Saturday, March 27, 2021

Cystic Fibrosis Case File

Posted By: Medical Group - 3/27/2021 Post Author : Medical Group Post Date : Saturday, March 27, 2021 Post Time : 3/27/2021
Cystic Fibrosis Case File
Eugene C.Toy, MD, William E. Seifert, Jr., PHD, Henry W. Strobel, PHD, Konrad P. Harms, MD

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 exam 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?

Summary: A 3-year-old Caucasian male has a history of chronic pulmonary and gastrointestinal problems and has a positive chloride sweat test. A family history of similar symptoms is also present.

Likely diagnosis: Cystic fibrosis, which is an autosomal recessive disorder resulting in defective chloride ion channels of exocrine glands and epithelial tissues of the pancreas, sweat glands, and mucous glands in the respiratory, digestive, and reproductive tracts.

Gel electrophoresis: Separates deoxyribonucleic acid (DNA) chains of varying length and can allow identification of a specific gene sequences.

Cystic fibrosis is an inherited condition affecting approximately 1 in 2500 white individuals. Affected patients usually have abnormal mucus secretion and eccrine sweat glands leading to respiratory infections, gastrointestinal obstruction, pancreatic enzyme dysfunction leading to malabsorption of nutrients, and excessive electrolyte secretion. The protein cystic fibrosis transmembrane conductance regulator (CFTR) is defective, leading to abnormal chloride transport. Approximately 70 percent of mutations are accounted for by deletion of three specific base pairs at the F 508 position of the CFTR. Oligonucleotide probes can be used to assay for this mutation, but other tests are required for the less common mutations.

1. Understand the process of gel electrophoresis.
2. Know the difference between various types of blots.
3. Be familiar with DNA sequencing using the Sanger dideoxynucleotide method.
4. Understand the process and uses of cloning DNA.

Blotting: The transfer of proteins or nucleic acids from an electrophoresis gel to a membrane support (such as nitrocellulose or nylon). The membrane blot is then incubated with probes that bind the molecules of interest.
Complementary DNA (cDNA): A sequence of DNA copied from messenger ribonucleic acid (mRNA), which does not contain introns that were present in native DNA.
CFTR: Cystic fibrosis transmembrane conductance regulator; a ligandgated chloride channel that is regulated by phosphorylation. It is a member of the adenine nucleotide binding cassette (ABC) family of transport proteins.
Electrophoresis: The technique by which charged molecules in solution are separated in an electric field according to their different mobilities in the supporting medium.
Gel electrophoresis: Electrophoresis using a gel as the supporting medium. Common gels are polyacrylamide and agarose.
Northern blot: The technique by which molecules of ribonucleic acid (RNA) are separated by gel electrophoresis, transferred to a membrane support, and incubated with labeled oligonucleotide probes. Specificity is obtained by using oligonucleotide probes that have sequences complementary to the target RNA.
Southern blot: The technique by which molecules of DNA are separated by gel electrophoresis, transferred to a membrane support, and incubated with labeled oligonucleotide probes. Specificity is obtained by using oligonucleotide probes that have sequences complementary to the target DNA.
Western blot: The technique by which protein molecules are separated by gel electrophoresis, transferred to a membrane support, and incubated with labeled antibodies. Specificity is obtained using antibodies that will bind the protein molecule of interest.

Cystic fibrosis is the most common lethal autosomal recessive disease affecting the Caucasian population. It has a frequency of approximately 1 in 2500 and a carrier frequency of approximately 1 in 25. The protein affected is the cystic fibrosis transmembrane conductance regulator (CFTR), which is a chloride ion channel. There are over 1000 mutations that have been discovered in the CFTR gene and over 80 percent of these mutations lead to disease. The mutations lead to (1) defective or decreased protein production, (2) defective processing of the protein, (3) protein that is defective in the regulation of the chloride channel, or (4) defect in the transport of chloride ions. The most common mutation, a deletion of a phenylalanine residue at amino acid position 508 (ΔF508), results in misfolding of the protein; it consequently does not traffic to the membrane.

