Question

A 3-year-old Caucasian boy is brought to the clinic for a chronic productive cough not responding to antibiotics given recently. He has no fever or sick contacts. His medical history is significant for abdominal distention, failure to pass stool, and emesis as an infant. He continues to have bulky,foul-smelling stools. No diarrhea is present. He has several relatives with chronic lung and “stomach” problems, and some have even died at a young age. The examination reveals an ill appearing, slender male in moderate distress. The lung physical reveals poor air movement in the base of lungs bilateral and coarse rhonchi throughout both lung fields. A chloride sweat test was performed and was positive, indicating cystic fibrosis (CF). What is the mechanism of the disease? How might gel electrophoresis assist in making the diagnosis?

Answer 1

Mutations in CFTR (cystic fibrosis transmembrane conductance regulator) are the primary cause of cystic fibrosis. In CF, there is defective CFTR channels formed in the apical membrane of the mucus secreting epithelial cells. These epithelial cells excrete sodium, chloride ions along with water, to dilute the mucus secreted by goblet cells. CFTR channel allows export of chloride ions and to a certain extent bicarbonate ions out of the epithelial cells via the transcellular pathway (through the epithelial cells). Epithelial cell absorb sodium ions through the apical membrane by ENaC channels. Basolateral transport of sodium from cells to blood occurs via sodium potassium pump. The net transport of chloride also causes the sodium ions to be excreted out via the paracellular pathways along with water into the lumen of the epithelial cells. Water moves into lumen via aquaporin channels and paracellular pathway (through junction between two epithelial cells). Mucus therefore gets diluted and the mucus viscosity is maintained. However, in CFTR there is defective transport of chloride which affects water and chloride transport. This leads to congestion of lungs, pancreatic ducts and hepatic and biliary systems along with other effects. Thus, the person has digestive issues causing foul smelling stools, defective oxygenation and inflammation of lungs, fertility issues etc. Sweating by skin is also affected.

Gel electrophoresis can be used to detect cystic fibrosis along with PCR or RFLP analysis. CF is an autosomal recessive condition caused by inheritance of two copies of the mutant allele. There are different mutations that create the CF mutant allele. Differences in molecular Sizes of the mutant and wild type PCR products after amplification can be used to detect the cystic fibrosis. The wild type and CF samples will have only one type of band, specific to their sizes. Carriers (heterozygous) will have both wild type and mutant PCR products.

In case of Delta F508 mutation of CF, which is very common in CF, there is a shorter exon 10 generated in CF patients. The exon 10 of wild type allele on PCR amplifies to a larger fragment PCR product with specific primers that are used. However, when the mutant exon 10 is amplified, it generates a shorter PCR product. Thus, difference in sizes of PCR product can detect the CF sample. Normal homozygous dominant sample show only high molecular weight band, the carrier show both high and low molecular weight bands while CF samples show low molecular weight bands on gel electrophoresis. In this instance, a single PCR can detect both wild type and mutant bands.

Similarly, two different PCR can be carried out with two different sets of primers, one primer set each for Wild type and mutant allele in case of Delta F508 mutation. Thus, if both wild type and mutant PCR product is detected in the sample after PCR and gel electrophoresis, the sample is from a carrier. If only wild type band is seen and no mutant band, it is from homozygous dominant normal person. A CF sample will show only PCR product of mutant PCR. There will be no band detected in the wild type PCR.

Restriction fragment length polymorphism can be used which combines restriction enzyme digestion of PCR product followed by gel electrophoresis to detect CF. One of the mutations is the G551D mutation of CFTR, which eliminates the ability of CFTR to bind ATP, which is essential for its function. This mutation creates an MBo1 recognition site in codon 551. This site is not present in normal allele. Thus, when the PCR product of exon 11 (that has codon 551) of mutant allele is cleaved by Mbo1, there will be two fragments created of 243 bp and 182 bp. The normal allele has a size of 425 bp. When these digests are run on gel via electrophoresis, the CF patient sample will show the two bands of 243 bp and 182 bp. Unaffected homozygous samples show one band of 425bp. Unaffected heterozygote samples show three bands of 425 bp, 243 bp and 182 bp. Thus, CF can be detected. RFLP has been used to detect H63D and C282Y mutation as well.

Another way to detect the G551D mutation as well as R553X mutation in CF again by using PCR amplification of 425 bp exon 11. However, in this case there is digestion done with Hinc II restriction enzyme. There is no such site in mutant allele. Thus, wild type samples give two bands of 239 and 186 bp. The mutant allele will only give 425 bp. Hence, these mutations can be detected by gel electrophoresis.

Allele specific oligonucleotides can be used to detect G542X mutation via radioactive gel electrophoresis. These oligonucleotides will be radiolabeled and are specific to the mutant allele or to the wild type allele. If after electrophoresis, positive signal are observed with only wild type allele, the patient is only homozygous dominant. If the sample shows positive sample with mutant oligonucleotide probes, it is homozygous recessive and hence, the patient suffers from CF. If there is positive signal for both mutant and wild type allele, the sample is from a heterozygous carrier.