Detection of diseased genes

Question 1

What are the three main classes of searching for diseased genes?

Answer 1

• Position independent
• Positional cloning
• Use of chromosomal aberrations

Question 2

What are the methods of position independent gene detection?

Answer 2

• Proteomic analysis
• Animal models
• Position independent DNA sequence knowledge (e.g. succession)

Question 3

How are genes detected using position independent proteomic analysis?

Answer 3

• Mass spectrometry: A powerful method for analysis is mass spectrometry which allows for protein analysis in detail. Modern methods can detect small amounts of protein using this method. If the sequence of cDNA can be worked out, an oligonucleotide probe can be used to screen libraries to recover cDNA, however this is open to inaccuracy from the degeneracy of the genetic code. Alternatives are probing using antibodies.
• 2D gel electrophoresis: proteins are sorted based upon pH and charge. Proteins are obtained through cell extracts using mild deteregents, which creates a crude protein extract. This extract can then be purified, which can be loaded into a gel. The first dimesion is isoelectric focusing which sorts molecules based upon pH, and the second dimension is done through creating an electric current which sorts proteins based on size. It is hard to estimate the complete number of proteins, but there are estimates as high as 3 million. Each cell will have a different set of proteins. Proteins can be cut from the gel and analysed using mass spectrometry. Protein digested, analysed, histogram created, and then the histogram is matched against a database, which can be used to identify proteins. This can be done through either peptide mass or fragment ion masses. The protein can be used to estimate the DNA code, but this is not reliable because the genetic code is degenerate.

Question 4

How are animal models used to detect diseased genes?

Answer 4

Animal models are only used by chance, not deliberately.

In isolated cases diseased genes have been identified, such as Waardenburg-Shah syndrome (WS4) (Pingault et al., 1998).

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung’s disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon).

The identification of Sox10 as the gene mutated in Dom mice prompted the analysis of the role of its human homologue SOX10 in neural crest defects. Patients from four families with WS4 have mutations in SOX10, whereas mutations are absent in patients with HSCR alone.

Question 5

How can knowledge independent of position about DNA sequence be used in identifying diseased genes?

Answer 5

There have been attempts to clone genes based on triplet repeats. Anticipation means that diseases present earlier and with increased severity with successive generations, for example Huntington disease. If diseases present this way, it is suspected that there is a trinucleotide expansion. Two classes:

• Promoter (outside coding) expansion: Fragile X syndrome is a rare, but well characterised disease. It is the most common form of inherited mental retardation, in which FMR1 is mutated and the gene cannot produce enough protein for normal brain function. In a vast majority of patients, FMR1 is silenced by the expansion of an unstable triplet CGG-repeat motif in the 5′UTR that occurs in the maternal germ line. Between 6-40 repeats is normal, but anything above 40 is in diseased territory. Anything above 200 is full blown disease. This expansion can be seen in karyotype analysis as a pinch near the end of the X chromosome.
• Coding expansion: in particular CAG becomes expanded heavily, all nine of which are neurodegenerative diseases. This creates polyglutamine. When there are too many amino acids in one place, this harms the cell causing protein aggregration and the protein changes conformation and damages other proteins. There are thresholds for when the number of repeats cause damage, and the larger the number of repeats the earlier the onset. For example: Huntington disease, in which a CAG trinucleotide repeat expansion encodes an abnormally long polyglutamine tract in the huntingtin protein. In normal individuals, the number of CAG repeats is 35 or fewer, with 17–20 repeats found most commonly. The disease range is 36 and above. Most adult-onset cases have 40–50 CAGs, whereas expansions of 50 and more repeats generally cause the juvenile form of the disease. Pedigree charts can be used coupled with gel electrophoresis to define thresholds. Agarose gel for larger amount of DNA, polyacramide gel electrophoresis can distinguish specific base number differences.

Question 6

Outline positional cloning.

