrepresented among those affected with arecessive disease (and since the overrepre-sentation is more pronounced the rarer thedisease), it should often be easier to gatherenough inbreds for homozygosity mappingthan to gather enough families with multipleaffecteds for traditional family studies (11).

Bloom's syndrome (12), for example, af-fects roughly 100 living individuals, butthere are only eight known families withtwo living affecteds and one with three

@ 30-0A

0.

0.0 20'C

E 10z

Distance between consecutive RFLPs (cM)

c@ 30,oQ

n 200

.01-

2 10z

affected (13). These resources are insuffi-cient for traditional linkage analysis. By con-trast, at least 24 affecteds are children ofmarriages between cousins (with F _ 1/64).Even for a more common recessive diseasesuch as Werdnig-Hoffmann syndrome[q 0.006 in England (14)], multiplex fam-ilies may be difficult to collect because affect-ed children die at a very young age.Homozygosity mapping may therefore be

the method of choice for mapping the chro-mosomal location of most of the 1420 sus-pected recessive disorders listed in McKu-sick's catalog Mendelian Inheritance in Man(12). Of course, multiplex families shouldalso be collected where available, since LODscores can be combined.

Cases of affected inbreds can be collectedin the United States and Britain, eventhough overall consanguinity rates are low.However, population geneticists have exten-sively studied many regions in which geo-graphic isolation or cultural norms have ledto a considerably higher rate of consanguin-eous marriages (15). Among the regionsdeserving special attention for homozygos-ity mapping are: parts of Italy, where con-

n

v4)_

_

la

L.~C

0 E

00

m 0_ -

0a-u

0 10 20 30 40Distance between consecutive RFLPs (cM)

Distance between consecutive RFLPs (cM)

.0001 .001 .01Frequency q of disease allele

.1

Fig. 2. Number of inbred progeny needed to map a rare (q 0) recessive disease via homozygositymapping, as a function of the spacing between consecutive RFLPs and the degree of polymorphism ofeach RFLP. The four curves refer to RFLPs that are found homozygous with probability 50%, 30%,10%, and 0% (limiting case) in the general population. (A) Progeny of a sibling mating; (B) progeny ofa first-cousin mating; (C) progeny of a second-cousin mating. If the disease is not rare, the requirednumbers must be multiplied by the approximate correction factor in (D). [The correction factor given isexact when the distance between consecutive markers d = 0 and the rate of homozygosity h = 0; itdiffers, but only slightly, for other cases.]

REPORTS 1569

on

Sep

tem

ber

21, 2

008

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

O-~~~~~~_. , . ,-.

E h_I h=.5 /

0 10 1-CD H ogt aio heteroge-hog % 3 %, an 0% t

.0

z

0'0 10 20 30 40Distance between consecutive RFLPs (M)

Fig. 3. Homozygosity mapping of a heteroge-neous rare recessive trait by the method of simul-taneous search (19). Number offirst-cousin prog-eny required to identify a set of two loci at whichdisease alleles can occur, by means of RFLPshomozygous 50%, 30%, 10%, and 0% in thepopulation, is shown in solid curves. Analogousresult for a set of three loci is shown in dashedcurves (unlabeled). Frequency of disease alleles ateach of the loci is assumed to be equal.

sanguineous marriages are not uncommonand can be traced through records of thespecial dispensation that such marriages re-quire from the Catholic Church (16, 17);Andhra Pradesh in India, where one-third ofall Hindu marriages are between a niece andher matenal uncle (F = 1/8) (18); andmany Middie Easten populations (15).Note that the homozygosity rate h for anRFLP refers to the population from whichthe inbred is ascertained (19).Homozygosity mapping clearly requires a

reasonably complete genetic map of thehuman genome. An adequate map mightconsist of about 330 RFLPs evenly spacedevery 10 cM, each homozygous at most50% of the time in the population. Lessdense maps would still be useful, whiledenser maps would aflow linkage mappingwith even fewer cases. Since more than 1000RFLPs have already been discovered (in ahuman genome of about 3300 cM), such amap seems within reasonable prospect. Al-though the ideal map for homozygositymapping is perhaps twice as dense as that fortraditional family-based linkage studies (20),the smaller number of affected cases re-quired for mapping should compensate forany increased effort in applying more mark-ers. Moreover, one may first screen bymeans of a subset of the RFLPs and thenconfirm a suggestion of linkage by using ahigher density of RFLPs in the region,ofinterest.The DNA of affected inbred children

offers advantages not just for the initialdetection of linkage, but also for the molec-ular cloning of the disease gene. While thesurrounlding region of homozygosity by de-scrent is fairly lnare in any griven child (mei-i-an length -28 cM for the affected progeny

1570

of a first-cousin marriage), the search for thegene may be confined to the overlap of theseregions (median length -28/n cM, if naffected first-cousin progeny are studied).

