Question

To answer the following questions you may use any resource however the material is primarily derived from Introduction to Bioinformatics, Lesk 2014. In particular refer to chapter 2 on tracing inheritance patterns to identify genes responsible for disease (Box 2.3and 2-4). Describe how positional cloning can be used to identify genes responsible for specify diseases. Describe the process of identifying the gone responsible for cystic fibrosis. What types of evidence were used for this analysis? How did this evidence lead to the dentil nation of a cystic fibrosis gone? The gene for cystic: fibrosis was isolated and sequenced in 1989. How would the process for Isolating and sequencing this gene be different in 2017?

Answer 1

1. Positional Clonining can be used for identifying genes responsible for specific diseases:

The process by which a disease gene is identified based on its location on a chromosome is called Positional cloning. It consists of different consecutive steps like:

• Linkage- where the chromosome area is located;
• Fine Mapping- narrowing the initial area to a smaller genomic region;
• Candidate Gene Analysis- where the smaller genomic region is looked for mutations.

Through positional cloning, one can identify or "clone" a gene by tracing its location on a chromosome, via linkage analysis step. The identification and characterization of abnormalities like translocations, deletions and duplications on the chromosome either by cytogenesis methods or by micro array-based comparative genomic hybridization (array CGH) technique may prescribe the extensions and borders of the small genomic regions. The following step is the identification of candidate genes by analyzing available databases from the genome browsers. Positional cloning identifies the causative gene mutation and traces its role in the pathogenesis of the specific disorder, via cell-based or animal-based experiments. ​

2. Process of identifying the gene responsible for Cystic Fibrosis:

In understanding the defects and pathophysiology of cystic fibrosis (CF), the positional cloning of the gene responsible for the disease was the primary step. The cloning of the gene responsible for cystic fibrosis (CF) is a standard example of disease-gene identification via genetic linkage analysis, where the first CF linkage was detected with a serum enzyme marker, Paraoxonase (PON).

Evidence used in analysis:

• The initial characterization of the CFTR gene was based on the isolation and alignment of genomic clones with the cDNA clones derived from the T84 human colonic adeno-carcinoma cell line and those containing the p.Phe508del mutation isolated from a CF sweat gland cDNA library.
• It is now well established that the full-length CFTR mRNA consists of 6128 nucleotides. Alignment of this sequence with the genomic DNA sequence derived from the Human Genome Project has provided the precise exon–intron structure and intronic sequences of the CFTR gene.
• Based on the recent sequencing and functional data, the transcription module of CFTR was established to be >216 kb in size. The CFTR gene itself spans only 189.36 kb; however, the immediate promoter can be extended as far as 20.9 kb upstream, where the CTCF- dependent insulator element is located—the expanded promoter region includes the regulatory binding element required for proper gene expression .
• To understand the pathophysiology of CF, one of the approaches is to discover the CFTR expression pattern in different tissue surveys. CFTR is found to be expressed in the epithelial cells of a variety of tissues and organs, whose functions are significantly affected in CF patients: lung and trachea, pancreas, liver, intestines, and sweat glands.