Search

Current approaches to characterize the mutational profile of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are based on targeted mutation analysis or whole gene studies derived from short-read next generation sequencing (NGS). However, these methods lack phasing capability which, in certain scenarios, can provide clinically valuable information. In the present work, we performed near-full length CFTR using Single-Molecule Real-Time Sequencing to produce haplotype-resolved data from both homozygous and heterozygous individuals for mutation c.1521_1523delCTT (p.Phe508del, F508del). This approach utilizes target enrichment of the CFTR gene using biotinylated probes, facilitates multiplexing samples in the same sequencing run, and utilizes fully-automated bioinformatics pipelines for error correction and variant calling. We show a remarkable conservation of F508del haplotype, consistent with the single gene founder effect, as well as diverse mutational profiles in non-F508del alleles. By the same method, 105 single nucleotide polymorphisms (SNPs) exhibiting invariant linkage to F508del CFTR (which better define the founder haplotype) were identified. High level homology between F508del sequences derived from heterozygotes, and those obtained from homozygous individuals, demonstrate accuracy of this method to produce haplotype resolved sequencing. The studies provide a new diagnostic technology for detailed analysis of complex CFTR alleles linked to disease severity.

Current approaches to characterize the mutational profile of CFTR are based on targeted mutation analysis (TMA) or whole gene studies derived from short-read next generation sequencing (NGS). However, these methods lack phasing capability which, in certain scenarios, can provide clinically valuable information. In the present work, we performed near-full length CFTR using Single-Molecule Real-Time Sequencing to produce haplotype resolved data from F508del homozygous and F508del compound heterozygous individuals. This approach utilizes target enrichment of the CFTR gene using biotinylated probes, facilitates multiplexing samples in the same sequencing run, and utilizes fully-automated bioinformatics pipelines for error correction and variant calling. We show a remarkable conservation of F508del haplotype, consistent with the single gene founder effect, as well as diverse mutational profiles in non-F508del alleles. By the same method, 105 single nucleotide polymorphisms exhibiting invariant linkage to F508del CFTR (which better define the founder haplotype) were identified. High level homology between F508del sequences derived from heterozygotes, and those obtained from homozygous individuals, demonstrate accuracy of this method to produce haplotype resolved sequencing. The studies provide a new diagnostic technology for detailed analysis of complex CFTR alleles linked to disease severity.

During antigen-driven affinity maturation germinal center B lymphocytes undergo multiple rounds of somatic mutagenesis in the rearranged IgHV gene segment. A direct consequence of this process is the generation of multiple antibody mutants that are, in general, specific for antigen. Survival of these mutants is contingent on preserving or improving antigen-specificity in the complementarity-determining regions (CDRs) as well as retaining a functional framework region (FWR). Broadly, somatic mutations in germinal center B cells can be classified as templated or untemplated. In animals such as rabbits and chickens, somatic mutations are mostly templated. Conversely, in mice and humans it is widely accepted that mutations are untemplated. Here, we argue in favor of the alternative, that a predominant number of somatic mutations in IgHV gene segments in mice and humans can be traced to germline IgHV genes and occur through gene conversion. Using the NP hapten model, we demonstrate that mutations in germinal center B cells and plasma cells are traceable to other germline IgHV genes. Subsequently we also demonstrate using the novel V-LAIR1-DJ antibodies described by Tan et al (Nature 2016) that mutations in human antibody IgHV rearrangements, including those in non-immunoglobulin sequences (LAIR1), are traceable to the human IgHV germline repertoire. Together, this suggests that the plethora of diversity generated in antigen specific B cells is primarily directed by the germline repertoire of IgHV genes.