Abstract
Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing.
Highlights
Diagnostic testing for monogenic forms of diabetes and obesity is of paramount importance for the patient and his/her family as it can lead to personalized medicine associated with a better life
The patients presenting with neonatal diabetes mellitus (NDM), maturity-onset diabetes of the young (MODY), or adult onset atypical type 2 diabetes, who carry a heterozygous mutation in ABCC8 or KNCJ11, can be successfully switched from an expansive and demanding insulin therapy to easy and cheap oral sulfonylureas, with much better result on glucose control [1]
We first compared the mean percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in coding regions of the 43 targeted genes, according to each enrichment method (Fig 1)
Summary
Diagnostic testing for monogenic forms of diabetes and obesity is of paramount importance for the patient and his/her family as it can lead to personalized medicine associated with a better life. The patients presenting with neonatal diabetes mellitus (NDM), maturity-onset diabetes of the young (MODY), or adult onset atypical type 2 diabetes, who carry a heterozygous mutation in ABCC8 or KNCJ11 (encoding subunits of the ATP-dependent potassium channel at the membrane of pancreatic beta cells), can be successfully switched from an expansive and demanding insulin therapy to easy and cheap oral sulfonylureas, with much better result on glucose control [1]. Among the patients with severe, early-onset obesity, the carriers of a homozygous mutation in the leptin gene (LEP) can be treated with recombinant leptin therapy, which leads to a marked and sustained reduction in weight [5]. The development of quick, reliable and cost-effective protocols for genetic diagnosis of diabetes and obesity are limiting factors for precision medicine in metabolic diseases
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