Orofacial clefts are common congenital defects with a complex etiology that involves both genetic and environmental factors. They can be classified as either syndromic or nonsyndromic (isolated) cases, based on the presence of other developmental defects. The latter can be further classified into cleft lip only (CLO), cleft lip with cleft palate (CL+P) or cleft palate only (CPO).1
CPO is considered etiologically distinct from cleft lip with or without cleft palate (CL/P) and is less common, occurring in approximately 1/1500-2000 births, compared to 1-2/1000 births for CL/P.2
They also differ in their sex ratios with CL/P occurring more frequently in males than females, but the reverse is observed for CPO.3
While the etiology of clefting is still poorly understood, genetic mapping approaches and evaluation of candidate genes has found important roles for IRF6,4
and some other genes,6
but disease-causing mutations are yet to be identified. The gene identification process is hindered by the complex nature of clefting, which includes genetic heterogeneity. In this study, we examined the role of skewed X chromosome inactivation (XCI) in females with isolated clefting.
XCI occurs early in female embryogenesis by the random inactivation of one X chromosome, to achieve equal X-linked gene dosage between females and males. This process is generally random, but a skewed XCI pattern in which the same X chromosome is inactivated in the majority of the cells in a tissue can occur due to selection during development, mutations in genes involved in the X-inactivation process, or stochastic processes.7, 8
In a large study, Amos-Landgraf et al
. examined the distribution of XCI patterns in blood samples from 1,005 phenotypically unaffected females, and found that only 8% of the samples had >80:20 skewing. Since highly skewed XCI is rare, a finding of significantly skewed XCI is unlikely to be a purely stochastic event.9
Female carriers of X-linked recessive mutations do not usually express the phenotype, but highly skewed XCI favoring gene expression from the chromosome with the mutant allele can result in manifestation the disease phenotype, and has been reported for various X-linked diseases. Similarly, XCI can modulate expression of X-linked dominant traits.
Differences in XCI have been shown to underlie phenotypic discordance for X-linked diseases in monozygotic (MZ) female twins such as Duchenne muscular dystrophy 10
and red-green color blindness.11
This results from the preferential inactivation of the normal chromosome in the affected twin, while the unaffected twin has predominant inactivation of the mutant chromosome or random XCI.12
Twin studies have previously confirmed a genetic component of clefting, with a higher concordance rate in monozygotic (25–50%) compared to dizygotic (3–6%) twins.13
Female MZ discordant twins provide a valuable resource to examine the role of XCI in clefting. In addition, non-random XCI could result in expression of X-linked diseases in carrier females due to predominant inactivation of the normal chromosome.14
It has also been suggested that XCI may contribute to disease variability between males and females,7
such as the observed altered gender ratios for clefting phenotypes3
. Evidence for linkage of clefting to the X chromosome,15
and the possible contribution of genes for X-linked clefting syndromes further support this proposition.
In this study, we have compared XCI pattern in a sample set of MZ female twins and sister pairs discordant for clefting. Our results show that differences in XCI are not likely etiologic in the discordance for clefting in the MZ female twins. However, significantly greater XCI skewing in unaffected females compared to their affected sisters suggests a role for XCI in orofacial clefting, particularly the CL+P phenotype.