Finding Needles in Haystacks — IRF6 Gene Variants in Isolated Cleft Lip or Cleft Palate
http://www.100md.com
《新英格兰医药杂志》
Human geneticists have been on a long joyride. The power of contemporary genetics and genomics has allowed us to identify the specific causal gene in a dizzying variety of mendelian (single-gene) and chromosomal disorders that affect every single human organ system.1 This information has transformed our knowledge of the pathophysiology of genetic diseases in humans, including cleft lip with or without involvement of the palate.
Three specific genes are known to harbor mutations that lead to cleft lip or palate and additional anomalies: MSX1 (also known as HOX7), which encodes muscle-segment–specific homeobox 12; FGFR1, which encodes fibroblast growth factor receptor 13; and IRF6, which encodes interferon regulatory factor 6.4 Cleft lip or palate occurs with dental anomalies as a consequence of MSX1 mutation, with infertility and anosmia in the case of the FGFR1 mutation and with lower-lip pits in the case of the IRF6 mutation. Disease-causing sequence changes in each of these three genes are individually rare in the population and cumulatively account for no more than 5 percent of all patients with clefts. Nevertheless, the discovery of each of these genetic causes is a major success, because each has led to specific molecular diagnoses and improved the genetic counseling of patients with cleft lip or palate and their families (including the possibility of antenatal diagnosis, when appropriate).
More important, these discoveries have led to identification of the biochemical pathways that are involved in embryonic development of the palate.5 This knowledge is not purely of academic interest, since many cases of cleft lip or palate arise from environmental triggers and exposures as well. It is well documented that a variety of teratogens — such as maternal smoking, phenytoin, benzodiazepines, and dioxin — can increase the risk of cleft lip or palate.5 Moreover, there is strong suspicion that maternal exposure to infections during pregnancy, exposure to statin drugs during the first trimester, and other factors can also increase the risk.5 Although the precise connections between genes and environmental exposures are still elusive, it is likely that the environmental triggers, like the genetic mutations, affect the same developmental pathways.6 If so, the identification of even rare mutations can significantly affect our knowledge of therapeutic targets.
Not all is sunlight and sweetness in the field of human genetics, however. We have known for a very long time that, for most disorders, mendelian patterns are the exception rather than the rule. Indeed, the most common nonsyndromic forms of each human disorder have complex inheritance patterns.6 Dissecting the genetic components of these complex diseases has remained distressingly difficult; we are still in the dark regarding their genetic causation. Therefore, the international study by Zucchero et al. in this issue of the Journal, 7 which demonstrates that a common polymorphism in the IRF6 gene is a major susceptibility factor for isolated cleft lip or palate in multiple human populations, is an important advance in genetic medicine.
In over 70 percent of cases, cleft lip or palate occurs as an isolated birth defect. These cases must involve genes, because of the clustering of cleft lip or palate in families and because siblings of affected children have a 3 to 5 percent risk of being affected. The environment must also be a factor, since identical twins are not always concordant for the phenotype.8 It has been known for a long time that cases of isolated cleft lip or palate probably involve the action of many genes, each harboring a susceptibility allele that makes only a small, yet perceptible, contribution to risk. But another hypothesis also explains the familial observations, though it is not a favorite explanation among geneticists. Environmental exposures common to family members can explain the clustering of cleft lip or palate in some families, since many of the exposures, such as those mentioned above, might be expected to occur in multiple pregnancies.
The identification of a gene that underlies a common form of isolated cleft lip or palate is crucial to the genetic hypothesis of the causation of complex disorders. But the process has been technically challenging, since the individual components of the genetic and nongenetic effects are small. Geneticists have shown that the great success of linkage analysis of mendelian traits, in which DNA-based markers are used to identify a small segment of the human genome shared by all affected members of a pedigree, is greatly outweighed in efficiency by association studies of complex traits, in which DNA-based markers are used to identify a smaller segment of the human genome shared by a larger proportion of affected persons in the population than of unaffected controls.9
Zucchero et al. hypothesized that, because mutant IRF6 causes Van der Woude's syndrome (in which cleft lip or palate is accompanied only by lip pits), minor variations in IRF6 may affect the risk of isolated cleft lip or palate as well. They carried out family-based association studies using 36 single-nucleotide polymorphisms in IRF6. (Single-nucleotide polymorphisms are specific nucleotides that vary from individual to individual; for example, a single-nucleotide polymorphism at position 274 of IRF6 results in a codon for a valine [V] residue in some chromosomes and an isoleucine [I] residue in others.) They identified the polymorphisms by comparing the IRF6 sequences of affected persons with those of unaffected controls and also IRF6 sequences from public databases. They show that the polymorphism that results in either a V residue or an I residue at position 274 in the protein-binding domain of IRF6 is the most likely susceptibility factor. The V residue is the high-risk allele.
