Abstract
We sought to establish rapid and specific genotyping methods for G360R mutation and for seven tightly linked markers in the homogentisate dioxygenase gene to address the question of whether G360R is a mutational hot spot or the result of a founder effect, as it has been repeatedly found in alkaptonuric patients from a geographic isolate in Italy.
For G360R and single nucleotide polymorphism genotyping, high-resolution melting analysis was performed. Microsatellites were analysed by multiplex PCR and capillary electrophoresis. To investigate the natural history of the G360R mutation, we genotyped markers in 52 controls and in 8 unrelated patients from the UK and USA, who also segregated the G360R mutation, and calculated its age using DMLE+2.3 software.
A distinct G360R-bearing haplotype was identified in all patients of Caucasian descent. Estimated mutation age was 545 generations (95% credible set, 402–854), suggesting that G360R arose in an ancestor who lived 8,000–10,000 years BC. Archaeological, historical and demographic data support that a G360R carrier has settled the remote valley where present-day population might have a heterozygote frequency of at least 6%.
Given the late health-threatening complications of alkaptonuria and a cure within reach, inhabitants of this isolate would benefit from screening and genetic counselling.
Competing interests: None declared
This paper is dedicated to the memory of Dr. Claudio Castellan
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Acknowledgements
The study was supported by a research grant (Ricerca d’Ateneo 2014) from the University of Florence, Italy, to BP.
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Communicated by: Verena Peters
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Synopsis
Inhabitants of a geographic isolate, where a founder mutation of the homogentisate dioxygenase gene was characterised, would benefit from screening and genetic counselling for alkaptonuria.
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All authors, Berardino Porfirio, Roberta Sestini, Greta Gorelli, Miriam Cordovana, Alessandro Mannoni, Jeanette L. Usher, Wendy J. Introne, William A Gahl and Thierry Vilboux, declare that they have no conflict of interest.
Informed Consent
The Italian subjects involved gave written informed consent in accordance to the Helsinki Declaration, and the study was approved by our institutional review board. Proof that informed consent was obtained will be available upon request. The US and UK DNA samples were provided upon material transfer agreements between the respective Institutions.
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This article does not contain any studies with animal subjects performed by the any of the authors.
Details of the Contributions of Individual Authors
BP planned, conducted and reported the work described in the article with the substantial contribution of RS, GG, MC, JLU and TV for data gathering, analysis and interpretation and of AM, WJI and WAG for revising the manuscript critically for important intellectual content.
BP serves as guarantor for the article, accepts full responsibility for the work and/or the conduct of the study, had access to the data and controlled the decision to publish.
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Electronic-Database Information
Accession numbers and URLs for data in this article are as follows:
http://bioinfo.ut.ee/primer3/ (for guidance in the identification of PCR primers)
http://census.gov/ (for current US population census data)
http://demo.istat.it/ (for current population census of the present-study South Tyrolean valley)
http://ec.europa.eu/eurostat/ (for current European population census data)
http://enzyme.expasy.org/ (the enzyme nomenclature database for HGD [EC 1.13.11.5])
http://hgddatabase.cvtisr.sk/ (a Leiden Open Variation Database [LOVD] v.2.0 on HGD mutations and polymorphisms curated by A. Zatkova)
http://stephenslab.uchicago.edu/phase/download.html (to get PHASE v.2.1, a programme for reconstructing haplotypes from population data)
http://www.dmle.org/ (to get the DMLE+ v2.3 linkage disequilibrium mapping software)
http://www.istat.it/it/archivio/ (for historical population censuses of the present-study South Tyrolean valley)
http://www.ncbi.nlm.nih.gov/nuccore/ (for genomic sequences of HGD and its transcript, accession numbers NG_011957.1 and NM_000187.3, respectively)
http://www.provinz.bz.it/astat/it/ (for data on language usage, migration rates, marriages in the present-study South Tyrolean valley)
https://www.dna.utah.edu/umelt/umelt.html (for HRMA pattern prediction)
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/omim/ (for AKU [OMIM 203500] an HGD [OMIM 607474])
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Porfirio, B. et al. (2016). A Founder Effect for the HGD G360R Mutation in Italy: Implications for a Regional Screening of Alkaptonuria. In: Morava, E., Baumgartner, M., Patterson, M., Rahman, S., Zschocke, J., Peters, V. (eds) JIMD Reports, Volume 30. JIMD Reports, vol 30. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8904_2016_534
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DOI: https://doi.org/10.1007/8904_2016_534
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