American Journal of Medical Genetics 42:lO-14 (1992)
Cleft Lip (t Cleft Palate) Etiology: A Search for Solutions Michael Melnick Craniofacial Biology, University of Southern California,Los Angeles, California INTRODUCTION Cleft lip with or without cleft palate (CL-tP) is a major public health problem worldwide, with an incidence a t birth of about 1in 500-1,000 that varies by race and nationality; Asians are at higher risk than Caucasians or Blacks [Chung et al., 1974; Hu et al., 1982; Koguchi, 1975; Melnick et al., 1980, 1986; Myrianthopoulos and Chung, 1974; Tanaka et al., 19691. While it is increasingly clear that the same cause probably does not obtain across races or even across national groupings within races [Marazita et al., 1986a1,it is also certain that there is an important genetic component to the etiology in all populations, if not in all individual cases. Despite a half-century of intense study, the etiology of C L k P is still largely an enigma. The precise roles played by genes, environment, and chance are not even within focus. In our search for solutions to this puzzle, we have failed primarily because we have attempted to elucidate etiologic correlates to the wrong phenotype(s). CL-tP is a distant consequence of more relevant and proximate “inborn errors of morphogenesis.” As with any other genetic disorder, if the phenotype is vaguely or incorrectly defined, the chance of understanding its etiology is minimal. The purpose of this communication is to briefly review the history of our search for etiologic solutions and to suggest potentially more profitable avenues of investigation. HISTORY OF OLD SOLUTIONS Nearly 50 years ago, Fogh-Andersen 119421published the first comprehensive study of CL r P. His conclusion, after analyzing nearly 500 pedigrees, was that CL -t P was an autosomal dominant disorder with greatly reduced penetrance. While the study was first rate, the etiologic explanation was more than a little unsatisfying, for it offered clinicians little more than the empiric risk estimates they were already using. Two decades later, the multifactorial/threshold model (MF/T) was introduced to the problem [Carter, 19691. The MF/T provided a clever and innovative first approximation to a solution of what was clearly a difficult Received for publication November 13, 1990; revision received June 7, 1991. Address reprint requests to Michael Melnick, DDS, PhD, Craniofacial Biology, DEN 4242, MC-0641, ZJniversity of Southern California, Los Angeles, CA 90089-0641.
0 1992 Wiley-Liss, Inc.
problem; for it recognized that the genetic component was likely non-Mendelian and that environment played an important role as well. The model postulates that the occurrence of CL P depends on the additive effects of several “minor” mutant genes (polygenes)and environmental factors that may also be multiple; the accumulation of these aberrant genetic and environmental factors can be tolerated by the developing embryo to a point (termed the threshold), beyond which there is a risk of malformation [Carter, 19761. The model logically gave rise to a series of testable predictions of population and family data. Unfortunately, these predictions were never satisfied when subjected to statistical analysis [Melnick et al., 1977,1980,1986; Marazita et al., 1984, 1986bl. The flaw in the argument of the model’s partisans went far deeper than a failure to test rigorously the predictions or ignoring the results of tests that were made; it went to the heart of scientific hypothesis testing. Even those who tested and rejected MFiT did not understand this: “Testing the predications of the MF/T model does not constitute a test of the hypothesis” [Marazita et al., 1986133. The presentation and discussion of the data [Carter, 1977; Fraser, 19761, from the outset [Carter, 19691, took on the form of a hypothetical confirmatory argument [Hempel, 19661:
If H is true, then so are predictions 11,I,, . . . , I, (As the evidence shows) Zl,I,, . . . , Z, are all true H is true Even if the facts established by statistical analysis were contingent with 11,12,. . . ,I,, all being true (which was not the case), this form of argument succumbs to the fallacy of affirming the consequent (like proving the null hypothesis) and is deductively invalid, for its conclusion may be false even if its premises are true [Hempel, 19661. The only argument relevant to MFiT that is deductively valid is the modus tollens:
If H is true, then so are predictions ZI,I,, . . . , I , (As the evidence shows) 11,12,. . . , I , are not true H is not true In this form, if the premises are true (which was the case), then its conclusion is unfailingly true as well. All this notwithstanding, MF/T was an important step in the right direction. More than a little impatient with the situation that