Defects in the CFTR decrease the ability of cells to transport Cl- in a number of tissues, particularly the pancreas, airway epithelia, and sweat glands. When Cl- transport is defective in the pancreas, it leads to decreased HCO3− secretion and decreased hydration that leads to thick secretions that block the pancreatic ducts and destruction of the organ. In the lungs, the decreased absorption of Cl- ions is thought to increase the absorption of the airway surface liquid thus increasing the viscosity of mucous, decreasing mucociliary clearance and increasing the incidence of airway infections. In sweat glands, defects in CFTR prevent the reabsorption of Cl- in the sweat gland duct, thus increasing the concentration of NaCl in sweat. The presence of salty sweat in children with CF gives rise to the Northern Europe adage: “Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die.”

Gel electrophoresis is a method routinely used in biochemistry and molecular biology to separate, identify, and purify peptides, proteins, and DNA fragments based on their size. Electrophoresis is a method by which charged molecules in a solution are separated in an electric field because of their different mobilities. The mobility of an ion in the electric field is dependent on the charge of the ionized molecule, the voltage gradient of the electric field, and the frictional resistance of the supporting medium. The supporting medium of choice for most protein and peptide analyses, as well as small DNA fragments, is polyacrylamide gel. For large DNA fragments, agarose gel gives the best results.

In general, DNA molecules have a high negative charge-to-mass ratio owing to their sugar-phosphate backbone. Therefore, when an electric field is applied, DNA will migrate through the agarose (or polyacrylamide) gel toward the positive pole in the electrophoresis apparatus. Because of the high charge-to-mass ratio, the relative electrophoretic mobility of DNA depends primarily on the size of the DNA molecule and porosity of the gel matrix, which can be varied by the concentration of agarose or polyacrylamide in the gel. Small DNA strands will move fast, whereas large fragments will lag behind smaller fragments as the DNA migrates through the gel. To estimate the size of DNA in the samples, molecular weight markers (a mixture of DNA fragments of known molecular size) are applied to one of the lanes in the gel. Gel electrophoresis separates DNA molecules based on their frictional coefficient, which is dependent on the length and conformation of the fragment. For most linear double-stranded DNA molecules, separation will be according to their molecular weight. However, circular DNA molecules (e.g., from bacteria or plasmids) can adopt a super coiled structure and therefore will pass through the gel with less resistance than the rod shape of linear DNA.

Gel electrophoresis can be applied as a method for detection of genetic diseases such as Cystic fibrosis (CF). Larger deletions in the cystic fibrosis transmembrane conductance regulator (CFTR) gene will cause it to move differently than the full-length wild type gene. In most cases, the DNA sample of the patient is amplified by a polymerase chain reaction (PCR) using specific primers. After the electrophoresis of the PCR samples the bands are visualized under ultraviolet (UV) light using ethidium bromide staining.

Bands of molecules separated by electrophoresis can be detected in the gel by various staining and destaining methods, such as the above mentioned ethidium bromide. However, more specific information can be obtained by application of blotting techniques. There are three different blotting techniques based on whether DNA, RNA, or protein is being analyzed. However, all blotting techniques have major steps in common. These include (1) the isolation of the biomolecules of interest (DNA, RNA, or protein); (2) separation of the mixture using gel electrophoresis; and (3) subsequent transfer of the sample molecules from the gel to a nitrocellulose or nylon membrane. The final identification step involves incubation of the membrane blot with probes that bind specifically to the molecule of interest. DNA is analyzed using Southern blotting, named after E. M. Southern, who first described this procedure in 1975. Based on this initial terminology, Northern and Western blotting were given as names referring to RNA and proteinblotting transfer, respectively. The major difference between the procedures involves the probes used for detection and identification of the molecules of interest. Southern and Northern blots take advantage of the ability of complementary nucleic acid strands to hybridize.

