Copy number determination of the gene for the human pancreatic polypeptide receptor NPY4R using read depth analysis and droplet digital PCR

Abstract

Background Copy number variation (CNV) plays an important role in human genetic diversity and has been associated with multiple complex disorders. Here we investigate a CNV on chromosome 10q11.22 that spans NPY4R , the gene for the appetite-regulating pancreatic polypeptide receptor Y4. This genomic region has been challenging to map due to multiple repeated elements and its precise organization has not yet been resolved. Previous studies using microarrays were interpreted to show that the most common copy number was 2 per genome. Results We have investigated 18 individuals from the 1000 Genomes project using the well-established method of read depth analysis and the new droplet digital PCR (ddPCR) method. We find that the most common copy number for NPY4R is 4. The estimated number of copies ranged from three to seven based on read depth analyses with Control-FREEC and CNVnator, and from four to seven based on ddPCR. We suggest that the difference between our results and those published previously can be explained by methodological differences such as reference gene choice, data normalization and method reliability. Three high-quality archaic human genomes (two Neanderthal and one Denisova) display four copies of the NPY4R gene indicating that a duplication occurred prior to the human-Neanderthal/Denisova split. Conclusions We conclude that ddPCR is a sensitive and reliable method for CNV determination, that it can be used for read depth calibration in CNV studies based on already available whole-genome sequencing data, and that further investigation of NPY4R copy number variation and its consequences are necessary due to the role of Y4 receptor in food intake regulation. Electronic supplementary material The online version of this article (10.1186/s12896-019-0523-9) contains supplementary material, which is available to authorized users.

Evaluation of the Genetic Association Between Adult Obesity and Neuropsychiatric Disease

“…Here we assessed the prevalence of protein truncating variants (PTV: stop gain, frameshift and splice variants) in genes previously associated with NPD as well as CNVs in regions previously implicated in NPD. We undertook further studies to assess the prevalence of CNVs in two other cohorts with microarray data: a cohort of patients referred for obesity management to University College London Hospital, London UK (UCLH cohort) comprising patients with EO and less extreme obesity (O, BMI [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] and a Swedish cohort of participants with predominantly O recruited from the community.…”

Digital droplet PCR versus quantitative PCR for lipoprotein (a) kringle IV type 2 repeat polymorphism genetic characterization

“…The number of studies in which this method is used for the determination of copy number variation is increasing, 32,[34][35][36][37]41,42 since copy number values are calculated in an absolute way directly by the software, without the need of standard samples for normalization. Data obtained from the present study are in keeping with those observed by Sallustio et al, 37 as a higher stability in KIV2 repeats measurement is achieved with ddPCR in comparison to that observed with qPCR technique.…”

“…ddPCR is rapidly gaining in popularity over standard real-time PCR assays for its relative ease of use, precise and accurate results, and ability to quantify nucleic acid concentration without the need for standard samples. 31,32 The number of publications in which this technology is employed for the investigation of CNVs, which can impact from single genes to regions of larger size, such as chromosomal rearrangements, continues to grow, [33][34][35][36][37][38] progressively opening up new avenues for the use of ddPCR in diagnostics.…”

Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injury

“…Amplicon lengths of SNP assays were 93, 70, 95, 67, 85 and 75 bp, respectively. RPP30 (amplicon length of 62 bp) and RPPH1 (amplicon length of 135 bp) assays were described previously (Oscorbin et al, 2019; Shebanits et al, 2019). RPPH1 and RPP30 were selected because they are single copy genes per haploid human genome and are commonly used as reference genes in copy number variation studies (Imaizumi et al, 2019; Oscorbin et al, 2019; Shebanits et al, 2019).…”

“…RPP30 (amplicon length of 62 bp) and RPPH1 (amplicon length of 135 bp) assays were described previously (Oscorbin et al, 2019; Shebanits et al, 2019). RPPH1 and RPP30 were selected because they are single copy genes per haploid human genome and are commonly used as reference genes in copy number variation studies (Imaizumi et al, 2019; Oscorbin et al, 2019; Shebanits et al, 2019). SRY assay for quantification of presence of chromosome Y was designed as follows: forward primer 5’-CGAAGTGCAACTGGACAACAG-3’, reverse primer 5’-TAGCTGGTGCTCCATTCTTGAG-3’ and probe 5’-ACAGGGATGACTGTACGAAAGCCACACA-3’ (HEX, ZEN-3IABkFQ) (Integrated DNA Technologies), with amplicon length of 76 bp.…”