CL * P Solutions MF/Thad become an unjustified ideology among clinical geneticists, two graduate students and their collaborating mentors proposed what they called a “biologic alternative” [Melnick and Shields, 1976; Melnick et al., 19771. They invoked “allelic restriction” as an explanation of the complex etiology of CL+P, a molecular genetic process that may be thought of as autosomal lyonization. The importance of allelic restriction as a potential biologic mechanism of cellular differentiation and morphogenesis had recently been carefully elaborated by Holliday and Pugh [19751 and alluded to by Sager and Kitchen [19751. Application of the allelic restriction model to CL 2 P data sets was found to be particularly useful in explaining the greatly reduced penetrance observed in familial cases [Melnick et al., 19771. Briefly, like classic lyonization in females, the probability of a heterozygous parent passing on the mutant allele to an offspring is 0.5 and the probability of a heterozygous offspring manifesting the trait (CL & P) is dependent upon the probability of allelic restriction occurring for either allele (usually 0.5) and the number of cells in the relevant tissue’s precursor pool at the time of restriction-this probability being distributed as a binomial. Using the simplest possible example, i.e., assuming that allelic restriction takes place at a time when there is a single precursor cell, then the frequency of affected individuals from an (affectedor unaffected) heterozygous parent would be 0.25. In this respect, a dominant mutation would then resemble a recessive one except that one would find an unexpected number of families showing vertical transmission through 2 and even 3 generations. The segregation frequency could be lowered further by several factors, including (1)cell selection during embryogenesis, (2) cell mixing during pattern formation prior t o organogenesis, and (3) the normal-to-mutant cell ratio necessary to produce the abnormal trait. This model was thought by Melnick et al. to have a great advantage over MF/T in explaining the population and family data; while retaining the factors of environmental effects and chance, it was monogenic and this meant one had a good opportunity to find the offending gene. Similar developmental and biomathematical implications can be found in a more recent variation on this same theme, parental gene imprinting during gametogenesis [Swain et al., 19871. Nevertheless, this allelic restriction model ultimately proved unsatisfying (as does imprinting) because in practice it was untestable in humans and somewhat circular. To prove allelic restriction, one had to locate the “CL 5 P gene” and that was the point of proposing the model in the first place. If one knew which gene it was, one would not be putting oneself through these intellectual contortions. One would put the clinical house in order, leave the molecular details to others, and move on to other intractable issues. In the end, those interested in the problem of CL + P etiology were forced to retreat to more complex segregation analyses to try and distinguish between several less detailed genetic models. Such studies of substantial size have been accomplished in both Caucasian and Asian populations. Marazita et al., [19841 studied 2,532 fami-
lies ascertained through CL k P probands born in Denmark between 1941 and 1971. Using classical [Morton, 19621 and complex [Morton and MacLean, 19741 segregation analysis, they were able to conclude: (1) the data provide no support for MF/T; (2) the data are consistent with a major gene (recessive or codominant) in a portion of the kindreds; (3) the data provide evidence for genetic heterogeneity and a significant proportion of sporadic cases (50-60%). Using a slightly different method of complex segregation analysis [Lalouel and Morton, 19811and essentially the same Danish data set, Chung et al.  confirmed the results of Marazita et al. [19841. They noted that while there was a major gene effect (probably recessive) in their Danish sample, one could not predict the etiology of a given case with certainty, for fully two-thirds of cases were sporadic. Finally, rigorous analysis of English data first published by Carter et al. U9821was conducted by Marazita et al. [1986b]. The results were consistent with those described for the Danish population, including a significant proportion of sporadic cases in the cleft lip sample (- 60%).