In a Southern blot, after the DNA fragments have been transferred from the electrophoretic gel to the membrane, they are allowed to interact with a singlestranded DNA oligonucleotide that contains the sequence of nucleic acids in the gene of interest. The oligonucleotide probe is usually radioactively labeled, which allows the DNA band to be identified following exposure to x-ray film. Northern blots are used to monitor the expression of a gene of interest by determining the amount of mRNA present. Then the mRNA is isolated, separated by electrophoresis, and, after transfer to a membrane, is allowed to interact with a radioactive DNA polynucleotide that has a sequence of the gene of interest. Visualization is by exposure to x-ray film. In the case of a Western blot of a protein mixture, after the proteins have been transferred to the membrane, the membrane is probed with an antibody that binds specifically to the target protein/antigen. The antibody may be radioactive or coupled to a fluorescent chromophore to allow easy detection.

The ability to sequence DNA and analyze genes at the nucleotide level has become a powerful tool in many areas of analysis and research. The Sanger dideoxynucleotide method of DNA sequencing was first developed in 1975 by Frederick Sanger, after whom the technique was named. The strategy used in this method is to create four sets of labeled fragments, corresponding to the four deoxyribonucleotides. It involves the enzymatic duplication of a DNA strand that is complementary to the strand of interest. In the Sanger method, 2',3'-dideoxyribonucleotide triphosphates (ddNTPs) are used in addition to deoxyribonucleotide triphosphates (dNTPs). Since the ddNTPs lack the 3'-OH group, they will terminate DNA chain elongation because they cannot form a phosphodiester bond with the next dNTP. Each of the four sequencing reaction tubes (A, C, G, and T) would then contain a singlestranded DNA template; a primer sequence; DNA polymerase to initiate synthesis where primer is hybridized to the template; mixture of the four deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend the DNA strand; one labeled dNTP (using a radioactive element or dye); and one ddNTP, which terminates the growing chain wherever it is incorporated. Tube A would have ddATP, tube C ddCTP, and so forth. Since the concentration of the ddNTP is low (approximately 1 percent of dNTP), the chain will terminate randomly at various positions throughout the chain, thus yielding an array of different length DNA fragments each terminated with the particular dideoxyribonucleotide. The fragments of varying length are then separated by electrophoresis in parallel lanes (each corresponding to the four deoxyribonucleotides) and the positions of the fragments analyzed to determine the sequence. The fragments are separated on the basis of size, with the shorter fragments moving faster and appearing at the bottom of the gel. The sequence is then read from bottom to the top, yielding the 5' to 3' sequence.

The ability to rapidly sequence DNA has become an important tool for molecular biology. For example, the polymerase chain reaction (PCR) method needs the information of the sequence flanking the region of interest to be able to design specific oligonucleotides (primers) to amplify the specific DNA region. Another important use is identifying restriction sites in plasmids, which is useful in cloning a foreign gene into the plasmid. Before DNA sequencing, molecular biologists had to sequence proteins directly, which was a challenging and laborious process. Now amino acid sequences can be determined more easily by sequencing a piece of cDNA and finding an open reading frame. In eukaryotic gene expression, sequencing made it possible to identify conserved sequence motifs and determine important sites in the promoter regions. Furthermore, sequencing can be used to identify the site of a point mutation or other changes in the genome.

Recombinant DNA technology allows for a transfer of a DNA fragment of interest into a self-replicating genetic element such as a bacterial plasmid or virus. This technology has allowed many human genes to be cloned in Escherichia coli, yeast, or even mammalian cells, leading to easy production of human recombinant proteins in vitro. These include insulin for diabetics, factor VIII for males suffering from hemophilia A, human growth hormone (GH), erythropoietin (EPO) for treating anemia, three types of interferons, several interleukins, adenosine deaminase (ADA) for treating some forms of severe combined immunodeficiency (SCID), angiostatin and endostatin for trials as anticancer drugs, and parathyroid hormone with many more in development.

