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Keywords:

  • gene expression;
  • left/right asymmetries;
  • micro-environment;
  • cleft lip;
  • quantitative genetics;
  • mitochondria

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

Discordance in monozygotic twins has traditionally been explained in terms of environmental influences. A recent investigation has found a difference in epigenetic markers in older but not in younger twins. However, phenotypic differences that depend on an individual's postnatal life style do not address the problem of discordance in congenital malformations, or the reason why malformations are frequently unilateral, often with a preference for one or the other side. One such condition, cleft lip with or without cleft palate, which is preferentially expressed on the left, is a multifactorial condition, that is caused by a failure of the critical timing necessary for different groups of cells to meet and develop into a normal face. This process is dependent on cell proliferation and migration, which are energy-dependent, while the additional requirement for apoptosis to allow cell fusion suggests the involvement of mitochondria. Recent progress in two separate areas of research could lead to a better understanding of the problem of facial clefts: (1) the recognition of an interaction between gene products and mitochondria in the aetiology of neurodegenerative diseases and (2) the discovery of an increasing number of genes, including transcription factors, growth factors and members of the TGF-β signalling family, that are differentially expressed on the left and right side, thus pointing to a difference in their micro-environment. These findings emphasize the importance of investigating the activity of candidate genes for complex developmental processes separately on the left and right sides. Data presented in this review suggest that differential growth rates may lead to an inversion of laterality. A method is described to test for a possible mitochondrial difference between left and right sides, using a mouse model with cleft lip.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

From the beginning of its coinage, the term “phenotype” included an environmental component. W. Johannsen, (1911), to whom we owe both the terms ‘genotype’ and ‘phenotype’ defined “genotype” as all the genes in a gamete or zygote, while all organisms that are distinguishable, either by direct inspection or more sophisticated methods, are characterized as ‘phenotypes’. The variability of organisms was thought to be determined by their genotypes interacting with the totality of all incident factors, including both external and internal variables. Almost a century later, attempts to understand the interplay between genotype and environment has become a subject of renewed attention.

A recent News feature in Nature (Qiu, 2006) has drawn attention to a new study on the as yet unsolved problem of discordance in monozygotic twins (Fraga et al. 2005). Whereas discordant phenotypes in monozygotic twins have traditionally been explained as environmental effects, Fraga et al. addressed the question whether epigenetic differences might be involved. Epigenetic differences in gene expression are not due to mutations in the DNA sequences but are marked by DNA methylation and histone modification which are retained through mitotic cell division (Jaenisch & Bird, 2003). An investigation of DNA methylation and histone acetylation in 80 monozygotic twin pairs, aged between three and 74 years revealed that whereas the youngest twins seemed epgenetically indistinguishable, the older twins showed remarkable differences in epigenetic markers, indicating that differences in gene expression accumulate with age (Fraga et al. 2005). On the other hand, a comprehensive account of genome-wide profiling for three human chromosomes by Eckhardt et al. (2006) failed to find a significant effect attributable to age.

Whitelaw & Martin (2001) postulated that discordance in monozygotic twins might be epigenetically mediated by transposons with the power to suppress transcription. The resetting of the process is thought to occur during early embryogenesis. Experimental evidence of prenatal effects on the epiphenotype has been provided by Waterland et al. (2006), who found that methyl donor supplementation of mice before and during pregnancy increased DNA methylation at AxinFused and reduced the incidence of tail kinks in the offspring.

In any event, age-related epigenetic effects would not address the problem of monozygotic twins who are discordant for congenital malformations, which, in contrast to diseases developing in later life, are unaffected by the life-style choices of their bearers. However, the prenatal environment can exert a decisive effect on the outcome of twin pregnancies, which is determined to a large extent by the type of placentation. Most monozygotic twins have monochorionic placentae, in which blood vessel fusion can occur and strongly influence intra-uterine development. One serious consequence is the formation of an acardiac fetus, whose development depends on being transfused by its normal co-twin (Benirschke, 1995). In less severe circumstances, one of the twins may develop a congenital heart defect, a condition that is two or three times more likely to occur in a monozygotic than a dizygotic twin or singleton (Burn, 1991).

In addition to problems of placentation, the time at which embryonic splitting occurs could affect the development of the two products, since gradients in the distribution of mitochondria in the egg have the potential to give rise to blastomeres containing different numbers of mitochondria (Dumollard, 2007). This implies that ‘identical’ twins could originate from cells with different numbers of mitochondria.

