Molecular diagnosis of Neonatal mellitus - Research Paper Example

Molecular diagnosis of Neonatal mellitus Neonatal diabetes mellitus (NDM) is less common form of non-autoimmune diabetes that affects few individuals in all diabetes cases. There is limitation on the efforts to contain this problem among the patients affected due to difficulty in searching for chromosome 6q24 abnormality and correspondence mutations in regions KCNJ11, ABCC8 and INS (Bonnefond et al, 2010). For diagnosis process to be successful, it is important to know the location of the chromosome with abnormality and this form the bulk of this technology. This paper discusses the feasibility of using next-generation sequencing as a whole exome in an attempt to relieve patients suffering from NDM problems. It also present the methodology used in testing the whole exome sequencing (WES) to patients and some of the achievements of this research method in relieving patients from diabetes mellitus (Carroll, Brilhante, and Suomalainen, 2014). Finally, the paper will explore inherent limitations and challenges hindering the implementation of this diagnosis. In this research study, the use of whole exome sequencing (WES) for patients with NDM provides a pertinent substitute for NDM molecular diagnosis. Initially, patients who suffered from diabetes mellitus were treated using insulin therapy. Furtherance on this, patients who carried a mutant gene in KCNJ11 or ABCC8 were treated using oral sulfonylurea drugs instead of insulin. Therefore, the adoption of whole exome sequencing (WES) for diagnosis of NDM at molecular level provided a remedy in general management of NDM as asserted by Bonnefond et al (2010). Molecular diagnosis of NDM has had several achievements such as it permitted a more rapid and cost-effective diagnosis problems. The analysis of patients with monogenic forms of diabetes is now very fast since the use of molecular diagnosis makes it possible of analysing many diabetes genes simultaneously (Bonnefond et al, 2010). Molecular diagnosis is applicable where the use of sanger-based sequencing was inefficient. Most importantly, the use of whole exome sequencing for molecular diagnosis is an exceptional tool for further genetic research. In addition, WES is important in further identification of new casual mutations (Ylikallio et al, 2014). For example, the use of WES has identified a new mutation of ABCC8. Therefore, this technology is more comprehensive, cheaper and less labour intensive as compared to other standard sequencing protocols. The Molecular Diagnosis of NDA Studies show that almost half of NDM cases are transient (TNDM) while the other forms are permanent (PNDM) (Carroll, Brilhante, and Suomalainen, 2014). The several cases of both the forms of NDM that were attend to since the adoption of WES use shows that, majority of all the cases handled had TNDM form. This form had abnormality in chromosome 6q24 and a diagnosis on the frequent cause of NDM shows there is mutation in the pancreatic beta cells. Bonnefond et al (2010) proposes that the presence of specific clinical features such as renal failure, thyroid disease, anaemia, cardiac disorders, pancreas agenesis or family history of diabetes suggest potential molecular aetiology of NDM. For this case, a molecular genetic diagnosis is important to anticipate the most appropriate treatment to help improve the quality of life. There prediction of the best method to use in treating an NDM patient is important and should be specific. This is because the mutation as well as chromosomal abnormality differs slightly for a particular form of NDM as explained by Bamshad et al (2011). Therefore, there is need to use technology during the diagnosis to help correct the problem distinctly and permanently. It is advisable for a country to offer DNA testing for NDM patients to help plan pharmacotherapy in a person molecular genetics especially when diagnosing a family (Chilamakuri et al, 2014).Periodic testing is important to anticipate whether NDM will be transient or permanent. The high concentration of hyperglycaemia usually prejudices the establishment of the form of NDM that is present in a patient. During diagnosis of a young patient, it is important to first check for the presence of extra pancreatic features and the history of the family on diabetes. The next step is to search for chromosome 6q24 abnormality and subsequently mutation on KCNJ11 genes then ABCC8 and finally on INS (Bonnefond et al, 2010).The gene sequencing may be negative. In that case, the performance of NDM analysis may be necessary. This is because the molecular sequencing of genes provide sequencing of limited known NDM genes. Therefore, a more comprehensive and efficient method of scrutinizing every NDM gene is necessary. The use of Sanger protocol for sequencing these genes was tedious, long and costly. Fortunately, with the use of molecular sequencing, the sequencing of a total of 42 coding exons altogether is possible. The