Efforts toward gene therapy make use of recombinant DNA technology. In the case of CF, which is the result of a defect in a single gene, direct insertion of the normal gene should theoretically restore the function of CFTR in the treated cells of the CF patient. Since respiratory failure is the major cause of deaths (95 percent) in CF, lung cells have become the primary target for efforts at treating CF with gene therapy. Another advantage is the ability of delivering vectors containing the functional CFTR gene directly into the patient’s airways by aerosol, without causing trauma to any other part of the body. To date, CF trials rely on adenovirus, adeno-associated virus (AAV), and cationic liposomes to mediate gene transfer to nasal epithelial cells and respiratory epithelial cells. Adenoviruses do not integrate into the host chromosome; therefore, the vectors derived from these viruses have the advantage of negligible oncogenic potential. The disadvantages are the development of inflammation and the transient expression of the recombinant genes. Adenoassociated viruses are unique among animal viruses, because they require coinfection with an unrelated helper virus (either adenovirus or herpesvirus) for productive infection in cell culture. However, AAV has the ability to integrate into the host genome in the absence of a helper virus, but because it integrates at a specific location, the risk of virus-induced mutagenesis and oncogenesis is reduced. Liposomes, however, are synthetic lipid bilayers forming spherical vesicles, with size ranging from 25 nm to 1 mm in diameter depending on how they are made. Liposomes can transfect a variety of cell types, are biodegradable and hypoimmunogenic, and can be manufactured to a drug standard. Theoretically, there is no restriction on the size of the DNA to be delivered by liposome. The disadvantages are short-term expression of the transferred gene, no specific targeting ability, and a low transfection rate in vivo.

[8.1] A 30-month-old female child whose growth rate has been in the lower 10th percentile over the last year presents with chronic, nonproductive cough and diarrhea with foul-smelling stools. She is diagnosed as having cystic fibrosis. For which of the following vitamins is this child most likely to be at risk of deficiency?
A. Ascorbic acid (vitamin C)
B. Biotin
C. Folic acid
D. Retinol (vitamin A)
E. Riboflavin (vitamin B2)

[8.2] Some forms of genetic diseases such as cystic fibrosis and sickle cell anemia can be diagnosed by detecting restriction fragment length polymorphisms (RFLPs). Which of the following is most likely to be used in an RFLP analysis?
A. Dideoxynucleotides
B. Mass spectrometry
C. Northern blot
D. Southern blot
E. Western blot

[8.3] Your patient has been diagnosed with cystic fibrosis and has been determined to have the most common mutation, the ΔF508 gene. Which of the following is the most cost- and time-effective method for testing family members to see who are carriers of the mutation?
A. Allele-specific oligonucleotide probe analysis
B. DNA fingerprinting analysis
C. DNA sequencing
D. Restriction length fragment polymorphism analysis

[8.1] D. Because cystic fibrosis leads to pancreatic damage and diminution of the ability to secrete HCO− and pancreatic digestive enzymes with the result that fat and protein are absorbed poorly. Retinol is a fatsoluble vitamin that must be absorbed along with lipid micelles; other fat-soluble vitamins are E, D, and K. The other vitamins listed are water-soluble and their absorption is not significantly affected.

[8.2] D. Restriction length fragment polymorphism analysis detects mutations in the DNA that either introduce or eliminate a recognition site for a restriction enzyme. This is detected by performing a Southern blot analysis on patient DNA after incubation with a specific restriction enzyme and comparing it to one performed on DNA obtained from a normal gene.

[8.3] A. If the mutation is known, radioactive or fluorescent probes can be produced for alleles that contain the mutation and for those that have a normal DNA sequence. The samples of DNA are spotted onto nitrocellulose paper in narrow bands. The paper is then incubated with the probe either for the normal or the mutant sequence. Since it does not involve an electrophoresis step, this type of analysis is much less time-consuming (and less expensive).

❖ Northern blot uses oligonucleotide probes to identify RNA, Southern blot identifies DNA, and Western blot identifies protein molecules.
❖ Recombinant DNA technology allows for a transfer of a DNA fragment of interest into a self-replicating genetic element such as a bacterial plasmid or virus.
❖ Restriction length fragment polymorphism analysis detects mutations in the DNA that either introduce or eliminate a recognition site for a restriction enzyme.
❖ Oligonucleotide probes can be directed against precise DNA sequences and yields a quick and accurate result.


Schwiebert LM. Cystic fibrosis, gene therapy, and lung inflammation: for better or worse? Am J Physiol Lung Cell Mol Physiol 2004;286: L715–L716. 

Welsh MJ, Ramsey BW, Accurso F, et al. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly WS, et al., eds. The Metabolic & Molecular Bases of Inherited Disease, 8th ed. New York: McGraw-Hill, 2001.


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