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Pathogenic or not ?

(last modified September 2009)

Introduction

Searching for a potential genetic cause of disease-X in a patient gene-Y was screened for sequence variants (mutations). Several variants were found and the question is "do these variants have a deleterious consequence or not ?". To answer this question, a range of things need to be checked;

was the variant reported before
sequence conservation (protein, protein domains, DNA in 5' and 3'UTR and directly gene flanking)
effects on splicing
effects on protein characteristics (folding, 3D-structure, localisation, etc.)

Note

Besides those mentioned below, the Bioinformatics Tools section from the Center for Human and Clinical Genetics might contain helpful hints to guide your analysis.

Task

Pathogenic or not

Seen before ?
1. has the variant been reported before and what is the conclusion according to these sources ?
  - extra for 1 group in 2009: use Mutalyzer to check correct description of the variant using the HGVS recommendations. Do you understand the output given by Mutalyzer? - discuss your experiences and wishes wit the developers (i.e. Jeroen Laros).
2. check to the list of gene variant databases (Waystation, LOVD), to check whether there is a gene variant database for the gene
3. is this gene / variant present in other databases; use OMIM, HMGD and dbSNP
4. are there publications describing this variant; use PubMed and GoogleScholar
Sequence conservation
1. generate a multiple sequence alignment (see Disease gene mutation analysis). Based on the MSA, does the variant identified change a conserved amino acid residue ?.
2. web-tools can also be used to determine the possible consequences of the amino acid change; determine the consequences according to SIFT (Sorting Intolerant From Tolerant), Align-GVGD (generates a Grantham score) and PolyPhen. When required, use the MSA generated.
Protein characteristics
1. proteins often contain several individual functional units, so called protein domains. Are their specific protein domains in this protein, where, and what is their (suggested) function ?
  NOTE: besides generating a MSA for the entire protein, making an MSA per protein domain may give helpful additional information; similar domains of related or different proteins are evolutionary further apart increasing knowledge on the functionally most important amino acids (= most conserved). The most distantly related domains can be identified using specific tools like e.g. PsiBlast (NCBI).
2. based on the amino acid sequence some general predictions can be made for the structure of the protein, incl. the presence of alpha-helices and/or beta-sheet, regarding hydrophobicity, etc. Analyse these general characteristics using both the normal and mutated sequence; are there prominent changes?
3. 3D-structure; is the 3D structure of protein-X, or a related protein, or from the mutated domain known ?. If yes, use this to analyse or build the 3D-model.
Effect on splicing
there are several ways sequence variants can disrupt splicing; they can generate aberrant (cryptic) splice sites, lower the strength of the normal splice site, or interfere with exon recognition by disrupting exonic-splicing-enhancer (ESE) sites or inducing exonic-splicing-silencer (ESS) sites. Analyze a putative effect on splicing using the Splice Sequence Finder; it will screen for all these mutations at the same time when you use the "analyze mutation(s)" option.
The rest
finally, by analysing sequence conservation at DNA-level (incl. DNA in 5' and 3' UTR, directly gene flanking and in the introns), check whether the variant changes a potential regulatroy sequence.
NOTE: using the genome browser (e.g. Ensembl, UCSC) it is possible to get a quick overview of conserved regions.

Result

What is your overall conclusion; "has the change detected, based on what evidence, a deleterious consequence or not ?".