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Copy number determination of the gene for the human pancreatic polypeptide receptor NPY4R using read depth analysis and droplet digital PCR.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Larhammar: Pharmacology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Human Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0001-9460-390x
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medicinsk genetik och genomik. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2019 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 19, article id 31Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2019. Vol. 19, article id 31
National Category
Cell Biology Genetics
Identifiers
URN: urn:nbn:se:uu:diva-356569DOI: 10.1186/s12896-019-0523-9ISI: 000470281900001PubMedID: 31164119OAI: oai:DiVA.org:uu-356569DiVA, id: diva2:1236206
Funder
Swedish Research CouncilThe Swedish Brain FoundationAvailable from: 2018-08-01 Created: 2018-08-01 Last updated: 2019-06-25Bibliographically approved
In thesis
1. The human pancreatic polypeptide receptor Y4: Genetic and functional variation
Open this publication in new window or tab >>The human pancreatic polypeptide receptor Y4: Genetic and functional variation
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Humans are evolutionarily adapted to an environment where food is scarce, but today many live in a world of food abundance. Paired with low physical activity, this may lead to weight gain and obesity. Efficient anti-obesity treatments require understanding of the mechanisms that control hunger, satiety, energy metabolism and body weight. This thesis investigates possible genetic and physiological mechanisms behind these processes.

Genetic correlation between body-mass index (BMI) and a highly polymorphic region on chromosome 10 was analysed with regard to single nucleotide polymorphisms (SNPs) and gene copy number variation (CNV). This region contains the gene NPY4R encoding the pancreatic polypeptide (PP) receptor Y4, which has been reported to reduce appetite.

The results show that the NPY4R gene was duplicated before the divergence of modern humans from the Neanderthals and the Denisovans (approximately to 400,000–800,000 years ago). The CNV of the NPY4R gene region was investigated by read depth analysis based on genome sequences and droplet digital PCR (ddPCR). The read depth results revealed a CNV range of 3-7 copies per genome, while the ddPCR results demonstrated a range of 2–11. Most humans have a total of 4–5 copies, in contrast to the two copies presumed by previous studies.

Investigation of an association between the NPY4R CNV and body mass index (BMI) led to interesting and ambiguous results. A study of 558 Swedish individuals with a wide range of BMI suggested, surprisingly, a positive correlation between NPY4R copy number and BMI for women. On the other hand, a study of 1009 individuals from Northern Sweden found no correlation between BMI and NPY4R copy number. These diverging findings may be due to geographical variation or lack of power in one of these studies.

Twelve naturally-occurring amino acid variants of the Y4 receptor were investigated pharmacologically in cell culture. Three of these showed no functional response, which may be explained by altered conformation of the receptors. For two receptor variants PP had a significantly decreased potency. A 3D model of the Y4 receptor was generated based on the crystal structure of the human Y1 receptor. The functional responses of the Y4 variants agree well with the 3D model and with the degree of evolutionary conservation of the positions.

In conclusion, these studies reveal unexpectedly large CNV as well as extensive SNP for the NPY4R gene and a possible correlation with BMI that may be due to the differing responses of the naturally occurring receptor variants.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 53
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1479
Keywords
NPY4R, Y4, obesity, CNV
National Category
Biochemistry and Molecular Biology Genetics
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-356573 (URN)978-91-513-0389-5 (ISBN)
Public defence
2018-09-18, C2:301, BMC, Husargatan 3, Uppsala, 09:15 (English)
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Supervisors
Available from: 2018-08-24 Created: 2018-08-01 Last updated: 2018-09-07

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Shebanits, KaterynaGünther, TorstenJohansson, Anna C. V.Maqbool, KhurramFeuk, LarsJakobsson, MattiasLarhammar, Dan

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