Melnick et al. 119861presented the results of a genetic analysis of 163 CL 5 P surgical proband families ascertained in Shanghai, China. There was again no support for MF/T, and classical segregation analysis for Mendelian inheritance showed a maximum likelihood estimate of the segregation ratio of 0.19 k 0.07, a value not significantly less than 0.25 (recessive inheritance) but significantly less than 0.50 (dominant inheritance). Unlike the European populations, the proportion of sporadic cases was not significantly different from 0. Expanding this preliminary study to 1,300 surgical proband families showed that the best-fitting, most parsimonious model for C L i P in Shanghai was again an autosomal recessive major locus with no evidence of a significant proportion of sporadic cases greater than 0 [Marazita et al., 19891. These results differ considerably from the complex segregation analysis of 627 Japanese families reported by Chung et al. [19861, which found no evidence of a major gene effect. Per contra, a recent genetic analysis of 331 CL ? P surgical proband families ascertained in Madras, India was consistent with the findings in the Shanghai population [Nemana et al., 19921.
From the weight of the evidence, it is reasonable to conclude: (1)there is a major gene component to CL 2 P etiology (most probably recessive); (2) there is genetic heterogeneity within and between populations; (3) there is, a t least in Caucasians, a very large number of truly sporadic cases (1/z to 2/31. On this last point, it should be noted that a reascertainment of Fogh-Andersen’s original proband families by David Bixler documented that only about 3% of sporadic cases had recurrence after 1or 2 generations hence [unpublished data]. Equally as bothersome as the high proportion of sporadic cases is the surprisingly high frequency of surgical probands with one or two affected parents (e.g., 11% in the Danish study reported by Chung et al. [19861). One is left to postulate a high gene frequency for the putative major gene. While large numbers of sporadic cases and affected parents in purportedly recessive families do not
mitigate against a major gene effect for some or all cases, it does suggest that the trait C L k P may not be Mendelian in character, even if the underlying gene transmission for the truly cognizant trait (see below) is.
POSSIBLE ALTERNATIVE SOLUTIONS While it is almost certain that clefts induced solely by the relatively few known human teratogens are to be found in any population (e.g., alcohol, phenytoin, retinoic acid),it is also quite likely that they account for only a very small number of the total nonsyndromicCL P. It is far more probable that some process of incomplete penetrance plays a major role in determining the character of the family data. This would be particularly so if manifestation of CL-CPdepends not only on a major gene effect but also in utero exposure to as yet undetermined harmful environmental agents during the critical period of development, as suggested by Melnick et al. [19801and demonstrated in an animal model of CL t P [Melnick et al., 19811. Rethinking the outcomes of the studies enumerated above, one is drawn to Penrose’s [19531formulations of 38 years ago. One may reasonably propose that all cases in any given population, worldwide, have the same genetic cause, but penetrance is quite low because the environmental exposure frequency is low; even with a strong gene-environment interaction, the recurrence risk would be low. Assuming recessive inheritance of a “susceptibility gene” and using the relationships derived by Penrose [19531 and the frequency of CL tP in the general population and sibs of probands in Denmark [Melnick et al., 19801, one would arrive a t a gene frequency ( q ) of 0.08 and a trait manifestation (-penetrance) of 21%; manifestation would depend on exposure to appropriate, as yet unknown, environmental agents and approximately 1 in every 6-7 persons would be a “susceptibility gene” carrier (homozygousor heterozygous). Further, the parent-child correlation for CL ? P would be about 0.02 and the sib-sib correlation would be about 0.06. These calculations serve to (1)explain the rather high gene frequency estimates in the segregation analyses [e.g., Marazita et al., 1984; Chung et al., 19861; (2) the unexpected number of probands with an affected parent; (3) the relatively low number of families with multiple affected persons. It should be noted here that Penrose’s formulations regarding penetrance or manifestation in the presence of genetic susceptibility to environmental factors have recently been confirmed by Khoury et al. . Seemingly, all that remains are linkage and other studies to identify the gene and then other studies to identify the environmental factors. It is all so tidy, maybe too tidy, for linkage studies to date have proven unsuccessful. Perhaps the problem is that CL 2 P at birth is the wrong phenotype for study, too far removed from the key error in morphogenesis. There are many processes in the genetically programmed construction of a lip. For example, neural crest cells must migrate from the closing neural tube to the presumptive face, multiply in sufficient numbers to fill the developing facial processes, and orient themselves to be useful in subsequent differ-