Another example of a mismatch between genotype and phenotype is provided by the asymmetric distribution between left and right sides. Here the causative agents must be confined to differences in the micro-environments between the two sides. As will be shown in the next section, this phenomenon is a frequent occurrence in medical genetics. The common malformation, cleft lip with or without cleft palate, will serve as an example.

Cleft Lip with or without Cleft Palate

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

The congenital malformation known as ‘cleft lip with or without cleft palate’ (CL(P) illustrates both discordance in monozygotic twins and of phenotypic divergence between the two sides. This condition would seem to provide excellent material for investigating the causes for differential gene expression by the same genotype in conditions that are independent of an individual's life style, so that data do not have to rely on the accuracy of self-reporting by patients.

Clefts in the lip, irrespective of whether they are associated with cleft palate, CL(P), are causally distinct from isolated cleft palate, as is evident from distinct patterns of recurrence in families (Cohen, 2002a). The formation of normal lips and palate depends on the migration of cells of neural crest origin into the facial region, followed by correctly timed growth to form lateral swellings, and finally a complex series of fusions of swellings in the mid line. These processes are completed by the end of the first trimester.

John Edwards (1960) wrote that ‘In man such developmental abnormalities as cleft palate, hare lip, anencephaly, spina bifida and most heart defects, which are probably related to the unpunctual fusion of the margins of various grooves and holes may be conditioned by continuous variation, innate and acquired, in developmental punctuality’ . More specifically, Cohen (2002a) pointed out that normal development of lips and palate is critically dependent on exact timing and positioning of facial prominences. Genetic or environmental factors, or both in combination, that inhibit the flow of neural crest cells or decrease their proliferation may lead to inadequate masses that inhibit contact between them. Failure of apoptosis in the epithelium that covers the mesenchyme can also prevent fusion from taking place, and failure of the disintegration of the nasal fin in human embryos aged 47–48 days likewise predisposes to cleft lip (Sperber, 2002). Since mitochondria, in addition to providing energy to the developing embryo, are also responsible for triggering apoptosis (Dumollard et al. 2007), the developmental evidence points to a likely involvement of mitochondria in the etiology of CL(P).

Concordance rates in twins provide strong evidence that genetic factors play an important role in CL(P): monozygotic twins.have a concordance rate of 40%, compared with only 4.2% in dizygotic twins. The most likely mode of inheritance is the multifactorial threshold model. Males are more often affected than females (Cohen, 2002b).

Based on allelic association or linkage studies, Cohen (2002b) lists the following candidate genes for orofacial clefting: TGFα. TGFβ, M/SX1, RARα, DLX2 and BCL3. By including data based on mouse mutants, Chong et al. (2002) arrive at a somewhat longer list of genes, comprising transcription regulators, growth factors and signalling molecules, that have been implicated in the complex process of lip/palate development and its abnormalities. Nevertheless, the substantial discordance rate in monozygotic twins demonstrates that facial clefts cannot be totally ascribed to the genotype, and this conclusion is further emphasized by the fact, which has been established on large series of patients, that most clefts are unilateral and more often on the left than the right side. (Fogh-Andersen, 1942; Fraser & Calnan, 1961; Chenevix-Trench et al. 1992). Cleft lip in some strains of mice has a similarly asymmetric distribution, with most clefts being left-sided (Trasler & Trasler, 1984); and a recent study by Juriloff et al. (2006) also suggests a possible left preponderance of unilateral clefts.

The occurrence of unilateral malformations could be explained on the basis of the idea put forward by Kurnit et al. (1987) that chance plays a major role in the development of many common malformations. Although the Kurnitt model is based on a single gene that predisposes to an abnormality, it could be extended to apply also to multiple genes interacting with one another. However, an additional problem arises in cases when the asymmetry is biased. A study of unilateral birth defects in 102 anomalies found that 15 were significantly more frequent on one or the other side, suggesting that laterality may be a strong modifier of the effects of other exposures (Paulozzy & Lary, 1999). The following were predominately left-sided: preauricular tag, cleft lip, cleft lip with cleft palate, fused lip and cleft gum, congenital hip dysplasia, unstable hip, absent forearm or hand, anomaly of knee, and skin tags; whereas inguinal hernia, incarcerated inguinal hernia, microtia, preauricular sinus, talipes calcaneovalgus, and lambdoidal craniosynostosis were predominantly right-sided. These abnormalities are typically affected by quantitative trait loci (QTLs). Chase et al. (2004) have identified two QTLs that have differential effects on laxity in right versus left hip joints in dogs.

