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Disease gene mutation analysis

(last modified April, 2008)

Introduction

Your research strongly suggests that mutations in gene-X may cause the disease-Y. Before you start a scan for mutations, you first want to determine what parts of the gene need to be analysed. A similar situation might arise when during mutation scanning you originally focused on the protein coding region and intron/exon borders of gene-X but failed to detect pathogenic changes in a significant fraction of the patients. The question is then; did I analyse all important regions ?. In addition we need to design primer pairs to be able to screen the new regions your analysis might have identified.

Task

Gene structure

Determine, based on all available information, the structure of gene-X. Good starting points are;

EntrezGene (annotated transcripts)
the genome browsers (transcribed DNA, alternative splicing, conserved regions, etc.); Ensembl, UCSC
Blast searches (see also task Mutation analysis)

Using the retrieved gene-X cDNA (mRNA) sequence, preferably from the RefSeq databases, perform Blast searches.When using Blast always include dbEST, starting with human ESTs only. When the number of hits is large (more then displayed), split the cDNA sequence into segments and Blast these individually. Give specific attention to the start and end of the gene transcript (e.g. using 100-200 nucleotide query sequences); genes often contain several different promoters / first exons as well as several last exons / polyA-addition sites.

When you have generated a reliable cDNA sequence based on Blasting human sequence, Blast this sequence against other species (again incl. dbEST) to check its conservation; are the transcripts you have identified conserved ?, do other species contain transcripts that you have missed in human ?.

NOTE: new findings can be reported to the ResSeq database (NCBI), after curation they will be added as a GeneRif.

Sequence conservation

Determine which segments, besides the part of the DNA sequence encoding the protein are evolutionary conserved (pointing to functional importance). Start with the protein coding regions, then the transcript (cDNA) giving specific attention to the 5' and 3' UTR, then go to the introns and complete your analysis with the direct gene flanking regions.

There are many ways to obtain a good MSA;

NOTE: using the genome browser (e.g. Ensembl, UCSC) it is possible to get a quick overview of conserved regions.

from a genome browser (Ensembl or UCSC)
through HomoloGene (NCBI)
through SwissProt
generate one yourself
- download the sequence to analyse, perform a BlastP search to identify similar proteins from other organisms, retrieve the files of these proteins and finally generate a MSA (e.g. use BioEdit, ClustalW or 3DCoffee)

Problematic sequences

Some peculiar sequences are known to potentially cause problems. A clear example are so the called 'repeat expansion diseases' like Huntington's disease (CAG expansion in the gene's protein coding region), Fragile-X syndrome (CGG expansion in 5' UTR [Verkerk 1991]), Myotonic dystrophy (CTG expansion in 3' UTR [Fu 1992]) and Friedreich's ataxia (intronic GAA repeat expansion [Campuzzo, 1996]).

Does gene-X contain regions with simple sequences that need to be checked for expansion ?.

Result

What is your overall conclusion; which regions of gene-X have to been screened for mutations in patients with gene-Y ?.

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