entiation. Each of these processes requires several important molecules which are, in turn, regulated in time of appearance and amount by other molecules. An allelic variant could alter the shape and function of any one of these proteins [Bowie et al., 19901.At the very least, the initial conditions for a given process, say cell patterning, would be altered, leading to “embryologic chaos” and, subsequently, in our case, to cleft lip. Using CL t P as a phenotypic marker would likely prove useless, for the outcomes from any set of initial conditions is probabilistically determined, not necessarily predictable or certain. Hence, the high occurrence rate of CL tP and the low recurrence rate. One very interesting approach to this embryologic complexityis the stochastic single-gene model described by Kurnit et al. [19871. They performed computer simulations of endocardial cushion outgrowth and fusion using randomly walking endocardial cells that were allowed to migrate, divide, and adhere with probabilities set in advance. By altering the probability of adhesion, they were also able to simulate allelic variants at the “adhesion locus,” demonstrating significant changes in the risk for malformation. Further, the population structure derived from the stochastic single-gene model was consistent with actual population data for common malformations like congenital heart defects and CL+P. What is disturbing about this solution is that, while it more accurately incorporates what is known of embryologic development, it remains an oversimplification of the process. To wit, though the model considers only a single locus, reality requires consideration of genetic heterogeneity. With so many embryologic processes and so many molecules involved in any particular organogenesis, the participating locus in any given affected family is likely to differ from another randomly selected affected family. Consequently, establishing linkage to DNA markers in populations is likely to prove confusing and of limited clinical usefulness. Another fascinating, but troubling, aspect of the simulations by Kurnit et al. [19871was the effect of stochastic variability alone. Using identical parameters and probabilities, multiple simulations produced significant phenotypic variation, from normal endocardial cushions to severe atrial and ventricular septa1 defects. Thus, genetic variation was not required for phenotypic variation, merely chance. Should this be an important phenomenon in embryogenesis, it has interesting implications for CL k P. Let us say there are 10 processes associated with 10 different proteins in the making of a lip; each macromolecule is normally distributed and has a 0.025 probability of being more than 2 standard deviations (SD) below the mean concentration. The chance that 0 or 1of these molecules will have a concentration below 2 SD is about 98%;the chance that 0,1, or 2 will be below 2 SD is about 99.8%. Thus, it is unlikely that more than 2 of these molecular concentrations will be below 2 SD; that malformation will result from one or two is also unlikely given embryonicbuffering. However, if, we imagine that a threshold exists in which the embryo cannot compensate for 3 molecules below 2 SD, then the chance of this occurring and there being abnormality is about 0.2% or
CL * P Solutions 1in 500 embryos. The important thing to remember in these cases is that there are no gene mutations and no abnormal environments, just bad luck. Search as we might, we will neuer find the cause. Such cases will rarely, if ever, be associated with familial recurrence and may, for example, account for nearly all of the 1/2 to 2/3 sporadic cases seen in the Danish and English populations [Chung et al., 1986; Marazita et al., 1984,19861 as well as those in other populations. A concern for etiologic solutions among these cases would be misplaced and misleading. Perhaps the most interesting alternative model to consider, from the perspectives of both embryology and genetics, is “emergenic” inheritance or emergenesis [Li, 19871. An emergenic trait is one that is determined by a particular combination of alleles at a number of gene loci, a so-called gene constellation. When any one allele is absent, it destroys the constellation and the emergenic trait disappears. Meiosis will ensure that any constellation will not be preserved as such. Thus, an emergenic trait will usually not be familial, even though the phenotype itself is genetically determined; an emergenic trait mostly appears as isolated or sporadic cases, with little sib-sib or parent-child correlation. Emergenesis is particularly interesting as a model because it allows us to consider together the multigenic correlates of a given embryologic process, say cell patterning. Cell patterning during morphogenesis is a complex process that requires a minimally sufficient number of cells and cell-cell communication; this communication is mediated by large cell surface glycoproteins termed CAMS (cell adhesion molecules), a primary one being NCAM [Edelman, 19881. NCAM is specified by a single gene; changes in synthesis or