Across mammalian evolution, the V allele at position 274 has been highly conserved; thus, the new I allele, specific to humans, appears to be a protective (or less risk prone) allele. This observation implies that the protective allele, or resistance, was selected for in human evolution and may have arisen along with orofacial changes in our remote ancestors. But it also suggests that the majority of pregnancies are at risk for cleft lip or palate. The genetic effect of the V allele is small, as expected, associated with an odds ratio of cleft lip or palate of about 2. However, because the V residue is so common in human populations, the attributable risk is 12 percent for all common forms of cleft lip or palate. Clearly, then, genetic changes in IRF6 are the most common known genetic cause of this defect.
Are we close to a better understanding of the common forms of cleft lip or palate? We have made a substantial step forward but we are still far from the finish line. Zucchero et al.7 correctly point out that their findings have immediate implications for the genetic counseling of families with children who have cleft lip or palate. Their data suggest that, in families in which the parents are capable of producing a child who is homozygous for the V allele, the risk of having a child with cleft lip or palate would be as high as 9 percent — several times the average in the general population. These families would require careful genetic counseling with regard to diagnosis, patient care, and treatment options. However, it is unlikely that families would react very differently to a risk of recurrence of 3 to 5 percent than to a risk of 9 percent. The most important implication of this study is that it provides a promising lead for identifying other genes linked to cleft lip or palate and elucidating the mechanisms of environmental exposure.
Another implication of the IRF6 polymorphism is that patients with isolated cleft lip or palate should no longer be considered a monolithic group but should be stratified according to their individual genotypes (V/V, V/I, and I/I) in future studies. It is imperative that, in new studies, we examine environmental exposures in women who give birth to a child with cleft lip or palate according to their IRF6 genotypes so as to increase the statistical power of the study. It is possible that environmental exposure will have a greater effect in some pregnancies (e.g., those in which the fetus has the V/V genotype) than in others, resulting in an increased chance of expression in fetuses with specific genotypes. If environmental factors do interact with the IRF6 pathway, it becomes possible to identify the mechanism of action of specific environmental triggers. Knowledge of the IRF6 genotype will help to identify additional genes related to cleft lip or palate by stratifying the patient population.
The findings of Zucchero et al., together with the International HapMap Project (a resource of millions of single-nucleotide polymorphisms across the human genome in multiple populations)10 and advances in genotyping technology, will lead scientists to the many other genes that, together with IRF6, cause one of the most common human birth defects.
Source Information
From the McKusick–Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore.
References
Johns Hopkins University. OMIM — Online Mendelian Inheritance in Man. (Accessed July 30, 2004, at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM.)
van den Boogaard M-JH, Dorland M, Beemer FA, et al. MSX1 mutation is associated with orofacial clefting and tooth agenesis in humans. Nat Genet 2000;24:342-343.
Dode C, Levilliers J, Dupont J-M, et al. Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. Nat Genet 2003;33:463-465.
Kondo S, Schutte BC, Richardson RJ, et al. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat Genet 2002;32:285-289.
Murray JC, Schutte BG. Cleft palate: players, pathways, and pursuits. J Clin Invest 2004;113:1676-1678.
Chakravarti A, Little PFR. Nature, nurture and human disease. Nature 2003;421:412-414.
Zucchero TM, Cooper ME, Maher BS, et al. Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N Engl J Med 2004;351:769-780.
Mossey PA, Little J. Epidemiology of oral clefts: an international perspective. In: Wyszynski DF, ed. Cleft lip and palate: from origin to treatment: New York: Oxford University Press, 2002:127-58.