Finally, we have assumed above that thedisease is homogeneous (is caused by muta-tions at a single locus), although it is hard toknow this in advance. If linkage is not foundunder the assumption of homogeneity,however, one may adapt the strategy ofsimultaneous search, which we recently pro-posed elsewhere (21). Briefly, simultaneoussearch would consist of searching for a set ofseveral loci with the property that at leastone is homozygous by descent in most ofthe inbreds. Figure 3 shows the number ofinbred individuals needed to map a hetero-geneous trait by means of homozygositymapping; for maximum efficiency, a higherresolution RFLP map would be preferred.The study of inbred children will be more

likely than traditional family studies to re-veal rare trait-causing loci in the case of aheterogeneous trait, since loci will be repre-sented in proportion to their allele frequen-cy (ql:q2:.:qk) among inbreds, but in pro-portion to the square of the allele frequency(qI2:q22:...qk2) among the general popula-tion.Homozygosity mapping may greatly ex-

tend the range ofrecessive diseases amenableto linkage mapping, because affected inbredsare not uncommon and relatively few areneeded. Homozygosity mapping becomespractical only with the advent of a humangenetic map. It exemplifies the sort of strate-gies for human genetics that will becomeavailable, we believe, as the human genomebecomes more thoroughly explored (20).

REFERENCES AND NOTES

1. A. E. Garrod, Lancet 1902-11, 1616 (1902).2. W. Bateson, Mendel's Principles of Heredity (Cam-

bridge Univ. Press, Cambridge, 1902).3. F. Lenz, Munch. Med. Wochenschr. 66, 1340

(1919); W. Weinberg, Z. Morphol. Anthropol. 46,293 (1920).

4. This point was apparently first noted by C. A. B.Smith [J R. Stat. Soc. B 15, 153 (1953)], whorecognized the theoretical efficiency of studyinyinbred affected children, given a tightly linkehighly polymorphic marker. The paucity of suchhighly polymorphic markers and the inability at thattime to construct complete linkage maps of lesspolymorphic markers led Smith to conclude that"the method is impractical." We thank an anony-mous referee for calling our attention to this pre-scient insight.

5. D. Botstem, R. L. White, M. H. Skolnick, R. W.Davis, Am. J. Hum. Genet. 32, 314 (1980).

6. S.W Am. Nat. 56, 330 (1922).7. N. E orton,Am. J. Hum. Genet. 7, 277 (1955).8. G. M. Lathrop and J. M. Lalouel, ibid. 36, 460

(1984).9. E. S. Lander and P. Green, Proc. Natl. Acad. Sci.

U.SA. 84, 2363 (1987).10. J. B. Stanbury, J. B. Wyngaarden, D. S. Frederick-

son, J. L. Goldstein, M. S. Brown, The MctabolicBasis ofInheritcd Disease (McGraw-Hill, New York,ed. 5, 1983).

11. In practice, we would recommend preparing "im-mortal" lymphoblastoid cell lines from both theinbred, affected individual and his parents. Availabil-ityofparental DNA makes it easier correctly to inferthe alleles at each locus, in order to detect homozy-gosity. Also, the expected LOD scores in the textincrease slightly if parental DNA is included in themultipoint lnage analysis. If convenient, DNAfrom other relatives, such as grandparents, may alsobe induded.

12. V. McKusick, Mendelian Inheritance inMan (JohnsHopkins, Baltimore, ed. 7, 1986).

13. J. German, personal communication.14. J. H. Pearn, J. Med. Genet. 10, 260 (1973).15. N. Morton, Curr. Dep. Anthropol. Genet. 2, 449

(1982).16. A. Moroni, AttiAssoc. Genet. Ital. 9, 220 (1964).17. G. Romeo et al., Am. J. Hum. Genet. 37, 338

(1985).18. P. S. S. Sundar Rao et al., Indian J. Med. Res. 59,

294 (1971).19. L. L. Cavalli-Sforza et al., Cold Spring Harbor Symp.

Quant. Biol. 51, 411 (1986).20. E. S. Lander and D. Botstein, ibid., p. 63.21. Proc. Natl. Acad. Sci. U.SA. 83, 7353

(1986).22. We thank R. Fitts and D. Page for helpful discus-

sions. Supported by grants from the National Sci-ence Foundation (DCB8611317), the National In-stitutes of Health (GM30467), and the SystemDevelopment Foundation.

10 February 1987; accepted 8 May 1987

Thrombospondin Promotes Cell-SubstratumAdhesionGEORGE P. TuszYNsIK, VICKI ROTHMAN, ANDREW MURPHY,KATHERINE SIEGLER, LINDA SMITH, SENA SMITH, JERZY KARCZEWSKI,KAREN A. KNUDSEN

The physiological role ofthe platelet-secreted protein thrombospondin (TSP) is poorlyunderstood, although it has been postulated to be involved in platelet aggregation andcellular adhesion. In this report, TSP isolated from human platelets was found topromote, in vitro, the cell-substratum adhesion of a variety of cells, including platelets,melanoma cells, muscle cells, endothelial cells, fibroblasts, and epithelial cells. Theadhesion-promoting activity ofTSP was species independent, specific, and not due tocontamination by fibronectin, vitronectin, laminin, or platelet factor 4. The cell surfacereceptor for TSP is protein in nature and appears distinct from that for fibronectin.

T ~* HE PLATELET-SECRETED PROTEIN scribed by Baenziger et al. (1) and laterthrombospondin (TSP) is thought shown to have a molecular weight ofto participate in platelet aggregation Lankenau Medical Research Center, Lancaster Avenue

and cellular adhesion. TSP was first de- west of City Line Avenue, Philadelphia, PA 19151.

SCIENCE, VOL. 236

on

Sep

tem

ber

21, 2

008

ww

w.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om