What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity?

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

Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in UTRs of targeted genes, according to each enrichment method.

<p>Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in UTRs of targeted genes, according to each enrichment method.</p

Number of UTRs (<i>n</i>_<i>total</i> = 197) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.

<p>Number of UTRs (<i>n</i>_<i>total</i> = 197) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.</p

Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in coding regions of targeted genes (with 10 bp of intronic flanking regions), according to each enrichment method.

<p>Percentage of base-pairs covered by 8, 20, 50 or 100 sequence reads in coding regions of targeted genes (with 10 bp of intronic flanking regions), according to each enrichment method.</p

Number of coding exons (with 10 bp of intronic flanking regions; <i>n</i>_<i>total</i> = 576) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.

<p>Number of coding exons (with 10 bp of intronic flanking regions; <i>n</i>_<i>total</i> = 576) exhaustively covered by 8, 20, 50 or 100 sequence reads, according to each enrichment method.</p

Venn diagram displaying the total number of variants in the targeted genes (coding exons and UTRs, with 10 bp of intronic flanking regions), which were identified by each enrichment method, in Patient #1 and Patient #2.

<p>The presence of mutations colored in red was not confirmed by Sanger sequencing while the presence of mutations colored in green was confirmed by Sanger sequencing (see also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143373#pone.0143373.s001" target="_blank">S1 Fig</a></b>).</p

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Nature genetics

Study of monogenic forms of obesity has demonstrated the pivotal role of the central leptin-melanocortin pathway in controlling energy balance, appetite and body weight (1) . The majority of loss-of-function mutations (mostly recessive or co-dominant) have been identified in genes that are directly involved in leptin-melanocortin signaling. These genes, however, only explain obesity in <5% of cases, predominantly from outbred populations (2) . We previously showed that, in a consanguineous population in Pakistan, recessive mutations in known obesity-related genes explain ~30% of cases with severe obesity(3-5). These data suggested that new monogenic forms of obesity could also be identified in this population. Here we identify and functionally characterize homozygous mutations in the ADCY3 gene encoding adenylate cyclase 3 in children with severe obesity from consanguineous Pakistani families, as well as compound heterozygous mutations in a severely obese child of European-American descent. These findings highlight ADCY3 as an important mediator of energy homeostasis and an attractive pharmacological target in the treatment of obesity