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British Journal of Healthcare and Medical Research - Vol. 11, No. 4

Publication Date: August 25, 2024

DOI:10.14738/bjhmr.114.17294.

You, S., Bergmann, M., Lessel, J., Hart, M., Belyue, A., Conley, E., Baker, A., Nguyen, S., Newmeyer, L., Romanelli, J., & Luckie, D.

(2024). Identification of the W1282X-CFTR SNP Mutation in CF Patient Cells via AS-PCR. British Journal of Healthcare and Medical

Research, Vol - 11(4). 220-231.

Services for Science and Education – United Kingdom

Identification of the W1282X-CFTR SNP Mutation in CF Patient

Cells via AS-PCR

SunYoung You

Michigan State University

Mallory Bergmann

Michigan State University

Jessica Lessel

Michigan State University

Makayla Hart

Michigan State University

Alexandria Belyue

Michigan State University

Eva Conley

Michigan State University

Alexa Baker

Michigan State University

Sydney Nguyen

Michigan State University

Laurence Newmeyer

Michigan State University

Joseph Romanelli

Michigan State University

Douglas Luckie

Michigan State University

ABSTRACT

The W1282X cystic fibrosis transmembrane conductance regulator (CFTR) gene

mutation causes a severe form of cystic fibrosis which accounts for 1.2% of the CF

worldwide. This single nucleotide polymorphism (SNP) CFTR mutation is located on

exon 20, consisting of a change from guanine to adenine at the 3846th base pair,

creating a premature stop codon. The purpose of this study was to develop a

polymerase chain reaction (PCR) based diagnostic assay to identify the W1282X

mutation using allele-specific primers. We hypothesized that the Yaku method,

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You, S., Bergmann, M., Lessel, J., Hart, M., Belyue, A., Conley, E., Baker, A., Nguyen, S., Newmeyer, L., Romanelli, J., & Luckie, D. (2024). Identification

of the W1282X-CFTR SNP Mutation in CF Patient Cells via AS-PCR. British Journal of Healthcare and Medical Research, Vol - 11(4). 220-231.

URL: http://dx.doi.org/10.14738/bjhmr.114.17294.

which introduces an additional intentional base pair mismatch, would reduce non- specific binding in allele-specific PCR (AS-PCR). To increase accuracy, we also

employed a “nesting” amplification approach. A published reverse primer (PRP1)

and a universal forward primer (UFP1) were designed to first amplify a 718 bp

"template" region. After which, a published forward primer (PFP1) and Yaku

reverse primers (RPMT1 and RPWT1) were paired to produce a 316 bp region

diagnostic of the presence of either mutant W1282X-CFTR or wild-type CFTR in DNA

samples. PCR products were analyzed via agarose gel electrophoresis, revealing

expected bands of approximately 316 base pairs, supportive of successful W1282X

detection with Yaku primers.

Keywords: cystic fibrosis, genetic diagnosis, SNP, PCR, gel electrophoresis.

INTRODUCTION

Cystic Fibrosis (CF) is an autosomal recessive disorder distinguished by the buildup of thick

mucus in the lungs, digestive tract, and salty sweat from sweat glands [1]. There are thousands

of documented mutations within the Cystic Fibrosis Transmembrane Conductance Regulator

(CFTR) gene known to cause CF [2]. Typically, a healthy, non-mutated CFTR gene produces a

CFTR protein that functions as a chloride ion channel, transporting chloride across epithelial

cells [2]. A mutated CFTR gene, however, produces a misfolded, defective CFTR protein or fails

to produce the protein entirely. Consequently, a buildup of thick mucus due to high chloride ion

concentration, occurs within the pancreas and lungs, leading to persistent lung infections in CF

patients [3]. Treatments for CF include but are not limited to antibiotics such as tobramycin or

penicillin, postural drainage, physiotherapy, oral pancreatic enzymes, and more recently,

correctors and potentiators [4, 5]. Sweat tests are commonly used to diagnose CF due to high

levels of sodium and chloride in patients’ sweat [4]. In addition, polymerase chain reaction is

used to diagnose CF, particularly in infants and adolescents, improving quality of life through

early therapeutic and medicinal treatment [6]. Polymerase chain reaction (PCR) is a technique

developed to amplify specific DNA sequences [7]. The PCR process replicates a segment of

genomic DNA utilizing forward and reverse primers which mark the ends of the desired region

of amplification [8]. In a single thermal cycle of PCR, the sample is heated up to 95°Cto denature

and separate dsDNA in lieu of the role of helicase used in vitro DNA replication, breaking the

hydrogen bonds holding the DNA strands together [9]. The temperature then drops to around

50-60oC, allowing primers to anneal to complementary DNA sequence sites by forming

hydrogen bonds to single strand DNA template [9]. In the final step, the temperature rises to

72°C to enable a thermostable DNA polymerase to replicate the segment of DNA between

primers [9]. When the cycle is repeated multiple times, the exponential DNA replication

produces large amounts of target DNA (i.e. 30 cycles yield over 1 billion copies) [10]. Gel

electrophoresis is frequently performed to visualize and analyze the PCR products [11]. Since

DNA is negatively charged as a result of the presence of phosphate groups, an applied electrical

field moves DNA fragments through the gel matrix separating them by size [12].

This study focused on designing a PCR diagnostic assay for the W1282X mutation, a nonsense

mutation that causes CF [13]. The W1282X-CFTR mutation is prominent in those who are of

Ashkenazi Jewish descent and the second most common CF-mutation in the general population

[13, 14]. The W1282X mutation is caused by a single nucleotide point or polymorphism (SNP),

leading to a transition of guanine to adenine, at the 3846th base pair in exon 20. The codon TGG

is changed to a stop codon, TGA, causing translation to terminate prematurely [15].