Identification of novel genes in Hirschsprung disease pathway using whole genome expression study.
OBJECTIVE
This study aims to identify new genes not described previously that may be relevant in the etiology or pathophysiology of patients with Hirschsprung disease (HD). This was done by identifying differences in gene expression between normal and abnormal segments of bowel in HD patients compared with controls.


METHODS
Full-thickness colonic tissue samples were taken from HD patients, both from the diseased (Ds) and normal segment of the colon (Nr), and from controls (Ct). Samples were further dissected into mucosa (MUC) and muscle (MUS). RNA was extracted and analyzed on Affymetrix Gene Chip Human Gene 1.0 ST arrays. Statistical analyses using ANOVA with a fold change cut off of 2 was applied to detect a number of differentially expressed genes. Selected genes were revalidated by quantitative real-time reverse transcriptase polymerase chain reaction.


RESULTS
Thirty-four samples (18 MUS and 16 MUC) were analyzed. MUC (1.64 ± 0.46 μg/mg) and MUS (0.83 ± 0.48 μg/mg) showed good RNA extraction yield and quality. Of the 24,987 filtered on expression genes, MUS showed 220 genes with expression difference of 2-fold, out of which 120 genes were significant with P ≤ .05. Similarly, MUC demonstrated 206 genes with 2-fold changes and 9 had P ≤ .05. Some genes showing differential expression between groups and therefore subject to further analysis were RELN, GAL, GAP43, NRSN1, and GABRG2.


CONCLUSIONS
Analyzed data showed significant differences in expression of above sets of genes with up- and down-regulation, which has not been described before in HD and could have a role in pathogenesis of this condition.
Department of Paediatric Surgery, Box 267, Addenbrookes Hospital, Cambridge University Hospitals NHS Foundation Trust, Hills Road, Cambridge CB2 0QQ, UK. atif.saeed@addenbrookes.nhs.uk
Atif
A
Saeed
Lenka
L
Barreto
Sudhesna Guha
SG
Neogii
Andrea
A
Loos
Ian
I
McFarlane
Adil
A
Aslam
D015816:Cell Adhesion Molecules, Neuronal / Q000096:biosynthesis / Q000235:genetics; D003106:Colon / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D019922:GAP-43 Protein / Q000096:biosynthesis / Q000235:genetics; D019004:Galanin / Q000096:biosynthesis / Q000235:genetics; D020869:Gene Expression Profiling; D055106:Genome-Wide Association Study; D006627:Hirschsprung Disease / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007223:Infant; D007413:Intestinal Mucosa / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D009130:Muscle, Smooth / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011963:Receptors, GABA-A / Q000096:biosynthesis / Q000235:genetics; D000091042:Reelin Protein; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics
D015816:Cell Adhesion Molecules, Neuronal; D016326:Extracellular Matrix Proteins; C546279:GABRG2 protein, human; C056567:GAL protein, human; D019922:GAP-43 Protein; D008565:Membrane Proteins; C046375:NRSN1 protein, human; D009419:Nerve Tissue Proteins; D011963:Receptors, GABA-A; D000091042:Reelin Protein; D019004:Galanin; C000717127:RELN protein, human; D012697:Serine Endopeptidases