It should be noted that congenital malformations like CL(P) develop at a time when the major anatomical asymmetries are already in place. As shown by Maclean & Dunwoody (2004), the left-sided position and internal architecture of the heart are complete in human embryos at 8 weeks, as is the left-sided position of the stomach and spleen, and the right-sided position of the liver. This is several weeks earlier than the time when the complex fusions of the palatal shelves and nasal septum take place (Cohen, 2002a; Sperber, 2002). Consequently, while malformations of the heart or spleen may reflect abnormalities of the early development of left/right patterning, the development of CL/P typically takes place in an environment of normal left/right asymmetry, and it affects organs that are not noticeably lateralized.

A brief consideration of some of the rapidly-growing evidence of the origin of left-right asymmetries will be given in the following section.

The Development of Left/Right Asymmetries in Vertebrates

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

The overall bilateral symmetry of the body plan of vertebrates is consistently broken by a directionally asymmetrical way of positioning the major viscera as well as parts of the brain (Opitz & Utkus, 2001; Cooke, 2003; Maclean & Dunwoody, 2004). Nearly all organs of the thorax, and particularly the abdomen, are left/right (L/R) asymmetric in anatomy, placement and in some cases, physiology, This asymmetry is highly conserved in all vertebrates – and indeed other chordates – even though there are some differences between different groups, for instance in the vascular system (Mercola, 2003).

Of the three axes presiding over vertebrate development, the dorsal/ventral, the anterior/posterior and the Left/Right, the L/R axis was the last to enter the realm of genetic investigations. However, in the last few years several dozens of genes have been described that are differentially expressed on the two sides of vertebrates. The genes include transcription factors, growth factors and TGF-β signalling molecules (Levin, 2005).

An example is provided by heparin-binding EGF-like growth factor (HB-EGF), a potent mitogen and chemoattractant, which is prominently expressed in skeletal muscle and kidney of adult mice. Golding et al. (2004) have examined the expression of Hb-EGF mRNA in mouse embryos and found it to be transiently expressed in mature somites between E9.25 and E11. The expression pattern was asymmetrical, with stronger expression in left than right members of a myotome pair.

Congenital hip dysplasia in humans occurs more often on the left than the right side, and the same asymmetry has been observed in dogs with hip joint laxity. Chase et al. (2004) identified two quantitative trait loci, both located on canine chromosome 1, that were differentially expressed on the two sides. The authors observed that separating left and right data sets provided more information that would have been gained by increased statistical power produced by combining them.

It has been postulated that a cascade of asymmetric gene expression during different developmental stages results in differential cell proliferation, adhesion and migration. Hecksher-Sørensen et al. (2006) have obtained evidence that a specialized cell type, the splanchnic mesoderm, is required to direct the spleen and pancreas in a leftward direction. Even though, as pointed out by Levin (2005), the molecular mechanism initiating the asymmetry is still unknown, there is increasing evidence that studying developmental processes on the left and the right separately may lead to new insights into normal and abnormal development.

Differential Growth Rates and Reversal in Laterality?

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

Some ‘laterality genes’ show a remarkable degree of concordance with regard to the side of increased expression throughout different classes of vertebrates. Thus Lefty and Nodal, both members of the TGF-β family of signalling molecules, are expressed on the left in mice, chicks and frogs, and the same applies to Pitx-2, a transcription factor. On the other hand, the transcription factor NKX3.2 is expressed on the right in mice and on the left in chicks (reviewed by Levin, 2005).

On the anatomical level, there is evidence that an inversion of biased asymmetry can sometimes be connected with organ size. In humans, the left kidney tends to be larger than the right (Gray, 1858), whereas in rats and mice it is the other way round. Figure 1 illustrates the difference in weight between right and left kidneys of wild mice, taken from seven populations that differed in marker genes as well as in body and kidney weights (Berry, 1978; Mittwoch, 1979). The data, comprising a total of 634 mice, were arranged in four groups, those with low body and kidney weights (from 3 populations) and those with high body and kidney weights (from 4 populations), as well as by age, younger and older, based on tooth category.

image

Figure 1. Plot of percentage differences between right and left kidney weights (g) versus mean kidney weights in 7 populations of wild mice, arranged in 4 groups according to low and high kidney weights (3 and 4 populations respectively), and younger and older ages (based on tooth categories). Percentage differences were calculated as (Right weight – Left weight)/Mean x 100%.

