Jeffrey S. Buguliskis Ph.D. Technical Editor Genetic Engineering & Biotechnology News

New Research Has Broadened the Number of Clinically Relevant CFTR Gene Mutations

Imagine yourself suspended a couple of hundred feet below the surface of the ocean. The sheer weight of the immense column of water pushing down on your chest makes each breath a harrowing task. Now picture that your only recourse to collect vital oxygen is to breathe laboriously throw a narrow straw that connects you to the atmosphere above. You slowly draw in air, cautiously trying not to collapse the straw from too forceful of suction—struggling just as much to exhale the expired air. Now repeat the entire cycle for the rest of your life. 

If you were able to envision how the immense difficulties of breathing in this manner would be for just a few minutes, let alone your entire life, then you may have a minute sense of what a person afflicted with cystic fibrosis (CF) endures. Gasping for air while thick, sticky mucus lines the pulmonary system, seemingly threatening to drown and suffocate patients with each inhale.

CF is the most common autosomal recessive life-limiting disease, with a high occurrence among peoples of northwestern European descent and an incidence rate of approximately 1 in every 2,500 individuals. CF arises due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a transmembrane ion channel protein that is a member of the ABC transporter-class—conducting chloride (Cl-) and thiocyanate (SCN-) ions across epithelial cell membranes.

While chronic respiratory issues represent the vast majority of morbidity and mortality associated with CF, the disease often encompasses multiple symptoms with pancreatic insufficiency manifesting in 85 percent of patients and fat malabsorption in 90 percent of infants by one year of age. Many consequences of CFTR abnormalities begin even before birth, leading to incomplete embryo formation and structural changes, causing infertility in virtually all males with CF.

Almost 2,000 mutations have been discovered for the CFTR gene resulting in varying degrees of disease severity. Mutations in CFTR are often classified into six, non-exclusive categories based on the cellular mechanisms that are affected. They can run the gamut of large frameshift and RNA splicing mutations, resulting in reduced protein expression and transport, to single base errors, which often lead to gating and conductance issues.

Don’t Sweat the Small Stuff

Early diagnosis of any disease is
critical to successful patient outcomes, but is of particular importance for sufferers of CF. For instance, in less than the five years the median life expectancy has increased ten years—to 37—due in no small part to recent advances in genetic sequencing and molecular diagnostics. This new generation of genetic screens not only allows clinicians to pinpoint afflicted individuals, but can also help identify at-risk patients, parents who carry mutated alleles, and whether certain therapeutics will be effective based on a particular genetic background.

While CF is known to be a genetic disease spawning from abnormalities in the CFTR gene, genetic screening is not currently the gold standard for positive diagnosis. The chloride sweat test, or quantitative pilocarpine iontophoresis as it is scientifically referred, measures the secreted chloride concentration on the skin after being induced by a chemical activator (pilocarpine). This diagnostic technique has been employed for decades with an overall accuracy rate around 98 percent. However, this method does little more than to provide the physician and patient with a positive or negative result. It provides no insight into the potential severity of symptoms or choice of appropriate therapy.

The primary objective of molecular diagnostic testing is to provide enhanced genetic characterization for individuals either with or suspected of having CF. These diagnostic screens are typically performed to provide improved understanding for a variety of reasons including prenatal diagnosis in a carrier couple, symptoms consistent with CF, family history of a relative with CF or with a CF-like condition, and newborn screening follow-up. The benefits of these diagnostic assays are numerous, but ultimately all lead to improved patient care. 

“CF newborn screening has important benefits as the disease can be caught much earlier before symptoms start, doctors can prevent and better manage clinical complications, e.g. respiratory therapy, prophylaxis against infection, improved growth, and extend survival,” notes Curt Scharfe, M.D., Ph.D., associate professor of genetics at Yale School of Medicine. “Our clinical lab has been performing CF newborn screening, CF carrier, and diagnostic testing.”

Dr. Scharfe adds that “newborn screening for CF is now implemented across the United States and in many other countries. It typically starts with an immunoreactive trypsinogen (IRT) testing from newborn dried blood spots. All IRT-screen positive specimens are then tested for a relatively small number of common CFTR mutations (there are 40 known CF mutations), followed by CFTR sequencing in all specimens identified with one common mutation. Thus, the current procedures could miss newborns with two rare CF mutations.”

In many clinical settings around the world, genetic tests for CF variants are being combined with sweat test diagnostics to provide a comprehensive disease overview that will provide physicians and clinical researchers information that can be vital to choosing appropriate drug therapies designed to combat the multitude of debilitating CF symptoms. Some recently developed CF drugs are extremely efficacious, but only to individuals with specific CFTR mutations. Knowing a patient’s genetic background will allow doctors to quickly apply the proper treatment course.

Molecular Advances Help Researchers Breathe Easier

For the past 20 years, a well-vetted panel containing 23 of the most common CFTR variants has been applied to diagnosing CF, as well as identifying carriers and affected newborns. In 2010, the Clinical and Functional Translation of CFTR (CFTR2) project was initiated, charged with the difficult task of increasing the number of annotated CFTR variants. As of 2016, the project has obtained data from 88,000 patients worldwide leading to 276 annotated CFTR variants.  

This project achieved many of its goals so rapidly due to the increased use of massively parallel sequencing techniques, which has opened up new avenues toward identifying genetic variations in the CFTR gene that lead to CF phenotypes. Next-generation sequencing can provide clinicians with essential variant data through multiplexing assays that can detect deletions and genomic duplications while concomitantly sequencing entire gene loci. 

However, since it has been previously established that there are approximately 2,000 possible variants for CFTR, this leaves more than 1,700 mutations—with varying degrees of disease symptoms—that remain uncategorized. Yet, in a recent study published in PLOS ONE, researchers at Children’s Hospital Los Angeles and the Genetic Disease Screening Program of the California Department of Public Health have begun to genetically classify the broad array of these unannotated mutations—helping to further understand the penetrance of CFTR mutations and the risk of CF for children with mutations of undetermined clinical significance. 

Additionally, the versatility of next-gen sequencing platforms allows investigators to tackle projects once only thought possible by large research consortiums. Moreover, the sequencing technology is amenable enough to allow scientists to quickly develop new methodology for addressing current assay insufficiencies.

“We recently developed a novel next-gen sequencing-based assay (CFseq) that’s more rapid, comprehensive, cost-effective and is done from dried blood spots—the common newborn screen specimen,” says Scharfe. “Assays like CFseq will help eliminate false negatives and help reduce false positive IRT screening tests.”

Biotech companies are also constantly looking to expand their CF screening panels through the addition of verified and clinically relevant DNA biomarkers. The rapid expansion of these panels over the past several years is a direct result of the exponential decrease in next-gen sequencing costs—allowing companies to continue to generate low-cost, reproducible, and highly accurate results. Furthermore, deep sequencing with extended coverage provides accuracy that can even supersede the sweat test.

With the current trend of rapid advancement for molecular diagnostics, it is not difficult to envision continued improvements for CF genetic testing. These advancements will most likely spread into other areas of CF genomics, such as improved drug discovery and design, as well as enhanced pharmacogenomics to identify appropriate target populations for novel therapeutic compounds.

This article was originally published in the July 2016 issue of Clinical OMICs. For more content like this and details on how to get a free subscription to this digital publication, go to

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