The goal of this analysis was to characterize the 72 PT specimens in NSQAP’s CFTR Mutation Detection repository, which are used to assist NBS laboratories in ensuring accuracy when they utilize a molecular second tier test for CF. This comprehensive analysis resulted in the identification of 34 classes of predicted haplotypes that span the CFTR gene promoter through intron 18. These haplotypes are consistent with the LD defined by the International HapMap project. Half (N=77) of the 152 chromosomes examined shared a common haplotype subgroup which was associated with three of the most prevalent CF-causing mutations, F508del, G542X, and N1303K.
To understand CFTR
mutations, a previous study assessing the origin of 27,177 CF chromosomes from 29 European countries and three North African countries described the five most common CF-causing mutations: F508del (66.8%), G542X (2.6%), N1303K (1.6%), G551D (1.5%) and W1282X (1.0%) [22
]. Similarly, Bobadilla et al. described the five most common CF-causing mutations in the U.S., which included F508del (68.6%), G542X (2.4%), G551D (2.1%), W1282X (1.4%) and N1303K (1.3%) [23
]. Hence, F508del, G542X, and N1303K are the more common mutations in Caucasians from Europe and the United States. A study on Spanish CF patients suggested an ancient common origin of these three mutations by showing that they all carried a common haplotype subgroup as defined by the IVS8CA, IVS17bCA and IVS17bTA microsatellites [24
]. The haplotypes identified in the present study were consistent with the Spanish patient findings, showing that F508del, G542X, and N1303K again share a common haplotype subgroup [24
]. Of our 62 F508del containing chromosomes (excluding the one probable recombinant), 29 predicted haplotypes are identical across 27 polymorphisms from the promoter to intron 21 to the G542X containing haplotypes (N=6) and the N1303K containing haplotypes (N=6). The remaining 33 F508del containing haplotypes are identical to this common haplotype except that they contain either 17 or 21 di-nucleotide repeats of the intron 9 IVS8CA microsatellite versus 23. Taken together, these data from a heterogeneous group of CF patients living in the United States support the existence of a common ancient European ancestral haplotype that independently gave rise to these three CF mutations.
A recent study examined the association of common CFTR
gene variants and their potential influence on body composition and survival in a non-CF population in rural Ghana [25
]. As expected, the LD across the CFTR
gene in this Ghana population was not as strong as that seen in a population of European descent; however, they reported specific intron 11 haplotype (comprised of 4 SNPs) associations with young versus old study participants as well as with lower or higher weight in children less than 5 years old. While further studies are necessary to understand if these associations are by chance or if they have any direct influence on health, it provides further motivation to understand the molecular framework of the CFTR
Molecular analysis of CF, as with many autosomal recessive disorders, is complicated by the imperfect correlation between mutations in the CFTR
gene and CF phenotype [26
]. In order to better understand the consequences of the greater than 1850 CFTR
mutations, Rowntree and Harris have classified CFTR
mutations into five groups, where each classification describes the mechanism by which a group of mutations disrupt CFTR function [27
]. To further explain the inability to correlate phenotype and genotype in a supposedly simple “single-gene” disorder, Dipple and McCabe have proposed that there are two functional thresholds relating mutant protein function to phenotype. When protein function is below the first threshold, a severe phenotype will always be observed, whereas if protein function is above the second threshold, the phenotype will be consistently mild. Between these two thresholds, mutations will not necessarily correlate with phenotype and thus should be viewed as “complex traits” [28
]. Complex traits are influenced by functional activity thresholds, modifier gene and system dynamics, thereby blurring the lines between genotype phenotype correlations [29
]. For CF, there are numerous studies that also describe modulatory effects of different genes on the severity of phenotype in CF patients (e.g. mannose-binding lectin 2 and transforming growth factor beta 1) [27
To further define the phenotypic heterogeneity in patients who have the same CF causing mutations, several molecular studies have analyzed the context of disease causing mutations. Researchers found that the phenotypic severity of a CF-causing mutation could be impacted by the genomic context of the CFTR
gene as seen when R117H is in cis
with the 5T variant and when S1251N is in cis
with F508C [33
]. Thus, the haplotypes defined in this study may assist newborn screeners and clinicians in predicting the cis
phase for mutations and poly T variants with variable expression without requiring parent studies. For example, when a newborn specimen is positive for R117H and either F508del, G542X or N1303K, and also carries both an 5T and a 9T variant, a clinician could use haplotype information to proceed with a strong probability that the 9T variant is in cis
with F508del, G542X or N1310K and not R117H (). This study also shows that R117H may reside on the opposite chromosome from nearly all of the listed mutations in because it has a different polymorphism background. Additionally, more extensive haplotype studies may allow researchers to determine what if any phenotypic effects results when a particular variant such as 5T are in cis
with an identified CF-causing mutation.
This study has defined 144 CFTR haplotypes associated with many CF-causing mutations, laying the groundwork for future research on the molecular structure of the CFTR gene and its potential influence on phenotypic heterogeneity in CF patients. In addition, these extensively characterized specimens will enhance the quality of the PT challenges offered by NSQAP to participating NBS laboratories.