Answer 6

The rough position of the gene in the genome is needed for positional cloning, such as the chromosome and arm. This is probably the most powerful method for gene detection, and is still used today. The first application was done in the 1980s, but the first major finding was in 1986 when the gene involved in DMD was discovered. CF and Huntington genes also discovered.

This is done in a series of steps:

1. The candidate region is defined, usually using linkage data from large families with genetic mapping markers. This is done via a genome scan where different markers spaced across the genome are tested for co-segregation with diseased phenotypes (determined using LOD-based linkage analysis, score of at least 3 needed for linkage). More spaced markers are used which are subsequently narrowed down to a local area. This was critical in mapping the DMD gene to Xp21.
2. Distinguishing the mutated gene from others in the area. The region is defined through multiple possible methods:
   
   • Gene functions from annotated databases (Ensembl) used to select candidate genes.
   • In the past Contig was used to search for transcripts (clone DNA fragments into vectors containing region of interest, get a transcription map, cDNA library screen and selection).
   • Expression analysis to detect genes preferentially expressed in diseased tissue using microarrays. Test by RT-PCR, Northern blotting, SAGE.
3. Once the gene has been identified, it needs to be shown to co-segregate with the diseased phenotype. However this creates problems when there are sequence variants are harmless polymorphisms. Candidates screened for mutation. In the past this was expensive, so ensurance of correct genes was needed.
   
   • A good indicator is if a mutation disrupts a highly conserved sequence in orthologs from other organisms.
   • Autosomal recessive mutant testing can be done via resuing the mutant gene in vitro by introducing the WT gene.
   • Autosomal dominant testing by introducing mutant allele into WT cell line to produce mutant phenotype.

Question 7

What are good model organisms to search for orthologs?

Answer 7

• Mouse (Mus muscus)
• Drosophila melanogaster
• Chaenorhabditis elegans
• Zebrafish
• Yeast (Candida albicans)

Question 8

How is homology used in gene identification?

Answer 8

30 years ago positional cloning took months, or even years. BRCA1 was identified in a two year process, involving dozens of labs. Various tricks were used to make searching easier, such as homology.

• This involves looking for similarity across the evolutionary range. Similarity between different organisms can be searched, and people used this.
• An example of this is using Drosophila melanogaster. This organism is good because mutants can be created easily, and they can be screened. A striking example is apterous, which produces wingless flies. In humans this gene (Lhx2) encodes a transcriptional regulatory protein involved in the control of cell differentiation in developing lymphoid and neural cell types. When human genes are inserted into the fly genome, it will develop with wings (ortholog). Between mice and humans, there are 20 blocks of homology in chromosome 6, for example. Homology varies between species.

Question 9

How can chromosomal abnormalities be used in gene identification?

Answer 9

Some diseases can be associated with chromosomal abnormalities, for example trisomy 21 and CML (Philadelphia chromosome). In some cases chromosomal rearrangements can cause disruption of genes at the break points, either by disrupting the gene sequence or separating it from the regulatory region. Sometimes there is a gain of function phenotype in which the splicing of exons from two genes together creates a chimeric gene poduct. Leukaemias sometimes involved breaks, the breakpoints of which can be identified using FISH.

Question 10

How can candidate genes be confirmed?

Answer 10

Once the selection process is done, i.e. postional cloning, identified candidates, the last thing to do would be to screen patients for mutation.

• Mutation screening
• Restoration of phenotype in vitro (can be done with cells). If disease can be seen in phenotype (under microscope), then this can be experimented on using restoration.
• Production of a mouse model. Most human genetic diseases have a mouse model. These are created through gene targeting, deleting genes from the genome. Sometimes this afflicts the embryos (embryonic lethal), so some cannot be created.
• Understanding the function of the identified gene.

Question 11

How were the diseased genes identified in Sotos syndrome and Treacher Collins syndrome?

Answer 11

• Sotos syndrome: identification through chromosomal abnormality.
• Treacher Collins syndrome: identification through positional cloning (pure transcript mapping).