Download figure to PowerPoint

As shown in Figure 1, the weight of right kidneys exceeded that of left kidneys in all groups, but the difference diminished from 8% at a mean kidney weight of 64 mg to 2.5% at a kidney weight of 200 mg. The difference between populations with small and large kidneys is highly significant, whereas that between younger and older mice does not reach significance in a two-tailed t-test (Table 1). It seems that kidney growth associated with different genotypes affects asymmetry by decreasing the size difference in favour of the right kidney, whereas evidence for a smaller effect in the same direction through growth during development, while suggestive, remains inconclusive.

Table 1.  Analysis of variance for difference between right & left kidney against weight (Data as in Fig. 1).
SourceDFSSMean SquareF Valuep Value
Total27248.34 
High vs low 1106.48106.4821.40<0.0001
Younger vs older 1 17.46 17.46 3.50 0.0730
Error25124.40  4.98 

Data on kidney weights in laboratory rats by Mackey and Mackey (1937) also show asymmetry in favour of the right kidney. The data comprised 930 rats aged between 35 and 760 days, and the authors state that the difference between right and left does not differ significantly with age. Nevertheless the observed difference, calculated as (Right- Left/Left)% was 3.1% for mean kidney weight of 0.72 g and 2.7% for mean kidney weight of 0.989 g. These findings go in the same direction as those observed in mice, but the differences are smaller.

In guinea pigs, the right kidney seems to have lost its superiority. Data by Shirley (1976) showed that for kidney weights ranging between 1 and 2.5 g, the left kidney was larger than the right, although the difference was significant only for females. These findings in mice, rats and guinea pigs suggest that with increasing kidney size, the originally faster-growing right kidney is left behind in favour of the originally slower left kidney, leading to an inversion of the original pattern of bilateral asymmetry.

It is tempting to invoke this phenomenon to explain the hitherto puzzling observation regarding the inverted laterality of ovaries and testes in true hermaphroditism between humans and mice: whereas in humans, testes are preferentially situated on the right and ovaries on the left, in mice the opposite situation prevails (reviewed by Mittwoch, 2000; Levin, 2005). Ovaries develop from the slower-growing gonad in both species (Mittwoch & Mahadevaiah, 1980; Mittwoch & Buehr, 1973), and in human fetal gonads, there was a significant asymmetry in favour of the right for weight, protein and DNA contents, whereas no consistent difference was detected in mouse embryos. Therefore, the bias in human hermaphroditism for ovaries to be positioned on the left could be simply related to the observed slower growth of fetal gonads on that side. While there are no corresponding data for fetal mice, it could be relevant that in embryos of the grey short-tailed opossum, Monodelphis domestica, in which gonad volume averaged less than 0.01mm3, there was a highly significant bias in favour of left gonads. In new-born opossums this difference was reduced and no longer significant (Baker, 1993).

These data lead to the possible conclusion that the decision as to which path of differentiation is to be followed reflects growth rates at an earlier stage of development, when they were influenced by the dominance relationship between the two sides that were operative at the time.

Mackey & Mackey (1937) have suggested a connection between the larger weight of the right kidney in rats and its better blood supply due to the asymmetric branching of the right renal artery. If the asymmetry of kidney weight is also affected by genotypically controlled growth of the kidney, the symmetry relation of the kidney could change in different species even though the bilateral disposition of blood vessels remains the same. This accords with data cited by Levin (2005) that the administration of cadmium leads to opposite-sided limb defects in rats and mice, while relative vessel size are similar in both species,

Differential Growth Rates and Energy Metabolism in Health and Disease

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

Mounting evidence points to a connection between rates of cell proliferation and mitochondrial activity controlling the cells' energy metabolism. The SRY gene, which plays an important, if short-lived, role in choosing the pathway of testis differentiation, causes an increase in cell proliferation in the coelomic epithelium (Schmahl et al. 2000). The search for a nuclear target remained fruitless for many years, leading to the suggestion that this transcription factor might interact with mitochondria (Mittwoch, 2004). In view of the wide-spread role played by temperature-dependent sex determination in reptiles (Pieau et al. (1999)– a mechanism no longer available for mammals – a mitochondrial target by the mammalian sex-determining gene, SRY, is in line with the proposal by Wallace (2005) that mitochondria provide a link between genes and environment. The finding by Matoba et al. (2005) of glycogen deposits in pre-Sertoli cells in mice immediately after the onset of Sry activity lends support to the hypothesis that the requirements for extra cell proliferation and migration involved a higher energy expenditure for testis than for ovary differentiation.