Risch N, Merkangas K. The future of genetic studies of complex human diseases. Science 1996;273:1516-1517.
The International HapMap Consortium. The International HapMap Project. Nature 2003;426:789-796.(Aravinda Chakravarti, Ph.)
Three specific genes are known to harbor mutations that lead to cleft lip or palate and additional anomalies: MSX1 (also known as HOX7), which encodes muscle-segment–specific homeobox 12; FGFR1, which encodes fibroblast growth factor receptor 13; and IRF6, which encodes interferon regulatory factor 6.4 Cleft lip or palate occurs with dental anomalies as a consequence of MSX1 mutation, with infertility and anosmia in the case of the FGFR1 mutation and with lower-lip pits in the case of the IRF6 mutation. Disease-causing sequence changes in each of these three genes are individually rare in the population and cumulatively account for no more than 5 percent of all patients with clefts. Nevertheless, the discovery of each of these genetic causes is a major success, because each has led to specific molecular diagnoses and improved the genetic counseling of patients with cleft lip or palate and their families (including the possibility of antenatal diagnosis, when appropriate).
More important, these discoveries have led to identification of the biochemical pathways that are involved in embryonic development of the palate.5 This knowledge is not purely of academic interest, since many cases of cleft lip or palate arise from environmental triggers and exposures as well. It is well documented that a variety of teratogens — such as maternal smoking, phenytoin, benzodiazepines, and dioxin — can increase the risk of cleft lip or palate.5 Moreover, there is strong suspicion that maternal exposure to infections during pregnancy, exposure to statin drugs during the first trimester, and other factors can also increase the risk.5 Although the precise connections between genes and environmental exposures are still elusive, it is likely that the environmental triggers, like the genetic mutations, affect the same developmental pathways.6 If so, the identification of even rare mutations can significantly affect our knowledge of therapeutic targets.
Not all is sunlight and sweetness in the field of human genetics, however. We have known for a very long time that, for most disorders, mendelian patterns are the exception rather than the rule. Indeed, the most common nonsyndromic forms of each human disorder have complex inheritance patterns.6 Dissecting the genetic components of these complex diseases has remained distressingly difficult; we are still in the dark regarding their genetic causation. Therefore, the international study by Zucchero et al. in this issue of the Journal, 7 which demonstrates that a common polymorphism in the IRF6 gene is a major susceptibility factor for isolated cleft lip or palate in multiple human populations, is an important advance in genetic medicine.
In over 70 percent of cases, cleft lip or palate occurs as an isolated birth defect. These cases must involve genes, because of the clustering of cleft lip or palate in families and because siblings of affected children have a 3 to 5 percent risk of being affected. The environment must also be a factor, since identical twins are not always concordant for the phenotype.8 It has been known for a long time that cases of isolated cleft lip or palate probably involve the action of many genes, each harboring a susceptibility allele that makes only a small, yet perceptible, contribution to risk. But another hypothesis also explains the familial observations, though it is not a favorite explanation among geneticists. Environmental exposures common to family members can explain the clustering of cleft lip or palate in some families, since many of the exposures, such as those mentioned above, might be expected to occur in multiple pregnancies.
The identification of a gene that underlies a common form of isolated cleft lip or palate is crucial to the genetic hypothesis of the causation of complex disorders. But the process has been technically challenging, since the individual components of the genetic and nongenetic effects are small. Geneticists have shown that the great success of linkage analysis of mendelian traits, in which DNA-based markers are used to identify a small segment of the human genome shared by all affected members of a pedigree, is greatly outweighed in efficiency by association studies of complex traits, in which DNA-based markers are used to identify a smaller segment of the human genome shared by a larger proportion of affected persons in the population than of unaffected controls.9
Zucchero et al. hypothesized that, because mutant IRF6 causes Van der Woude's syndrome (in which cleft lip or palate is accompanied only by lip pits), minor variations in IRF6 may affect the risk of isolated cleft lip or palate as well. They carried out family-based association studies using 36 single-nucleotide polymorphisms in IRF6. (Single-nucleotide polymorphisms are specific nucleotides that vary from individual to individual; for example, a single-nucleotide polymorphism at position 274 of IRF6 results in a codon for a valine [V] residue in some chromosomes and an isoleucine [I] residue in others.) They identified the polymorphisms by comparing the IRF6 sequences of affected persons with those of unaffected controls and also IRF6 sequences from public databases. They show that the polymorphism that results in either a V residue or an I residue at position 274 in the protein-binding domain of IRF6 is the most likely susceptibility factor. The V residue is the high-risk allele.
