Introduction
Hirschsprung disease (HSCR) is a condition characterized by the absence of ganglion cells within the gastrointestinal tract. It is considered the primary cause of functional intestinal obstruction and has an incidence rate of 1/5000 birth. Research studies indicate that the genetic aetiology of this condition arises from several gene mutations (Pan & Li, 2012). Changes in the REF proto-oncogene are closely associated with the development of HSCR in human beings. The development of therapeutic management approaches depends on an in-depth understanding of the molecular basis of the condition (Burzynski, Shepherd & Enomoto, 2009).
Techniques used in studying the molecular basis of Hirschsprung disease
A number of techniques have been developed for studying the molecular basis of the HSCR disease in human beings. Most of these approaches identify the presence of nonsense and missense mutation and other variations on the human genome.
MLPA technique
The Multiple ligation-dependent Probe Amplification-Hirschsprung kit was developed to detect the presence of gene alterations that predisposes people to the HSCR disease. The MLPA kit has over 41 different probes that are used in the identification of gene alteration. To amplify the regions within the genome that have been altered, the kit hybridizes five control fragments. This increases the population of the gene fragments that are analyzed within the DNA analyzer. Though the presence of nonsense/missense mutations has been described, copy number variations (CNV) studies have not been conclusively conducted (Pasternak, 2005).
The lack of reliable mutation analysis techniques has affected the description of the copy number variations in Hirschsprung disease. The multiple ligation-dependent probe amplification technique evaluates the presence of four genes that are related to the development of Hirschsprung disease in newborns. The four genes analyzed using this technique include the EDNB, GFRAI, NRTN and the PHOX2B genes. These genes have an implication on the ENS development in embryos which increase their risk factor to HSCR (Fernández, Bleda & Luzón-Toro, 2013).
SNP genotyping
SNP genotyping is a technique that is used to identify the presence of genetic variation within the single nucleotide polymorphism (SNPs). Single nucleotide polymorphism is considered one of the most common genetic variations that arise from missense or nonsense mutation in the genome. This mutation occurs at a specific locus which affects the functional ability of the ganglion cells within the intestine.
For the RET proto-oncogene, four SNPs of the human genome is evaluated with PCR restriction fragment length polymorphism (Brooks, Oostra & Hofstra, 2005). This approach has been extensively used in various research studies involving parents with high-risk factors for HSCR in Thai. From this study, two-thirds of the study participants showed a clear association with HSCR based on their RET proto-oncogene and the analysis of the NRG1 genes (Tang et al. 2014).
Restriction fragment length polymorphism (RFLP)
The final technique that can be employed in the molecular understanding of HSCR disease is the RFLP technique. With RFLP, SNP genotyping is conducted before the identification of the points of variations. The identification of the differences that exist within the homologous DNA is done in order to show the points of the mutation responsible for HSCR. In this technique, the DNA samples collected are subjected to digestion with restriction enzymes and the fragments are separated through gel electrophoresis (Bergeron, Silversides & Pilon, 2013).
References
Bergeron, K, Silversides, D & Pilon, N 2013, ‘The developmental genetics of Hirschsprung’s disease, Clinical Genetics, vol. 83, no. 1, pp. 15-22.
Brooks, A, Oostra, B & Hofstra, R 2005, ‘Studying the genetics of Hirschsprung’s disease: unravelling an oligogenic disorder’, Clinical Genetics, vol. 67, no. 1, pp. 6-14.
Burzynski, G, Shepherd, I & Enomoto, H 2009, ‘Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung’s disease’, Neurogastroenterology & Motility, vol. 21, no. 2, pp. 113-127.
Fernández, R, Bleda, M & Luzón-Toro, B 2013, ‘Pathways systematically associated to Hirschsprung’s disease’, Orphanet Journal of Rare Diseases, vol. 8, no. 1, pp. 1-27.
Pan, Z & Li, J 2012, ‘Advances in molecular genetics of Hirschsprung’s disease’, Anatomical Record, vol. 295, no. 10, pp. 1628-1638.
Pasternak, J 2005, An Introduction To Human Molecular Genetics: Mechanisms Of Inherited Diseases, John Wiley & Sons, New Jersey.
Tang, W, Li, H, Tang, J & Wu, W 2014, ‘Specific serum micro RNA profile in the molecular diagnosis of Hirschsprung’s disease, Journal Of Cellular & Molecular Medicine, vol. 18, no. 8, p. 1580.