Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.

Elevated sweat chloride levels, failure to thrive (FTT), and lung disease are characteristic features of cystic fibrosis (CF, OMIM #219700). Here we describe variants in CA12 encoding carbonic anhydrase XII in two pedigrees exhibiting CF-like phenotypes. Exome sequencing of a white American adult diagnosed with CF due to elevated sweat chloride, recurrent hyponatremia, infantile FTT and lung disease identified deleterious variants in each CA12 gene: c.908-1 G>A in a splice acceptor and a novel frameshift insertion c.859_860insACCT. In an unrelated consanguineous Omani family, two children with elevated sweat chloride, infantile FTT, and recurrent hyponatremia were homozygous for a novel missense variant (p.His121Gln). Deleterious CFTR variants were absent in both pedigrees. CA XII protein was localized apically in human bronchiolar epithelia and basolaterally in the reabsorptive duct of human sweat glands. Respiratory epithelial cell RNA from the adult proband revealed only aberrant CA12 transcripts and in vitro analysis showed greatly reduced CA XII protein. Studies of ion transport across respiratory epithelial cells in vivo and in culture revealed intact CFTR-mediated chloride transport in the adult proband. CA XII protein bearing either p.His121Gln or a previously identified p.Glu143Lys missense variant localized to the basolateral membranes of polarized Madin-Darby canine kidney (MDCK) cells, but enzyme activity was severely diminished when assayed at physiologic concentrations of extracellular chloride. Our findings indicate that loss of CA XII function should be considered in individuals without CFTR mutations who exhibit CF-like features in the sweat gland and lung.

Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions.

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585-1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools.

Increased male reproductive success in Ts65Dn "Down syndrome" mice.

The Ts65Dn mouse is trisomic for orthologs of about half the genes on Hsa21. A number of phenotypes in these trisomic mice parallel those in humans with trisomy 21 (Down syndrome), including cognitive deficits due to hippocampal malfunction that are sufficiently similar to human that "therapies" developed in Ts65Dn mice are making their way to human clinical trials. However, the impact of the model is limited by availability. Ts65Dn cannot be completely inbred and males are generally considered to be sterile. Females have few, small litters and they exhibit poor care of offspring, frequently abandoning entire litters. Here we report identification and selective breeding of rare fertile males from two working colonies of Ts65Dn mice. Trisomic offspring can be propagated by natural matings or by in vitro fertilization (IVF) to produce large cohorts of closely related siblings. The use of a robust euploid strain as recipients of fertilized embryos in IVF or as the female in natural matings greatly improves husbandry. Extra zygotes cultured to the blastocyst stage were used to create trisomic and euploid embryonic stem (ES) cells from littermates. We developed parameters for cryopreserving sperm from Ts65Dn males and used it to produce trisomic offspring by IVF. Use of cryopreserved sperm provides additional flexibility in the choice of oocyte donors from different genetic backgrounds, facilitating rapid production of complex crosses. This approach greatly increases the power of this important trisomic model to interrogate modifying effects of trisomic or disomic genes that contribute to trisomic phenotypes.

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants.

BACKGROUND: Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. METHODS: A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o-). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. RESULTS: Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. CONCLUSION: CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context.

Deep resequencing of CFTR in 762 F508del homozygotes reveals clusters of non-coding variants associated with cystic fibrosis disease traits.

Extensive phenotypic variability is commonly observed in individuals with Mendelian disorders, even among those with identical genotypes in the disease-causing gene. To determine whether variants within and surrounding CFTR contribute to phenotypic variability in cystic fibrosis (CF), we performed deep sequencing of CFTR in 762 patients homozygous for the common CF-causing variant, F508del. In phase 1, ~200 kb encompassing CFTR and extending 10 kb 5' and 5 kb 3' of the gene was sequenced in 486 F508del homozygotes selected from the extremes of sweat chloride concentration. In phase 2, a 510 kb region, which included the entire topologically associated domain of CFTR, was sequenced in 276 F508del homozygotes drawn from extremes of lung function. An additional 163 individuals who carried F508del and a different CF-causing variant were sequenced to inform haplotype construction. Region-based burden testing of both common and rare variants revealed seven regions of significance (α=0.01), five of which overlapped known regulatory elements or chromatin interactions. Notably, the -80 kb locus known to interact with the CFTR promoter was associated with variation in both CF traits. Haplotype analysis revealed a single rare recombination event (1.9% frequency) in intron 15 of CFTR bearing the F508del variant. Otherwise, the majority of F508del chromosomes were markedly similar, consistent with a single origin of the F508del allele. Together, these high-resolution variant analyses of the CFTR locus suggest a role for non-coding regulatory motifs in trait variation among individuals carrying the common CF allele.

Genome-wide association meta-analysis identifies five modifier loci of lung disease severity in cystic fibrosis.

The identification of small molecules that target specific CFTR variants has ushered in a new era of treatment for cystic fibrosis (CF), yet optimal, individualized treatment of CF will require identification and targeting of disease modifiers. Here we use genome-wide association analysis to identify genetic modifiers of CF lung disease, the primary cause of mortality. Meta-analysis of 6,365 CF patients identifies five loci that display significant association with variation in lung disease. Regions on chr3q29 (MUC4/MUC20; P=3.3 × 10(-11)), chr5p15.3 (SLC9A3; P=6.8 × 10(-12)), chr6p21.3 (HLA Class II; P=1.2 × 10(-8)) and chrXq22-q23 (AGTR2/SLC6A14; P=1.8 × 10(-9)) contain genes of high biological relevance to CF pathophysiology. The fifth locus, on chr11p12-p13 (EHF/APIP; P=1.9 × 10(-10)), was previously shown to be associated with lung disease. These results provide new insights into potential targets for modulating lung disease severity in CF.