Across mammalian evolution, the V allele at position 274 has been highly conserved; thus, the new I allele, specific to humans, appears to be a protective (or less risk prone) allele. This observation implies that the protective allele, or resistance, was selected for in human evolution and may have arisen along with orofacial changes in our remote ancestors. But it also suggests that the majority of pregnancies are at risk for cleft lip or palate. The genetic effect of the V allele is small, as expected, associated with an odds ratio of cleft lip or palate of about 2. However, because the V residue is so common in human populations, the attributable risk is 12 percent for all common forms of cleft lip or palate. Clearly, then, genetic changes in IRF6 are the most common known genetic cause of this defect.
Are we close to a better understanding of the common forms of cleft lip or palate? We have made a substantial step forward but we are still far from the finish line. Zucchero et al.7 correctly point out that their findings have immediate implications for the genetic counseling of families with children who have cleft lip or palate. Their data suggest that, in families in which the parents are capable of producing a child who is homozygous for the V allele, the risk of having a child with cleft lip or palate would be as high as 9 percent — several times the average in the general population. These families would require careful genetic counseling with regard to diagnosis, patient care, and treatment options. However, it is unlikely that families would react very differently to a risk of recurrence of 3 to 5 percent than to a risk of 9 percent. The most important implication of this study is that it provides a promising lead for identifying other genes linked to cleft lip or palate and elucidating the mechanisms of environmental exposure.
Another implication of the IRF6 polymorphism is that patients with isolated cleft lip or palate should no longer be considered a monolithic group but should be stratified according to their individual genotypes (V/V, V/I, and I/I) in future studies. It is imperative that, in new studies, we examine environmental exposures in women who give birth to a child with cleft lip or palate according to their IRF6 genotypes so as to increase the statistical power of the study. It is possible that environmental exposure will have a greater effect in some pregnancies (e.g., those in which the fetus has the V/V genotype) than in others, resulting in an increased chance of expression in fetuses with specific genotypes. If environmental factors do interact with the IRF6 pathway, it becomes possible to identify the mechanism of action of specific environmental triggers. Knowledge of the IRF6 genotype will help to identify additional genes related to cleft lip or palate by stratifying the patient population.
The findings of Zucchero et al., together with the International HapMap Project (a resource of millions of single-nucleotide polymorphisms across the human genome in multiple populations)10 and advances in genotyping technology, will lead scientists to the many other genes that, together with IRF6, cause one of the most common human birth defects.
Source Information
From the McKusick–Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore.
References
Johns Hopkins University. OMIM — Online Mendelian Inheritance in Man. (Accessed July 30, 2004, at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM.)
van den Boogaard M-JH, Dorland M, Beemer FA, et al. MSX1 mutation is associated with orofacial clefting and tooth agenesis in humans. Nat Genet 2000;24:342-343.
Dode C, Levilliers J, Dupont J-M, et al. Loss-of-function mutations in FGFR1 cause autosomal dominant Kallmann syndrome. Nat Genet 2003;33:463-465.
Kondo S, Schutte BC, Richardson RJ, et al. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat Genet 2002;32:285-289.
Murray JC, Schutte BG. Cleft palate: players, pathways, and pursuits. J Clin Invest 2004;113:1676-1678.
Chakravarti A, Little PFR. Nature, nurture and human disease. Nature 2003;421:412-414.
Zucchero TM, Cooper ME, Maher BS, et al. Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N Engl J Med 2004;351:769-780.
Mossey PA, Little J. Epidemiology of oral clefts: an international perspective. In: Wyszynski DF, ed. Cleft lip and palate: from origin to treatment: New York: Oxford University Press, 2002:127-58.
Risch N, Merkangas K. The future of genetic studies of complex human diseases. Science 1996;273:1516-1517.
The International HapMap Consortium. The International HapMap Project. Nature 2003;426:789-796.(Aravinda Chakravarti, Ph.)