expression on cell surfaces modulate the rate of hornophilic binding; for example, a 2-fold increase in surface concentration leads to a 30-fold increase in binding rate [Edelman, 19881. The relative expression of NCAM on mesenchymal cell surfaces changes with progressive morphodifferentiation of craniofacial processes and other structures [Chuong, 19901. There are two ways NCAM are thought to affect cellcell adhesion in order to facilitate multicellular collectives with properties different from groups of isolated cells: (1)spatiotemporal changes in the number of homophilic bonds between NCAMs on apposing cell membranes; (2) posttranslational reduction of NCAM polysialic acid (PSA) content, the more removed the greater the binding [Rutishauser et al., 19881. Development of the medial nasal process is critical for lip development. The mesenchymal cells of this process are largely derived from neural crest cells which, after arrival from the neural tube, must undergo cell division and subsequent patterning as multicellular collectives. Should there be inadequate cell division or cell-cell adhesion it is not unreasonable to expect lip malformation. If cell division, NCAM expression, and PSA removal are all specified by single genes, then the emergenic trait is medial nasal process mesenchymal cell patterning, not cleft lip. Further, studies of linkage of DNA markers to the phenotypic marker C L k P are again likely to be confusing. While all this is, of course,
hypothetical, it does suggest to us again that humans may be a poor choice for most studies of clefting. Nevertheless, let us return to emergenic inheritance and its relation to CL k P population data. As already noted, emergenic inheritance is quite different from both single-locus Mendelian inheritance and patterns that include the additive effects of many loci. Based on the foregoing discussion and Li’s [19871derivations one might propose the following: 1. Assume the emergenic trait is abnormal medial nasal process mesenchymal cell patterning, and this inevitably results in cleft lip several developmental steps later. 2. Assume 3 loci associated with three key components of the phenotype: cell division, NCAM synthesis, PSA removal. 3. Each locus has two alleles (Aa; Bb; Cc); aabbcc is called affected (decreased cell division + decreased NCAM synthesis + decreased PSA removal = abnormal cell patterning, the emergenic trait); all other genotypes are normal. 4. Instead of 1CL +- P locus with a = 0.06 (approximated from the studies cited above), consider each of 3 loci for the emergenic trait with q = 0.02. Based on (1)--(4),the segregation probability for affected X normal matings (A x N) would be: 1S = (2 - 4 ) (2 4 ) 444 (2 - 4 ) - 444
and for normal x normal matings (N x N):
(1 - 444l2 2 [(2 - 4 ) (2 - 4 ) (2 - 4 ) - qqq)1
These low segregation probabilities are consistent with the preponderance of families having only one affected child and there being few families in which there is both an affected child and an affected parent. While one may still see an occasional Mendelian pattern in certain rare families (e.g., Aabbcc x Aabbcc, N x N), the overall pattern in families is that the trait appears and disappears. If emergenesis is truly the case, human linkage studies are likely to prove unproductive, if not maddening. CL P, in essence, is both a complex trait and not the relevant phenotype. A more germane phenotype, say medial nasal process mesenchymal cell patterning, is also a complex trait and not unlike a quantitative trait. The only extant method for mapping emergenic trait loci is not applicable to humans because it depends on carefully constructed breeding studies [Lander and Botstein, 19891. Identification of emergenic trait loci using genetically defined mice will, nevertheless, permit an informed search for human homologs.
CONCLUSION Four alternative etiologic models have been proposed and discussed (1)environmental susceptibility single-
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gene inheritance, (2) stochastic single-gene inheritance, (3) pure chance, and (4) emergenic inheritance. Of the four, the first would seem the most parsimonious, and, thus, the most reasonable to pursue. However, the fact that linkage studies with DNA markers have been unsuccessful is not surprising since the first model is also the most unrealistic embryologically,and thus linkage is probably being sought to the wrong phenotype (CL +- P). Per contra, while the other models do account for the realities of embryogenesis, they are marginally, if at all, amenable to human studies. One is left to contemplate the possibility that we have been placing the cart before the horse. Perhaps we have far more work to do in the mouse, searching for more embryologically and genetically appropriate analogies and homologies before we return to human studies. However, even the mouse studies will prove to be frustrating. If Conway’s game “Life”teaches us anything, it is that while a given phenotypic configuration has only a single future it can have several different pasts [Eigen and Winkler, 19811.To be sure, this search for etiologic solutions will provide dissertation topics for many years to come.