Evidence is also accumulating that an increasing number of pathological conditions may be caused by abnormal interactions between nuclear genes and mitochondria, often leading to insufficient energy being available to the cells to perform their normal function. Attempts to discover the genes which, when over-expressed on a trisomic chromosome 21, are responsible for the phenotypic manifestations in Down syndrome have proved difficult. At a workshop set up to tackle this question (Gardiner, 2005), Jorge Buscilio reported that mitochondrial morphology and function are altered in Down syndrome patients, suggesting that chronic energy deficits may contribute to their pathology.

There is much recent evidence of mitochondrial involvement in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease (Lin and Beal) 2006, in which specific interactions of disease-related proteins have recently been discovered. These changes could originate either by mutations in mitochondrial DNA or by mutated nuclear genes interacting with mitochondria. Data by Roses et al. (2007) suggested that Alzheimer patients carrying the APOE4 allele, which predisposes to the disease, required a higher dose of rosiglitazone, a drug shown to increase mitochondrial biogenesis, than did patients not carrying the allele (Roses et al. 2007). The results of new research on a Drosophila ortholog of the human NF1 gene, that is responsible for the neurogenetic disease, neurofibromatosis-1, indicate that neurofibromin acts via mitochondrial regulation, rather than the Ras/Raf pathway, as previously thought (Tong et al. 2007).

Inevitably, the major thrust of mitochondrial research has been focused on pathological conditions, but it seems an almost foregone conclusion that much normal variation within and between individuals is likewise caused by subtle differences in mitochondrial action.

Quantitative trait loci

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

The completion of the Human Genome Project has provided reliable reference points for the location of genes causing so-called ‘monogenic’ diseases, which segregate according to Mendelian rules. This type of segregation implies that any environmental effect on gene expression is minimal, and that almost any environment that is compatible with life will permit the appropriate genotype/phenotype correlation. By contrast, the genetic causation of common diseases is said to be ‘multifactorial’, consisting of many genes as well as environmental effects, and such conditions do not follow clear-cut rules of segregation. It is, nevertheless, a long-established basic premiss of quantitative genetics that the inheritance of quantitative differences is based on genes subject to the same laws of transmission and having the same general properties as genes responsible for qualitative traits, and the first part of the premiss has been confirmed by the mapping of quantitative loci, QTLs, to specific regions of chromosomes (Falconer & Mackay, 1996).

If complex traits are caused by multiple genes of small effect interacting with environmental factors, it follows that common disorders might represent the quantitative extreme of the same genetic and environmental factors that operate in the normal population (Craig and Plomin, 2006). This implies that when many of the same genes and environmental effects find themselves in associations giving rise to less extreme quantitative effects, they contribute to the normal quantitative variation that is seen in populations. For example, QTLs that are involved in mental retardation also contribute to the normal variation in IQ (Craig & Plomin, 2006).

It has already been mentioned that mental and physical impairment in Down Syndrome seems to be associated with mitochondrial abnormalities that might lead to impaired energy metabolism (Busciglio et al. 2002) and that much new evidence points to mitochondria playing a central role in ageing-related neurodegenerative diseases (Lin & Beal (2006). The latter include Huntington's disease, which is caused by an expansion of a CAG trinucleotide repeat in the huntingtin, HTT, gene, which in turn gives rise to an expanded polyglutamine stretch in the corresponding protein. However, the number of CAG repeats, though lower than in patients, is also variable in unaffected individuals, and therefore might give rise to subtle differences in mitochondrial function, leading to quantitative variation in the normal population

While the exact mechanism by which the mutation in HTT causes mitochondrial dysfunction remains to be worked out, a direct interaction between HTT and mitochondria appears to be a distinct possibility (Lin & Beal, 2006). This suggests the likelihood that there may be many other proteins that target mitochondria. Such an activity could indeed be a characteristic of many QTLs that modulate quantitative traits by controlling the energy available to cells; and an interaction between nuclear genes and mitochondria would explain the dependence of their phenotypic expression on environmental factors, which is such a common feature in genetically complex diseases.

Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

A suitable model for human CL(P) would be a mouse strain with a high incidence of cleft lip preferentially situated on the left side, such as CL/Fr. In this strain, Trasler & Trasler (1984) reported that in embryos aged 13–16 days of gestation, 15% had clefts, and that among unilateral ones, there were three times as many on the left as on the right. In view of the high degree of asymmetry shown by unilateral clefts, it may be worth-while to examine laterality also in bilateral clefts.

In order to compare numbers of mitochondria on the left and the right, mitochondrial volume fractions can be estimated using confocal microscopy, as described by Casley et al. (2002). Small samples of tissues are removed from both facial sides of embryos with facial clefts and the cells grown on poly-L-ornithine-coated cover slips. By comparing the number of pixels containing mitochondrial material with the number of empty pixels, the fraction of the cell occupied by mitochondria can be calculated.

Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

Much effort is presently expended in the search for a better understanding of the genotype/phenotype correlation of common diseases and anomalies that originate from genetic as well as environmental causes. The Human Epigenome Project seeks to establish rules underlying gene silencing by documenting the interplay of DNA methylation, histone modifications and the expression of non-coding RNAs in the regulation of gene expression (Brena et al. 2006). Two other sets of data are relevant to the question of gene expression versus silencing. One of them is the fact that many external birth defects are unilateral, and of these, some are more common on the left and others on the right side (Paulozzi & Lary, 1999). These anomalies are clearly related to the normal left/right asymmetry that is characteristic of the human (and vertebrate) anatomy (Cohen, 2001; Opitz & Utkus, 2001; Maclean & Dunwoody, 2004; Cooke, 2004; Levin, 2005). Genes that are expressed differentially on the two sides are known, but their metabolic effects remain to be discovered. The other set of data refers to growing evidence between pathology, QTLs and abnormal interaction between nuclear genes and mitochondria.

CL(P) is a common birth defect, in which the embryological evidence points to an energy deficit and mitochondrial involvement as possible causative agents. Inhibition of migration or proliferation of cells of the neural crest, as well as failure of apoptosis in the epithelium that covers the mesenchyme (Cohen, 2002a), and of nasal fin disintegration at the correct time (Sperber, 2002), all predispose to cleft lip. The condition also illustrates bilateral asymmetry, since most clefts are unilateral, and of these most are on the left (Cohen, 2002b; Sperber, 2002). The higher failure rate of groups of cells from diverse origin to meet and fuse at the correct time indicates and important difference between the two sides that is waiting to be discovered.

Since a large part of quantitative variation must be dependent on rates of cell proliferation, it is to be expected that the amount of energy available to the cells plays an important role, and it would hardly be surprising if genes that are responsible for quantitative variation were active in providing the necessary energy by their association with mitochondria. This view point is further supported by recent findings of the interaction of nuclear genes with mitochondria in the aetiology of neuronal diseases (Lin & Beal, 2006; Tong et al. 2007), suggesting that the relationship between genes and phenotypes in quantitative genetics should be viewed in terms of more dynamic processes than was customary in classical Mendelian genetics.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References

I thank Sue Povey for her interest and helpful co-operation, Nancy Mendell, at Stony Brook University, for the statistical analysis of the kidney weight data, and Michelle Bush for transferring the results into Excel. I am also grateful to Fred Biddle, at the University of Calgary, and Diana Juriloff, at the University of British Columbia, for information on cleft lip in mice, and to Michael Duchen for discussions about mitochondria. Finally, I acknowledge the constructive input made by three anonymous referees to an earlier version of this manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Cleft Lip with or without Cleft Palate
  5. The Development of Left/Right Asymmetries in Vertebrates
  6. Differential Growth Rates and Reversal in Laterality?
  7. Differential Growth Rates and Energy Metabolism in Health and Disease
  8. Quantitative trait loci
  9. Testing the hypothesis that bilaterally asymmetrical malformations have a mitochondrial basis
  10. Conclusion
  11. Acknowledgements
  12. References