present study, demonstrate the feasibility of next generation whole exome sequencing (WES) for the molecular diagnosis of patients with NDM. The supposition in this condition is that the patients in question do not have any extra pancreatic features of family history of diabetes (Carroll, Brilhante, and Suomalainen, 2014). The use of WES identifies a novel non-synonymous mutation, which the result was negative with the use of Sanger sequencing of ABC8. This is an indication that the novel technology is a more comprehensive, cheaper and less labour intensive for diagnosis of NDM as compared to other standard sequencing protocols The use of WES made it possible of identification of 55202-targeted DNA variants from human genome sequence. In an endeavour to validate data analysis from use of WES, the DNA of an NDM patient was genotyped as proposed by Bamshad et al (2011). WES identified 7969 exomic of single nucleotide polymorphism (SNPs). The result suggests that WES use based on the use of two channels of the genome analyser has a low false negative rate. The low rate is an important consideration in achieving molecular diagnosis for patients having monogenic disorders. In precedent to the adoption of next generation WES, a single-stranded Sanger sequencing analysis of patient’s DNA in a laboratory showed no evidence for coding mutations in ABCC8. In addition, there were no abnormalities in KCNJ11, INS and chromosome 6q24 (Chilamakuri et al, 2014). Therefore, this demonstrates for the first time that WES is a relevant alternative for molecular diagnosis of NDM. Accurate molecular diagnosis of NDM condition can lead to improvement in patients’ health care hence there is need to develop a reliable method for quick and accurate DNA analysis (Bonnefond et al, 2010).The use of WES is not only a cost-effective tool for molecular diagnosis of NDM, but it is also an excellent tool to further genetic research and identification of novel casual mutation. Limitations with the Molecular Diagnosis of NDM Although next-generation sequencing (NGS) technology aimed at targeted sequencing is gaining adoption for use in molecular diagnosis, there are several limitations delaying its application. The study requires ethical approval by the local committees making the process of experiment and study to be difficult as explained by Ylikallio et al (2014). The clinical trials of the exome require the approval of government or local authorities among the regions visited. It was also a requirement of technicians and physicians to obtain a written consent from the parents who gave their children for genetic testing. It is difficult to standardize the procedures used in sequencing due to the frequent update of enrichment methods. This brings many variations in procedures, which in turn compromise the validity of the research procedure. Another limitation is non-definition of the cut-off threshold for accurate or minimum read depth identification. Finally, the coverage of the variable depth across target region between pseudogenes and homologous sequences affect the accuracy of the data sequenced. In conclusion, next generation sequencing (NGS) technology is comprehensive, less labour intensive and cost effective method of whole exome sequencing (WES). The WES has made it possible the identification of novel de novo ABCC8 mutation in a NDM patient’s. There are suggestions that WES will be a near future tool of choice by most technicians for further molecular diagnosis of NDM cases. WES is therefore an accurate method compared to the current Sanger protocol method. It also a better method compared to other standard protocol of using insulin therapy or treatment using oral sulfonylurea drugs. References Bamshad, M. J., Ng, S. B., Bigham, A. W., Tabor, H. K., Emond, M. J., Nickerson, D. A., & Shendure, J. (2011). Exome sequencing as a tool for Mendelian disease gene discovery. Nature Reviews Genetics, 12(11), 745-755. doi:10.1038/nrg3031 Bonnefond, A., Durand, E., Sand, O., De Graeve, F., Gallina, S., Busiah, K., & ... Froguel, P. (2010). Molecular Diagnosis of Neonatal Diabetes Mellitus Using Next-Generation Sequencing of the Whole Exome. Plos ONE, 5(10), 1-5. doi:10.1371/journal.pone.0013630 Carroll, C. J., Brilhante, V., & Suomalainen, A. (2014). Next-generation sequencing for mitochondrial disorders. British Journal of Pharmacology, 171(8), 1837-1853. doi:10.1111/bph.12469 Chilamakuri, C. R., Lorenz, S., Madoui, M., Vodák, D., Jinchang, S., Hovig, E., & ... Meza-Zepeda, L. A. (2014). Performance comparison of four exome capture systems for deep sequencing. BMC Genomics, 15(1), 1-24. doi:10.1186/1471-2164-15-449 Ylikallio, E., Johari, M., Konovalova, S., Moilanen, J. S., Kiuru-Enari, S., Auranen, M., & ... Tyynismaa, H. (2014). Targeted next-generation sequencing reveals further genetic heterogeneity in axonal Charcot-Marie-Tooth neuropathy and a mutation in HSPB1. European Journal of Human Genetics, 22(4), 522-527. doi:10.1038/ejhg.2013.190 Read More